Sterilization method and device for cephalosporin C fermentation medium

Abstract

The present invention discloses a method for sterilizing a cephalosporin C fermentation medium, comprising the following steps: step 1, mixing a nitrogen source component and an inorganic salt component of the medium with a first group of water to form a first feed liquid, and mixing the remaining components with a second group of water to form a second feed liquid; step 2, respectively transporting the first feed liquid and the second feed liquid to two sterilizing tanks for steam sterilization; step 3, transporting the first feed liquid and the second feed liquid to a fermentation tank, and simultaneously transporting the third group of water to the fermentation tank, and cooling to the fermentation temperature; the destruction of nutrients in the medium can be reduced, the sterilization time can be shortened, and the amount of steam and cooling water can be reduced. The present invention also discloses a sterilizing device for a cephalosporin C fermentation medium, comprising two sterilizing tanks and a fermentation tank, both of which are connected to the fermentation tank through a pipeline, and the fermentation tank is connected to a sterile water introduction pipe; it is suitable for the sterilization method of the present invention, is simple to operate, and is easy to control.

Description

Technical Field

[0001] The present invention relates to the technical field of antibiotic fermentation production, and more specifically, to a method and device for sterilizing cephalosporin C fermentation medium. Background Art

[0002] Cephalosporin C is a product of the reproduction and metabolism of Cephalosporin Acremonium and a semi-synthetic precursor of cephalosporin antibiotics. Its production level affects the market supply of cephalosporin antibiotics. The production method of cephalosporin C is generally to place Cephalosporin Acremonium in a fermentation medium for fermentation. In order to ensure that the fermentation process is stable and reliable and not easy to be contaminated, the fermentation medium needs to be sterilized before inoculating Cephalosporin Acremonium.

[0003] The fermentation medium of cephalosporin C mostly adopts liquid medium. Commonly used sterilization methods include batch sterilization and continuous sterilization. Batch sterilization refers to the sterilization process in which all the prepared medium is transported to the fermentation tank, the medium is heated by steam, maintained for a certain period of time after reaching the sterilization temperature, and then cooled to the fermentation temperature. There are problems such as long sterilization time and more destruction of the nutrients in the medium. Although continuous sterilization has a short sterilization time and less destruction of the nutrients in the medium, the fermentation medium of cephalosporin C contains more solid materials, oils and proteins, has a large viscosity, and an unstable flow rate, which will affect the sterilization effect and increase the probability of contamination. Therefore, it is very important to provide a better sterilization method for cephalosporin C fermentation medium. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a method for sterilizing a cephalosporin C fermentation medium, which can reduce the destruction of nutrients in the medium, shorten the sterilization time, and thereby reduce the amount of steam and cooling water used;

[0006] Also provided is a sterilizing device for cephalosporin C fermentation medium, which is suitable for the sterilization method of the present invention, is easy to operate and control, and is beneficial to further reduce the destruction of nutrients in the medium and shorten the sterilization time.

[0007] In order to achieve these objects and other advantages according to the present invention, a method for sterilizing a cephalosporin C fermentation medium is provided, comprising the following steps:

[0008] Step 1: prepare the nitrogen source component and inorganic salt component of the culture medium with the first group of water into a first liquid, and prepare the remaining components of the culture medium with the second group of water into a second liquid; wherein the amount of the first group of water is 10-20% of the amount of water used in the culture medium, and the amount of the second group of water is 10-20% of the amount of water used in the culture medium;

[0009] Step 2: transporting the first liquid feed and the second liquid feed to two sterilization tanks respectively for steam sterilization;

[0010] Step three, conveying the sterilized first liquid and the sterilized second liquid into a fermentation tank, and conveying the third group of water into the fermentation tank at the same time, cooling to the fermentation temperature; wherein the amount of the third group of water is the remaining water in the culture medium, and the third group of water is sterile water.

[0011] Preferably, the pressure of steam sterilization is 0.09-0.11 MPa, the temperature is 120-125° C., and the insulation time is 20-30 min.

[0012] Preferably, the temperature of the third group of water is room temperature.

[0013] Preferably, the volume of one of the sterilization tanks for containing the first liquid is 1.2 to 1.3 times the volume of the first liquid, and the volume of the other sterilization tank for containing the second liquid is 1.2 to 1.3 times the volume of the first liquid.

[0014] The present invention also provides a sterilization device for cephalosporin C fermentation medium, characterized in that it comprises:

[0015] Two sterilization tanks, either of which is connected to a steam introduction pipe, a feeding port and a steam exhaust valve are provided on the top of either sterilization tank, and a discharge port is provided on the bottom; the first feed liquid is transported to one of the sterilization tanks, and the second feed liquid is transported to the other sterilization tank;

[0016] The fermentation tank is provided with two feed ports, one feed port is correspondingly provided for each sterilization tank, and the discharge port of any sterilization tank is connected with the corresponding feed port through a pipeline; the fermentation tank is connected with a sterile water inlet pipe, and the third group of water is transported to the fermentation tank through the sterile water inlet pipe.

[0017] Preferably, any sterilization tank comprises:

[0018] The first tank body is a hollow cylindrical structure, with a feeding port and a steam exhaust valve on the top and a discharge port on the bottom; a steam inlet is provided on the first tank body, and a steam introduction pipe is connected to the steam inlet;

[0019] A first rotating shaft, which is coaxially disposed in the first tank body and is rotatably connected to the first tank body;

[0020] A pair of first vertical rods are vertically arranged on both sides of the first rotating shaft, and a plurality of triangular spikes are arranged at intervals on any of the first vertical rods;

[0021] A pair of first connecting rods, one first connecting rod is provided for each first vertical rod, and the two ends of any first connecting rod are respectively hinged to the upper end of the first vertical rod corresponding thereto and the first rotating shaft; a collar is sleeved on any first connecting rod, and any collar can move along the length direction of the first connecting rod corresponding thereto;

[0022] A pair of first cylinders, one first cylinder is correspondingly arranged for one collar, and any first cylinder is connected to the first rotating shaft; the free end of the piston rod of any first cylinder extends vertically downward and is hinged to the collar corresponding thereto;

[0023] Two groups of stirring mechanisms are arranged on both sides of the first rotating shaft. A group of stirring mechanisms is correspondingly arranged directly below a first connecting rod. Any group of stirring mechanisms includes a plurality of stirring mechanisms arranged at intervals in the vertical direction. Any stirring mechanism includes:

[0024] A pair of second connecting rods are arranged at intervals in the vertical direction, and two ends of any second connecting rod are respectively hinged to the corresponding first vertical rod and the first rotating shaft;

[0025] The second vertical rod is arranged between a pair of second connecting rods, and two ends thereof are respectively connected with the pair of second connecting rods in a sliding manner along the length direction of the second connecting rods; a plurality of triangular spikes are arranged at intervals on the second vertical rod.

[0026] Preferably, the fermenter comprises:

[0027] The second tank body is a hollow cylindrical structure, and is provided with two sterile water inlets and two feed ports, and any one of the sterile water inlets is connected to the sterile water introduction pipe; the side wall of the second tank body is an interlayer, and a cooling pipe is provided in the interlayer; the two feed ports are respectively connected to the two discharge ports through a pipeline; and the two sterile water inlets are both connected to the sterile water introduction pipe;

[0028] A second rotating shaft is coaxially disposed in the second tank body and is rotatably connected to the second tank body; a plurality of stirring blades are spaced apart on the second rotating shaft;

[0029] a stopper, which is a conical structure coaxially sleeved on the middle part of the second rotating shaft and with its vertex facing upwards; the stopper is fixedly arranged in the second tank body and is rotatably connected to the second rotating shaft; the diameter of the bottom surface of the stopper is smaller than the inner diameter of the second tank body;

[0030] The shell is a hollow frustum structure which is larger at the top and smaller at the bottom and open at both ends. The shell is arranged above the block. The upper end of the shell is sealed and slidably connected to the inner wall of the second tank body in the vertical direction. The diameter of the bottom surface of the shell is smaller than the diameter of the bottom surface of the block. The two feed ports are respectively located above and below the block, and the two sterile water inlets are respectively located above and below the block.

[0031] Preferably, for any stirring mechanism, a pair of second connecting rods are provided with a groove extending along the length direction on the side away from each other, and a pair of second connecting rods are provided with a through hole extending along the length direction on the side close to each other, any through hole is connected to the corresponding groove, and the longitudinal section of any groove is arc-shaped; the two ends of the second vertical rod respectively pass through the corresponding through holes and are provided with a ball, and any ball rolls in the corresponding groove.

[0032] Preferably, the axis of any triangular thorn is perpendicular to the first vertical rod / second vertical rod where it is located.

[0033] Preferably, it also includes: a pair of second cylinders, which are arranged at intervals along the radial direction of the second tank body, any second cylinder is arranged on the top of the second tank body, and the free end of the piston rod of any second cylinder extends vertically downward and is connected to the shell.

[0034] The present invention has at least the following beneficial effects:

[0035] 1. The sterilization method of the present invention transports the nitrogen source component and the carbon source component of the culture medium to two sterilization tanks for steam sterilization, which can effectively avoid the Maillard reaction between the ammonia substances in the first liquid and the reducing sugars in the second liquid, greatly reducing the destruction of the nutrients in the culture medium; the first liquid and the second liquid use less water and have a smaller volume, which can shorten the heating time of steam sterilization, thereby shortening the sterilization time and reducing the amount of steam used;

[0036] 2. The sterilization device of the present invention comprises two sterilization tanks and a fermentation tank, the two sterilization tanks are connected to the fermentation tank through a pipeline respectively, the fermentation tank is connected to the sterile water introduction pipe, the first feed liquid and the second feed liquid are respectively steam sterilized in the two sterilization tanks, and then transported to the fermentation tank through a pipeline respectively, and the third group of water is transported to the fermentation tank at the same time, and mixed evenly in the fermentation tank and cooled to prepare a sterilized culture medium; it is suitable for the sterilization method of the present invention, which is simple to operate and easy to control;

[0037] 3. The sterilizing tank of the present invention is provided with a first rotating shaft, a pair of first cylinders, a pair of collars, a pair of first connecting rods, a pair of vertical rods and two groups of stirring mechanisms. Under the cooperation of the first rotating shaft, a pair of first cylinders, a pair of collars and a pair of connecting rods, the pair of vertical rods and the two groups of stirring mechanisms stir the first liquid / second liquid in the sterilizing tank in multiple dimensions, and the stirring effect and efficiency are good, which can effectively avoid the heat transfer difficulty caused by the high concentration of organic matter forming a thin film around the microbial cells, the heat transfer speed is fast and the temperature rise is more uniform, which is conducive to shortening the sterilization time, reducing the amount of steam used and improving the sterilization effect;

[0038] 4. The fermentation tank of the present invention is provided with a second rotating shaft, a plurality of stirring blades, a block and a shell. The interior of the fermentation tank is divided into an upper cavity and a lower cavity which are not connected to each other by the movement of the shell. The sterilized first feed liquid and the sterilized second feed liquid are first respectively transported to the upper cavity and the lower cavity which are not connected to each other. When the temperature of the second tank body drops to below 60°C, the upper cavity is connected to the lower cavity again by the movement of the shell, and the first feed liquid, the second feed liquid and the third group of water are mixed evenly, and cooled to the fermentation temperature to prepare a sterilized culture medium, which can avoid the contact between the high-temperature first feed liquid and the high-temperature second feed liquid, further avoid the Maillard reaction of the ammonia substances in the first feed liquid and the reducing sugar in the second feed liquid, and reduce the destruction of the nutrients in the culture medium; at the same time, the third group of water is divided into two parts and transported to the upper cavity and the lower cavity respectively, and stirred. The sterile water at room temperature cools the first feed liquid in the upper cavity and the second feed liquid in the lower cavity, which can shorten the cooling time, which is conducive to shortening the sterilization time, and further reducing the amount of cooling water.

[0039] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic structural diagram of the sterilization device for cephalosporin C fermentation medium according to one of the technical solutions of the present invention;

[0041] Figure 2 This is a schematic diagram of the structure of the sterilization tank according to one of the technical solutions of the present invention;

[0042] Figure 3 This is a schematic structural diagram of the fermentation tank according to one of the technical solutions of the present invention;

[0043] Figure 4 A top view of a second connecting rod according to one of the technical solutions of the present invention;

[0044] Explanation of the reference numerals: sterilizing tank 1; steam inlet pipe 2; discharge port 3; fermentation tank 4; pipeline 5; sterile water inlet pipe 6; first tank body 7; first rotating shaft 8; first vertical rod 9; triangular thorn 10; first connecting rod 11; collar 12; first cylinder 13; second connecting rod 14; second vertical rod 15; second tank body 16; interlayer 17; cooling pipe 18; second rotating shaft 19; stirring blade 20; block 21; shell 22; groove 23; through hole 24; ball 25; second cylinder 26; valve 27; liquid pump 28; insulation layer 29; first motor 30; second motor 31; third connecting rod 32. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0046] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified; in the description of the present invention, the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the accompanying drawings, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0047] The present invention provides a method for sterilizing a cephalosporin C fermentation medium, comprising the following steps:

[0048] Step 1: prepare the nitrogen source component and inorganic salt component of the culture medium with the first group of water into a first liquid, and prepare the remaining components of the culture medium with the second group of water into a second liquid; wherein the amount of the first group of water is 10-20% of the amount of water used in the culture medium, and the amount of the second group of water is 10-20% of the amount of water used in the culture medium;

[0049] Step 2: transporting the first liquid feed and the second liquid feed to two sterilization tanks 1 respectively for steam sterilization;

[0050] Step 3: transport the sterilized first liquid and the sterilized second liquid to the fermentation tank 4, and at the same time transport the third group of water to the fermentation tank 4, cool it to the fermentation temperature, and prepare a sterilized culture medium; wherein the amount of the third group of water is the remaining water in the culture medium, and the third group of water is sterile water;

[0051] The sterilization method of the present invention transports the nitrogen source component and the carbon source component of the culture medium to two sterilization tanks 1 for steam sterilization, which can effectively avoid the Maillard reaction between the ammonia substances in the first liquid and the reducing sugars in the second liquid, greatly reducing the destruction of the nutrients in the culture medium; the water consumption of the first liquid and the second liquid is small, and the volume is small, which can shorten the heating time of steam sterilization, which is conducive to shortening the sterilization time and reducing the amount of steam; in addition, the third group of water is sterile water, so that it can be directly mixed with the sterilized first liquid and the sterilized second liquid to avoid contamination.

[0052] In another technical solution, the pressure of steam sterilization is 0.09-0.11 MPa, the temperature is 120-125°C, and the insulation time is 20-30 minutes, which can ensure the sterilization effect.

[0053] In another technical solution, the temperature of the third group of water is room temperature; the sterile water at room temperature can directly and quickly cool down the first liquid and the second liquid, which is beneficial to shorten the cooling time and thus reduce the amount of cooling water used.

[0054] In another technical solution, the volume of one of the sterilization tanks 1 for containing the first liquid is 1.2 to 1.3 times the volume of the first liquid, and the volume of the other sterilization tank 1 for containing the second liquid is 1.2 to 1.3 times the volume of the first liquid; the appropriate volume of the sterilization tank 1 can shorten the sterilization time and reduce the amount of steam used while ensuring the sterilization effect.

[0055] like Figures 1 to 4 As shown, the present invention provides a sterilization device for cephalosporin C fermentation medium, comprising:

[0056] Two sterilization tanks 1, either sterilization tank 1 is connected to a steam introduction pipe 2, a feeding port and a steam exhaust valve are provided on the top of either sterilization tank 1, and a discharge port 3 is provided on the bottom; a first feed liquid is transported into one of the sterilization tanks 1, and a second feed liquid is transported into the other sterilization tank 1;

[0057] The fermentation tank 4 is provided with two feed ports, one of which corresponds to one sterilization tank 1, and the discharge port 3 of any sterilization tank 1 is connected to the corresponding feed port through a pipeline 5; the fermentation tank 4 is connected to a sterile water introduction pipe 6, and the third group of water is transported to the fermentation tank 4 through the sterile water introduction pipe 6;

[0058] In the above technical scheme, valves 27 are provided on the discharge ports 3 of the two sterilizing tanks 1 and the discharge port of the fermentation tank 4, and a liquid pump 28 is provided on any pipeline 5; the valves 27 on the discharge ports 3 of the two sterilizing tanks 1 are closed, and the prepared first liquid is transported to one of the sterilizing tanks 1 through one of the feeding ports, and the prepared second liquid is transported to the other sterilizing tank 1 through the other feeding port, and saturated steam is introduced into the two sterilizing tanks 1 through the steam introduction pipe 2, and the pressure in the sterilizing tank 1 is controlled by the exhaust valve, so as to respectively control the first liquid and the second liquid. The feed liquid is steam sterilized to obtain a sterilized first feed liquid and a sterilized second feed liquid; the valve 27 on the discharge port of the fermentation tank 4 is kept closed, the valves 27 on the discharge ports 3 of the two sterilization tanks 1 and the liquid pumps 28 on the two pipelines 5 are opened, and the sterilized first feed liquid and the sterilized second feed liquid are respectively transported to the fermentation tank 4 through a pipeline 5, and at the same time, the third group of water is transported to the fermentation tank 4 through a sterile water introduction pipe 6, the first feed liquid, the second feed liquid and the third group of water are evenly mixed, and cooled to the fermentation temperature by cooling water to prepare a sterilized culture medium;

[0059] The sterilization device of the present invention comprises two sterilization tanks 1 and a fermentation tank 4. The two sterilization tanks 1 are connected to the fermentation tank 4 through a pipeline 5 respectively. The fermentation tank 4 is connected to a sterile water inlet pipe 6. The first liquid and the second liquid are respectively steam sterilized in the two sterilization tanks 1 and then transported to the fermentation tank 4 through a pipeline 5 respectively. Meanwhile, a third group of water is transported to the fermentation tank 4, mixed evenly and cooled in the fermentation tank 4 to prepare a sterilized culture medium. The sterilization device is suitable for the sterilization method of the present invention, is easy to operate, and is convenient to control.

[0060] In another technical solution, any sterilization tank 1 comprises:

[0061] The first tank body 7 is a hollow cylindrical structure. A feeding port and a steam exhaust valve are provided on the top of the first tank body 7, and a discharge port 3 is provided on the bottom. A steam inlet is provided on the first tank body 7, and a steam introduction pipe 2 is connected to the steam inlet.

[0062] A first rotating shaft 8 is coaxially disposed in the first tank body 7 and is rotatably connected to the first tank body 7; a heat-insulating layer 29 is disposed on the outer wall of the first tank body 7; a first motor 30 is disposed on the top of the first tank body 7, and an output end of the first motor 30 is connected to an upper end of the first rotating shaft 8;

[0063] A pair of first vertical rods 9, which are vertically arranged on both sides of the first rotating shaft 8, and a plurality of triangular spikes 10 are arranged at intervals on any first vertical rod 9;

[0064] A pair of first connecting rods 11, one first vertical rod 9 is provided with one first connecting rod 11, and the two ends of any first connecting rod 11 are respectively hinged with the upper end of the first vertical rod 9 corresponding thereto and the first rotating shaft 8; a collar 12 is sleeved on any first connecting rod 11, and any collar 12 can move along the length direction of the first connecting rod 11 corresponding thereto;

[0065] A pair of first cylinders 13, one first cylinder 13 is provided for each collar 12, and any first cylinder 13 is connected to the first rotating shaft 8; the free end of the piston rod of any first cylinder 13 extends vertically downward and is hinged to the corresponding collar 12;

[0066] Two groups of stirring mechanisms are arranged on both sides of the first rotating shaft 8. A group of stirring mechanisms is correspondingly arranged directly below a first connecting rod 11. Any group of stirring mechanisms includes a plurality of stirring mechanisms arranged at intervals in the vertical direction. Any stirring mechanism includes:

[0067] A pair of second connecting rods 14 are arranged at intervals in the vertical direction, and two ends of any second connecting rod 14 are respectively hinged to the corresponding first vertical rod 9 and the first rotating shaft 8;

[0068] The second vertical rod 15 is disposed between the pair of second connecting rods 14, and the two ends are respectively connected to the pair of second connecting rods 14 in a sliding manner along the length direction of the second connecting rods 14; a plurality of triangular spikes 10 are arranged at intervals on the second vertical rod 15;

[0069] After the first liquid feed and the second liquid feed are respectively transported to the two sterilization tanks 1, the first motor 30 is started to drive the first rotating shaft 8 to rotate, and the first rotating shaft 8 drives a pair of first vertical rods 9 and two groups of stirring mechanisms to rotate around the first rotating shaft 8, and the pair of first vertical rods 9 and the two groups of stirring mechanisms stir the first liquid feed / second liquid feed in the sterilization tank 1; at the same time, the pair of first cylinders 13 are driven to make the free end of the piston rod of any first cylinder 13 reciprocate upward and downward in the vertical direction, and under the action of the pair of collars 12 and the pair of first connecting rods 11, the pair of first vertical rods 9 swing up and down while rotating around the first rotating shaft 8, and any stirring mechanism also swings up and down. The stirring mechanism rotates around the first rotating shaft 8 and swings up and down at the same time, which can further improve the stirring effect and efficiency; and for any stirring mechanism, the pair of second connecting rods 14 simultaneously performs upward and downward tilting reciprocating motion under the action of the up and down swinging of the first vertical rod 9, and the second vertical rod 15 located between the pair of second connecting rods 14 slides back and forth along the pair of second connecting rods 14, stirring the first liquid / second liquid in the sterilizing tank 1, which is conducive to further improving the stirring effect and efficiency; in addition, any first vertical rod 9 and any second vertical rod 15 of the stirring mechanism are provided with a plurality of triangular spikes 10, which is conducive to further ensuring the stirring efficiency and effect;

[0070] A pair of vertical rods and two groups of stirring mechanisms stir the first liquid / second liquid in the sterilization tank 1 in multiple dimensions, with good stirring effect and efficiency, which can effectively avoid the heat transfer difficulty caused by the formation of a thin film around the microbial cells by high concentrations of organic matter. The heat transfer speed is fast and the temperature rises more evenly, which is conducive to shortening the sterilization time, reducing steam consumption and improving the sterilization effect.

[0071] In another technical solution, the fermentation tank 4 comprises:

[0072] The second tank body 16 is a hollow cylindrical structure. Two sterile water inlets and two feed ports are provided on the second tank body 16. Any sterile water inlet is connected to the sterile water introduction pipe 6. The side wall of the second tank body 16 is an interlayer 17. A cooling pipe 18 is provided in the interlayer 17. The second tank body 16 is cooled and maintained at the fermentation temperature by passing cooling water into the cooling pipe 18. The two feed ports are respectively connected to the two discharge ports 3 through a pipeline 5. The two sterile water inlets are both connected to the sterile water introduction pipe 6.

[0073] A second rotating shaft 19 is coaxially disposed in the second tank body 16 and is rotatably connected to the second tank body 16; a plurality of stirring blades 20 are arranged at intervals on the second rotating shaft 19; a second motor 31 is arranged on the top of the second tank body 16, and an output end of the second motor 31 is connected to an upper end of the second rotating shaft 19;

[0074] The stopper 21 is a conical structure coaxially sleeved in the middle of the second rotating shaft 19 and with the vertex facing upward. The stopper 21 is fixed in the second tank body 16 and is rotatably connected to the second rotating shaft 19. The diameter of the bottom surface of the stopper 21 is smaller than the inner diameter of the second tank body 16. Both sides of the stopper 21 are connected to the inner wall of the second tank body 16 through a third connecting rod 32 respectively.

[0075] The shell 22 is a hollow truncated cone structure with a larger upper portion and a smaller lower portion and open at both ends. The shell 22 is disposed above the stopper 21. The upper end of the shell 22 is sealed and slidably connected to the inner side wall of the second tank body 16 in the vertical direction. The diameter of the bottom surface of the shell 22 is smaller than the diameter of the bottom surface of the stopper 21. Two feed ports are respectively located above and below the stopper 21, and two sterile water inlets are respectively located above and below the stopper 21.

[0076] Before the sterilized first liquid and the sterilized second liquid are introduced, the valve 27 on the discharge port of the fermentation tank 4 is kept closed, and the shell 22 is moved downward to the limit in the vertical direction, and the lower end of the shell 22 is in sealing contact with the side wall of the stopper 21, so that the interior of the second tank body 16 is divided into an upper cavity and a lower cavity that are not connected to each other;

[0077] After the first liquid feed and the second liquid feed are respectively steam sterilized in the two sterilizing tanks 1, the valves 27 on the discharge ports 3 of the two sterilizing tanks 1 and the liquid pumps 28 on the two pipelines 5 are opened, and the sterilized first liquid feed and the sterilized second liquid feed are respectively introduced into a pipeline 5, and then transported to the upper cavity and the lower cavity through the two feed ports. At the same time, the third group of water at room temperature is evenly divided into two parts, respectively introduced into a sterile water introduction pipe 6, and then transported to the upper cavity and the lower cavity through two sterile water inlets; and the second motor 31 is started to drive the second rotating shaft 19 to rotate, and the multiple stirring blades 20 rotate accordingly. The multiple stirring blades 20 located in the upper cavity stir and mix the first liquid feed and a part of the third group of water in the upper cavity, and the multiple stirring blades 20 located in the lower cavity stir and mix the second liquid feed and another part of the third group of water in the lower cavity, and at the same time, cooling water is introduced into the cooling pipe 18 in the interlayer 17 to cool the second tank body 16;

[0078] When the temperature of the second tank body 16 drops below 60°C, the shell 22 is moved upward to the limit in the vertical direction, and the lower end of the shell 22 is separated from the side wall of the stopper 21 to form an opening, so that the upper cavity and the lower cavity in the second tank body 16 are connected, and the first feed liquid in the upper cavity and a first mixed liquid of a part of the third group of water are mixed with the second feed liquid in the lower cavity and another part of the second mixed liquid of the third group of water through the opening, and are stirred evenly by a plurality of stirring blades 20, and at the same time, the cooling water introduced into the cooling pipe 18 in the interlayer 17 cools the second tank body 16 to the fermentation temperature to prepare a sterilized culture medium;

[0079] The sterilized first liquid and the sterilized second liquid are first transported to the upper cavity and the lower cavity which are not connected to each other, respectively. When the temperature of the second tank body 16 drops to below 60°C, the upper cavity is connected to the lower cavity, the first liquid, the second liquid and the third group of water are mixed evenly, and the temperature is lowered to the fermentation temperature to prepare a sterilized culture medium, which can avoid the contact between the high-temperature first liquid and the high-temperature second liquid, further avoid the Maillard reaction between the ammonia substances in the first liquid and the reducing sugars in the second liquid, and reduce the destruction of the nutrients in the culture medium; at the same time, the third group of water is divided into two parts and transported to the upper cavity and the lower cavity respectively, and stirred. The sterile water at room temperature cools the first liquid in the upper cavity and the second liquid in the lower cavity, which can shorten the cooling time, which is beneficial to shorten the sterilization time and reduce the amount of cooling water.

[0080] In another technical solution, for any stirring mechanism, a groove 23 extending along the length direction is provided on the side away from each other of a pair of second connecting rods 14, and a through hole 24 extending along the length direction is provided on the side close to each other of a pair of second connecting rods 14, any through hole 24 is connected to the corresponding groove 23, and the longitudinal section of any groove 23 is arc-shaped; the two ends of the second vertical rod 15 respectively pass through the corresponding through holes 24, and are provided with a ball 25, and any ball 25 is rolled in the corresponding groove 23; the reciprocating sliding of the second vertical rod 15 between the pair of second connecting rods 14 along the pair of second connecting rods 14 is smoother and more stable, and under the action of the shaking of the first liquid / second liquid in the sterilization tank 1, the second vertical rod 15 will rotate at a certain angle, which is beneficial to further ensure the stirring effect and efficiency.

[0081] In another technical solution, the axis of any triangular spike 10 is perpendicular to the first vertical rod 9 / second vertical rod 15 where it is located, which is beneficial to ensure the stirring effect and efficiency.

[0082] In another technical solution, it also includes: a pair of second cylinders 26, which are arranged at intervals along the radial direction of the second tank body 16, and any second cylinder 26 is arranged at the top of the second tank body 16, and the free end of the piston rod of any second cylinder 26 extends vertically downward and is connected to the shell 22; the shell 22 is driven to move in the vertical direction by a pair of second cylinders 26, which is simple to operate and easy to control.

[0083] <Example 1>

[0084] The method for sterilizing cephalosporin C fermentation medium comprises the following steps:

[0085] Step 1, the composition of the cephalosporin C fermentation medium is as follows: sterile water as the medium, peanut powder 20g / L, cottonseed powder 16g / L, gluten powder 20g / L, corn steep liquor 50g / L, fructose 40g / L, gelatinized starch 30g / L, soybean oil 80g / L, methionine 6.0g / L, ammonium sulfate 6.0g / L, calcium sulfate 5.0g / L, calcium carbonate 5.0g / L, defoamer 0.5ml / L;

[0086] The nitrogen source components (peanut powder, cottonseed powder, gluten powder, corn steep liquor and methionine), the inorganic salt components (ammonium sulfate, calcium sulfate and calcium carbonate) are uniformly mixed with the first group of water (sterile water medium), and the pH value is adjusted to 7.2 with 20% sodium hydroxide solution to prepare the first feed liquid, and the remaining components (fructose, gelatinized starch, soybean oil and defoamer) are uniformly mixed with the second group of water (sterile water medium), and the pH value is adjusted to 7.2 with 20% sodium hydroxide solution to prepare the second feed liquid; wherein the dosage of each component of the fermentation medium and the sterile water are determined according to the amount of the prepared medium, the dosage of the first group of water is 15% of the amount of water used in the medium, and the dosage of the second group of water is 15% of the amount of water used in the medium;

[0087] Step 2, first sterilize the sterilization device by air, the sterilization includes two sterilization tanks 1 and a fermentation tank 4, any sterilization tank 1 is connected with a steam introduction pipe 2, any sterilization tank 1 is provided with a feeding port and a steam exhaust valve on the top, and a discharge port 3 on the bottom; the fermentation tank 4 is provided with two feed ports, one sterilization tank 1 is provided with a feed port, and the discharge port 3 of any sterilization tank 1 is connected with the corresponding feed port through a pipeline 5; the fermentation tank 4 is connected with a sterile water introduction pipe 6; valves 27 are provided on the discharge ports 3 of the two sterilization tanks 1 and the discharge port of the fermentation tank 4, and a liquid pump 28 is provided on any pipeline 5; wherein, the volume of one of the sterilization tanks 1 for holding the first liquid is 1.2 to 1.3 times the volume of the first liquid, and the volume of the other sterilization tank 1 for holding the second liquid is 1.2 to 1.3 times the volume of the first liquid;

[0088] After air sterilization, close the valves 27 on the discharge ports 3 of the two sterilization tanks 1, deliver the first liquid feed to one of the sterilization tanks 1 through one of the feed ports, deliver the second liquid feed to the other sterilization tank 1 through the other feed port, introduce saturated steam into the two sterilization tanks 1 through the steam introduction pipe 2, raise the temperature in the sterilization tank 1 to 120-125°C, and control the pressure in the sterilization tank 1 to 0.09-0.11MPa through the exhaust valve, keep warm for 25 minutes, and perform steam sterilization to obtain the sterilized first liquid feed and the sterilized second liquid feed;

[0089] Step 3: keep the valve 27 on the discharge port of the fermentation tank 4 closed, open the valves 27 on the discharge ports 3 of the two sterilization tanks 1 and the liquid pumps 28 on the two pipelines 5, and transport the sterilized first liquid and the sterilized second liquid to the fermentation tank 4 through a pipeline 5 respectively, and at the same time transport the third group of water at room temperature to the fermentation tank 4 through the sterile water inlet pipe 6, mix the first liquid, the second liquid and the third group of water evenly, and use cooling water to cool them to the fermentation temperature to prepare a sterilized culture medium; wherein, the amount of the third group of water is the remaining water in the culture medium.

[0090] <Example 2>

[0091] The difference from Example 1 is that in step 1, the amount of the first group of water is 15% of the amount of water used in the culture medium, and the amount of the second group of water is 10% of the amount of water used in the culture medium; in step 2, the temperature is kept for 20 minutes; the other steps and parameters are the same as in Example 1;

[0092] <Example 3>

[0093] The difference from Example 1 is that in step 1, the amount of the first group of water is 20% of the amount of water used in the culture medium, and the amount of the second group of water is 20% of the amount of water used in the culture medium; in step 2, the temperature is kept for 30 minutes; the other steps and parameters are the same as in Example 1;

[0094] <Example 4>

[0095] The difference from <Example 1> is that any sterilization tank 1 in the sterilization device comprises:

[0096] The first tank body 7 is a hollow cylindrical structure. A feeding port and a steam exhaust valve are provided on the top of the first tank body 7, and a discharge port 3 is provided on the bottom. A steam inlet is provided on the first tank body 7, and a steam introduction pipe 2 is connected to the steam inlet.

[0097] A first rotating shaft 8 is coaxially disposed in the first tank body 7 and is rotatably connected to the first tank body 7; a heat-insulating layer 29 is disposed on the outer wall of the first tank body 7; a first motor 30 is disposed on the top of the first tank body 7, and an output end of the first motor 30 is connected to an upper end of the first rotating shaft 8;

[0098] A pair of first vertical rods 9 are vertically arranged on both sides of the first rotating shaft 8, and a plurality of triangular spikes 10 are arranged at intervals on any first vertical rod 9; the axis of any triangular spike 10 is perpendicular to the first vertical rod 9 where it is located;

[0099] A pair of first connecting rods 11, one first vertical rod 9 is provided with one first connecting rod 11, and the two ends of any first connecting rod 11 are respectively hinged with the upper end of the first vertical rod 9 corresponding thereto and the first rotating shaft 8; a collar 12 is sleeved on any first connecting rod 11, and any collar 12 can move along the length direction of the first connecting rod 11 corresponding thereto;

[0100] A pair of first cylinders 13, one first cylinder 13 is provided for each collar 12, and any first cylinder 13 is connected to the first rotating shaft 8; the free end of the piston rod of any first cylinder 13 extends vertically downward and is hinged to the corresponding collar 12;

[0101] Two groups of stirring mechanisms are arranged on both sides of the first rotating shaft 8. A group of stirring mechanisms is correspondingly arranged directly below a first connecting rod 11. Any group of stirring mechanisms includes a plurality of stirring mechanisms arranged at intervals in the vertical direction. Any stirring mechanism includes:

[0102] A pair of second connecting rods 14 are arranged at intervals in the vertical direction, and the two ends of any second connecting rod 14 are respectively hinged to the corresponding first vertical rod 9 and the first rotating shaft 8; a groove 23 extending along the length direction is provided on the side away from each other of the pair of second connecting rods 14, and a through hole 24 extending along the length direction is provided on the side close to each other of the pair of second connecting rods 14, and any through hole 24 is connected to the corresponding groove 23, and the longitudinal section of any groove 23 is arc-shaped;

[0103] The second vertical rod 15 is arranged between the pair of second connecting rods 14, and the two ends are respectively connected to the pair of second connecting rods 14 in a sliding manner along the length direction of the second connecting rods 14; the two ends of the second vertical rod 15 pass through the corresponding through holes 24, and are provided with a ball 25, and any ball 25 is rolled in the corresponding groove 23; a plurality of triangular thorns 10 are arranged on the second vertical rod 15 at intervals; the axis of any triangular thorn 10 is perpendicular to the second vertical rod 15 where it is located;

[0104] The first liquid and the second liquid are respectively conveyed to two sterilizing tanks 1 for steam sterilization, and the first motor 30 is started at the same time to stir the first liquid / second liquid in the sterilizing tank 1; other steps and parameters are the same as in Example 1.

[0105] <Example 5>

[0106] The difference from <Example 4> is that the fermentation tank 4 in the sterilization device includes:

[0107] The second tank body 16 is a hollow cylindrical structure. Two sterile water inlets and two feed ports are provided on the second tank body 16. Any sterile water inlet is connected to the sterile water introduction pipe 6. The side wall of the second tank body 16 is an interlayer 17. A cooling pipe 18 is provided in the interlayer 17. The second tank body 16 is cooled and maintained at the fermentation temperature by passing cooling water into the cooling pipe 18. The two feed ports are respectively connected to the two discharge ports 3 through a pipeline 5. The two sterile water inlets are both connected to the sterile water introduction pipe 6.

[0108] A second rotating shaft 19 is coaxially disposed in the second tank body 16 and is rotatably connected to the second tank body 16; a plurality of stirring blades 20 are arranged at intervals on the second rotating shaft 19; a second motor 31 is arranged on the top of the second tank body 16, and an output end of the second motor 31 is connected to an upper end of the second rotating shaft 19;

[0109] The stopper 21 is a conical structure coaxially sleeved in the middle of the second rotating shaft 19 and with the vertex facing upward. The stopper 21 is fixed in the second tank body 16 and is rotatably connected to the second rotating shaft 19. The diameter of the bottom surface of the stopper 21 is smaller than the inner diameter of the second tank body 16. Both sides of the stopper 21 are connected to the inner wall of the second tank body 16 through a third connecting rod 32 respectively.

[0110] The shell 22 is a hollow truncated cone structure with a larger upper portion and a smaller lower portion and open at both ends. The shell 22 is disposed above the stopper 21. The upper end of the shell 22 is sealed and slidably connected to the inner side wall of the second tank body 16 in the vertical direction. The diameter of the bottom surface of the shell 22 is smaller than the diameter of the bottom surface of the stopper 21. Two feed ports are respectively located above and below the stopper 21, and two sterile water inlets are respectively located above and below the stopper 21.

[0111] The sterilized first liquid feed and the sterilized second liquid feed are respectively transported to the fermentation tank 4 through a pipeline 5, and the third group of water at room temperature is divided into two parts, which are respectively transported to the fermentation tank 4 through two sterile water inlets, and the temperature is cooled in the fermentation tank 4. At the same time, the second motor 31 is started to stir the first liquid feed, the second liquid feed and the third group of water in the fermentation tank 4 to prepare a sterilized culture medium; the other steps and parameters are the same as in <Example 4>.

[0112] <Comparative Example 1>

[0113] The fermentation medium of cephalosporin C was sterilized by batch sterilization method. The components of the fermentation medium were the same as those in Example 1.

[0114] <Comparative Example 2>

[0115] The fermentation medium of cephalosporin C was sterilized by continuous sterilization method. The components of the fermentation medium were the same as those in Example 1.

[0116] The amount of the fermentation medium prepared in Examples 1 to 5 and Comparative Examples 1 to 2 was 100 m 3 , record the sterilization time of Examples 1 to 5 and Comparative Examples 1 to 2, the sterilization time refers to the duration from the start of steam introduction to the first feed liquid and the second feed liquid to the cooling to the fermentation temperature, respectively detect the post-sterilization amino nitrogen and post-sterilization total sugar of the sterilized culture medium obtained in Examples 1 to 5 and Comparative Example 1, and judge the contamination, and calculate the contamination rate, the contamination rate (%) = fermentation tank 4 contamination batch / total tank batch × 100%, amino nitrogen is determined by formaldehyde method, total sugar is determined by Fehling's reagent method, and the contamination is determined by slant culture inspection method; the results are shown in Table 1;

[0117] Table 1 Sterilization results of Examples 1 to 5 and Comparative Examples 1 to 2

[0118]

[0119] As can be seen from Table 1, compared with Comparative Example 1, the sterilization time of the culture medium of Examples 1 to 5 is significantly shortened, and compared with Examples 1 to 3, the sterilization time of the culture medium of Examples 4 to 5 is shortened to a certain extent, and is not much different from the sterilization time of Comparative Example 2; compared with Comparative Examples 1 and 2, the contents of post-sterilization amino nitrogen and post-sterilization total sugars of the culture medium of Examples 1 to 5 are significantly increased, and compared with Examples 1 to 3, the contents of post-sterilization amino nitrogen and post-sterilization total sugars of the culture medium of Example 4 are slightly increased, and compared with Example 4, the contents of post-sterilization amino nitrogen and post-sterilization total sugars of the culture medium of Example 5 are significantly increased; compared with Comparative Example 2, the bacterial contamination rates of Examples 1 to 5 are significantly reduced, and compared with Examples 1 to 3, the bacterial contamination rates of Examples 4 to 5 are significantly reduced, and are not much different from the bacterial contamination rate of Comparative Example 1;

[0120] The results show that the sterilization method of the present invention can greatly shorten the sterilization time, reduce the destruction of the nutrients in the culture medium, and reduce the contamination rate. While overcoming the problems of long sterilization time of the batch sterilization method and high contamination rate of the continuous sterilization method, the destruction of the nutrients in the culture medium is further reduced, providing a sterilization method for cephalosporin C fermentation medium with better effect; the structure of the sterilization tank 1 in the sterilization device of the present invention can further slightly shorten the sterilization time and reduce the destruction of the nutrients in the culture medium, while further significantly reducing the contamination rate; the structure of the fermentation tank 4 in the sterilization device of the present invention can further slightly shorten the sterilization time and reduce the contamination rate, while shortening the sterilization time to the level of the sterilization time of the continuous sterilization method, reducing the contamination rate to the level of the contamination rate of the batch sterilization method, and further significantly reducing the destruction of the nutrients in the culture medium.

[0121] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for sterilizing a cephalosporin C fermentation medium, characterized in that: The following steps are involved: Step 1: prepare the nitrogen source component and inorganic salt component of the culture medium with the first group of water to form a first liquid, and prepare the remaining components of the culture medium with the second group of water to form a second liquid; wherein the amount of the first group of water is 10-20% of the amount of water used in the culture medium, and the amount of the second group of water is 10-20% of the amount of water used in the culture medium; Step 2: transporting the first liquid feed and the second liquid feed to two sterilization tanks respectively for steam sterilization; Step 3, transporting the sterilized first liquid and the sterilized second liquid into a fermentation tank, and at the same time transporting the third group of water into the fermentation tank, and cooling to the fermentation temperature; wherein the amount of the third group of water is the remaining water in the culture medium, and the third group of water is sterile water; The sterilization device used in the sterilization method includes: Two sterilization tanks, either of which is connected to a steam introduction pipe, a feeding port and a steam exhaust valve are provided on the top of either sterilization tank, and a discharge port is provided on the bottom; the first feed liquid is transported to one of the sterilization tanks, and the second feed liquid is transported to the other sterilization tank; A fermentation tank is provided with two feed ports, one feed port is provided for each sterilization tank, and the discharge port of any sterilization tank is connected with the corresponding feed port through a pipeline; the fermentation tank is connected with a sterile water introduction pipe, and the third group of water is transported into the fermentation tank through the sterile water introduction pipe; Any sterilization tank includes: The first tank body is a hollow cylindrical structure, with a feeding port and a steam exhaust valve on the top and a discharge port on the bottom; a steam inlet is provided on the first tank body, and a steam introduction pipe is connected to the steam inlet; A first rotating shaft, which is coaxially disposed in the first tank body and is rotatably connected to the first tank body; A pair of first vertical rods are vertically arranged on both sides of the first rotating shaft, and a plurality of triangular spikes are arranged at intervals on any of the first vertical rods; A pair of first connecting rods, one first connecting rod is provided for each first vertical rod, and the two ends of any first connecting rod are respectively hinged to the upper end of the first vertical rod corresponding thereto and the first rotating shaft; a collar is sleeved on any first connecting rod, and any collar can move along the length direction of the first connecting rod corresponding thereto; A pair of first cylinders, one first cylinder is correspondingly arranged for one collar, and any first cylinder is connected to the first rotating shaft; the free end of the piston rod of any first cylinder extends vertically downward and is hinged to the collar corresponding thereto; Two groups of stirring mechanisms are arranged on both sides of the first rotating shaft. A group of stirring mechanisms is correspondingly arranged directly below a first connecting rod. Any group of stirring mechanisms includes a plurality of stirring mechanisms arranged at intervals in the vertical direction. Any stirring mechanism includes: A pair of second connecting rods are arranged at intervals in the vertical direction, and two ends of any second connecting rod are respectively hinged to the corresponding first vertical rod and the first rotating shaft; A second vertical rod is arranged between a pair of second connecting rods, and two ends of the second vertical rod are respectively connected to the pair of second connecting rods in a sliding manner along the length direction of the second connecting rods; a plurality of triangular spikes are arranged at intervals on the second vertical rod; The fermentation tank comprises: The second tank body is a hollow cylindrical structure, and is provided with two sterile water inlets and two feed ports, and any one of the sterile water inlets is connected to the sterile water introduction pipe; the side wall of the second tank body is an interlayer, and a cooling pipe is provided in the interlayer; the two feed ports are respectively connected to the two discharge ports through a pipeline; and the two sterile water inlets are both connected to the sterile water introduction pipe; A second rotating shaft is coaxially disposed in the second tank body and is rotatably connected to the second tank body; a plurality of stirring blades are spaced apart on the second rotating shaft; a stopper, which is a conical structure coaxially sleeved on the middle part of the second rotating shaft and with its vertex facing upwards; the stopper is fixedly arranged in the second tank body and is rotatably connected to the second rotating shaft; the diameter of the bottom surface of the stopper is smaller than the inner diameter of the second tank body; The shell is a hollow frustum structure which is larger at the top and smaller at the bottom and open at both ends. The shell is arranged above the block. The upper end of the shell is sealed and slidably connected to the inner wall of the second tank body in the vertical direction. The diameter of the bottom surface of the shell is smaller than the diameter of the bottom surface of the block. The two feed ports are respectively located above and below the block, and the two sterile water inlets are respectively located above and below the block.

2. The method for sterilizing cephalosporin C fermentation medium according to claim 1, characterized in that: The pressure of steam sterilization is 0.09~0.11 MPa, the temperature is 120~125℃, and the holding time is 20~30 min.

3. The method for sterilizing cephalosporin C fermentation medium according to claim 1, characterized in that: The temperature of the third group of water is room temperature.

4. The method for sterilizing cephalosporin C fermentation medium according to claim 1, characterized in that: The volume of one of the sterilization tanks for containing the first liquid is 1.2 to 1.3 times the volume of the first liquid, and the volume of the other sterilization tank for containing the second liquid is 1.2 to 1.3 times the volume of the first liquid.

5. The method for sterilizing cephalosporin C fermentation medium according to claim 1, characterized in that: For any stirring mechanism, a pair of second connecting rods are provided with a groove extending along the length direction on the side away from each other, and a pair of second connecting rods are provided with a through hole extending along the length direction on the side close to each other. Any through hole is connected to the corresponding groove, and the longitudinal section of any groove is arc-shaped; the two ends of the second vertical rod respectively pass through the corresponding through holes and are provided with a ball, and any ball rolls in the corresponding groove.

6. The method for sterilizing cephalosporin C fermentation medium according to claim 1, characterized in that: The axis of any three-edged thorn is perpendicular to the first vertical rod / second vertical rod where it is located.

7. The method for sterilizing cephalosporin C fermentation medium according to claim 1, characterized in that: Also includes: A pair of second cylinders are arranged at intervals along the radial direction of the second tank body, any second cylinder is arranged on the top of the second tank body, and the free end of the piston rod of any second cylinder extends vertically downward and is connected to the shell.

Images