Method for improving bacitracin fermentation level

Abstract

The invention discloses a method for improving the fermentation level of bacitracin, and belongs to the technical field of veterinary drug production. A method for improving the fermentation level of bacitracin comprises the following steps: preparing a plurality of sterile fermentation tanks including a main fermentation tank, at least one propagation tank and a primary fermentation tank, inoculating a seed solution containing bacitracin producing bacteria into the primary fermentation tank, and inoculating the seed solution with thalli entering a logarithmic phase into the main fermentation tank sterilized in advance. According to the method for improving the fermentation level of bacitracin provided by the invention, the main fermentation tank is used for expanding culture towards the expanding culture tank, so that the dissolved oxygen of feed liquid in the fermentation tank can be effectively increased, the metabolic activity of hyphae is improved, the titer growth is further promoted, the concentration of bacitracin in unit volume of fermentation liquid is remarkably improved, and the fermentation titer is improved; part of feed liquid in the main fermentation tank is distributed into the expanding culture tank for culture, and the liquid level is lowered, so that the use of a defoaming agent can be reduced, the energy consumption and the raw material consumption are reduced, and the production cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary drug production, in particular to a method for improving the fermentation level of bacitracin. Background Art

[0002] There are a series of problems in the breeding process, such as feed shortage, low safety of veterinary drugs, frequent animal diseases, etc. Bacitracin can improve the utilization rate of animal feed, prevent and treat animal intestinal diseases, has no toxic side effects, and can promote the growth of animals. Bacitracin zinc and methylene salicylic acid bacitracin are two products of bacitracin, which are currently widely used as drug additives in animal breeding.

[0003] Traditional bacitracin fermentation processes mostly use a single-tank fermentation model, which has problems such as long fermentation cycles and low production efficiency. In order to overcome these problems, researchers have been exploring new fermentation processes and technologies to improve the yield and quality of bacitracin. Summary of the invention

[0004] The object of the present invention is to provide a method for improving the fermentation level of bacitracin, which can effectively increase the dissolved oxygen of the material liquid in the fermentation tank, improve the metabolic activity of mycelium, and then promote the titer growth, significantly increase the bacitracin concentration in the unit volume of fermentation liquid, improve the fermentation titer, and solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for improving the fermentation level of bacitracin, comprising the following steps:

[0006] Step 1: prepare several sterile fermentation tanks, including a main fermentation tank, at least one expansion tank and a primary fermentation tank. The main fermentation tank is connected to the expansion tank through a pipeline. Each fermentation tank is equipped with necessary fermentation equipment such as temperature control, pH adjustment, and stirring device;

[0007] Step 2: Inoculate the seed liquid containing bacitracin-producing bacteria into the primary fermentation tank. After the primary fermentation, inoculate the seed liquid in which the bacteria enter the logarithmic growth phase into the pre-sterilized main fermentation tank to start the fermentation process and control the fermentation conditions;

[0008] Step 3: After the main fermentation tank has been running for 16-20 hours, the sterile feed liquid after disinfection is added through the sterile pipeline flow, mainly carbon source and nitrogen source materials are added for mycelium growth metabolism, and the feeding rate is 0.5-1 ton / hour. Due to the feeding process, the fermentation volume and liquid level are gradually increased;

[0009] Step 4: After the expansion tank and the pipeline connecting the expansion tank and the main fermentation tank are sterilized, the valve of the main fermentation tank and the expansion tank are opened, and the partial fermentation fluid in the main fermentation tank is hydraulically pressed into the expansion tank by the pressure difference;

[0010] Step 5: After the material is hydraulically pumped into the expansion tank and the corresponding valve is closed, the expansion tank starts to operate and expand the culture. The flow rate, tank pressure, tank temperature and stirring process are the same as those of the main fermentation tank. After the tank discharge indicators meet the standards, it is discharged to the refining workshop in the same way as the main fermentation tank.

[0011] Preferably, the specific process of the primary fermentation is as follows: preparing a seed culture medium suitable for bacitracin-producing bacteria, heating the culture medium to dissolve, high-pressure sterilization, maintaining a temperature of 121°C for 15 minutes, cooling the sterilized seed culture medium to 30°C, and adding it to a primary fermentation tank, and the seed liquid is heated at a tank temperature of 34-38°C, a tank pressure of 0.03-0.06Mpa, and a ventilation flow rate of 8-30M 3 / min, and the stirring speed is 200rpm. Cultivate for 15-30 hours and then transfer to the main fermentation tank.

[0012] Preferably, the inoculation amount of the seed solution is 5%-10% of the volume of the culture medium.

[0013] Preferably, the fermentation conditions of the main fermentation tank are as follows: turn on the agitator, set the stirring speed to 250 rpm to ensure that the fermentation liquid is evenly mixed, turn on the air compressor, and set the ventilation volume to 50-150M 3 / min, ensure sufficient oxygen supply, adjust the temperature in the fermentation tank to 34-38℃, and the tank pressure to 0.02-0.05Mpa.

[0014] Preferably, the main fermentation tank is cultured for 25-50 hours and then released. After the expansion culture, the main fermentation tank continues the unfinished fermentation process, and the expansion culture tank completes the fermentation for the same period of time at the same time.

[0015] Preferably, the sterile liquid added in step three is sterilized by high pressure, and the sterile liquid includes a carbon source and a nitrogen source, the carbon source includes glucose and maltose, and the nitrogen source includes peptone and yeast extract.

[0016] Preferably, the fermentation liquid entering the expansion tank from the main fermentation tank in the step 4 accounts for 10%-30% of the fermentation liquid in the main fermentation tank, and the tank pressure of the main fermentation tank during the material transfer process does not exceed 0.06Mpa. By controlling the tank pressure, it can be ensured that the bacteria maintain complete morphology and function during the material transfer process, maintain their normal growth and metabolic capacity, and maintain positive pressure in the tank to prevent microorganisms in the outside air from entering the fermentation system, thereby avoiding contamination. By controlling the tank pressure, the speed and flow of material transfer can be better controlled to ensure the smooth progress of the material transfer process.

[0017] Preferably, the sterilization method of the main fermentation tank and the expansion tank is as follows: after cleaning the main fermentation tank or the expansion tank, the main fermentation tank or the expansion tank is disinfected and sterilized with steam, and the tank temperature is maintained at 121-125°C for 1-2 hours. After the sterilization is completed, positive pressure is maintained to ensure that the fermentation tank is well sealed and a sterile environment is guaranteed.

[0018] Preferably, the pipeline connecting the main fermentation tank and the expansion tank is sterilized with steam at 121-125°C for 1-2 hours, and the pipeline pressure is maintained at 0.2-0.4Mpa during sterilization, which can ensure that the inside of the pipeline is always in a positive pressure state, prevent microorganisms in the outside air from entering the pipeline, and further ensure a sterile environment.

[0019] Preferably, the expansion tank is connected to at least two main fermentation tanks through a pipeline, and the main fermentation tanks that are expanded into the same expansion tank are in the same fermentation operation cycle. Expanding the main fermentation tanks in the same fermentation operation cycle into one expansion tank can save the number of expansion tanks and avoid wasting space inside the expansion tanks.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention provides a method for improving the fermentation level of bacitracin. The method can effectively increase the dissolved oxygen of the feed liquid in the fermentation tank, improve the metabolic activity of mycelium, and further promote the titer growth by expanding the culture from a main fermentation tank to an expansion culture tank, significantly improve the bacitracin concentration in the fermentation liquid per unit volume, and improve the fermentation titer. The feed liquid in the main fermentation tank is divided into the expansion culture tank for culture, and the liquid level is lowered, so that the use of the defoaming agent can be reduced. A plurality of main fermentation tanks are connected with the expansion culture tank, and the feed is supplemented according to the same process at the same time, so that the overall tank volume can be increased, and further the fermentation level can be improved, thereby increasing the total output, improving the production efficiency, reducing the energy consumption and the raw material consumption, and reducing the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention is a process flow chart for improving the fermentation level of bacitracin. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] In order to solve the existing bacitracin fermentation process, most of them adopt the single tank fermentation mode, which has the problems of long fermentation cycle and low production efficiency, please refer to Figure 1 , this embodiment provides the following technical solutions:

[0025] A method for improving the fermentation level of bacitracin comprises the following steps:

[0026] Prepare three sterile fermentation tanks, one as the main fermentation tank, one as the expansion tank and one as the primary fermentation tank. The main fermentation tank is connected to the expansion tank through a pipeline. Each fermentation tank is equipped with necessary fermentation equipment such as temperature control, pH adjustment, and stirring device;

[0027] Prepare a seed culture medium suitable for bacitracin-producing bacteria, heat the culture medium to dissolve, sterilize under high pressure, maintain the temperature at 121°C for 15 minutes, cool the sterilized seed culture medium to 30°C, and add it to the primary fermentation tank. The inoculation amount of the seed liquid is 8% of the volume of the culture medium. The seed liquid is heated to 36°C, the tank pressure is 0.05Mpa, and the ventilation flow rate is 15M 3 / min, stirring speed of 200rpm, cultured for 20 hours and transferred to the main fermentation tank. The volume of liquid transferred to the main fermentation tank is 110 tons, and the stirring speed in the main fermentation tank is set to 250rpm to ensure that the fermentation liquid is evenly mixed, and the ventilation volume is 60M 3 / min, ensure sufficient oxygen supply, adjust the temperature in the fermentation tank to 36℃, and the tank pressure to 0.05Mpa.

[0028] After the main fermentation tank has been running for 18 hours, the sterile liquid is added through the sterile pipeline. The carbon source and nitrogen source are mainly added for the growth and metabolism of mycelium. The sterile liquid includes carbon source and nitrogen source. The carbon source includes glucose and maltose. The nitrogen source includes peptone and yeast extract. The feeding rate is 1 ton / hour, and 5 tons of sterile liquid are added. Due to the feeding process, the fermentation volume and liquid level are gradually increased. After cleaning the expansion tank prepared in advance, the expansion tank is sterilized with steam. The tank temperature is 121 Maintain -125℃ for 1-2 hours, maintain positive pressure after sterilization to ensure that the fermentation tank is well sealed and a sterile environment is guaranteed. The pipeline connecting the main fermentation tank and the expansion tank is disinfected with 121-125℃ steam for 1-2 hours. During disinfection, maintain the pipeline pressure at 0.2-0.4Mpa. After the disinfection is completed, open the main fermentation tank valve and the expansion tank valve, and use the pressure difference to press part of the fermentation liquid in the main fermentation tank to the expansion tank. During the material transfer process, the tank pressure of the main fermentation tank does not exceed 0.06Mpa.

[0029] After the corresponding valves are closed hydraulically in the expansion tank, the expansion tank starts to expand and culture. The flow rate, tank pressure, tank temperature and stirring process are the same as those of the main fermentation tank. The main fermentation tank and the expansion tank are fermented for another 15 hours. When the tank discharge index is met, the fermentation tank and the expansion tank are discharged to the refining workshop, and the discharge volume of the main fermentation tank is 91.8 tons and the discharge volume of the expansion tank is 34.3 tons.

[0030] Embodiment 2:

[0031] A expansion tank is connected with two main fermentation tanks through a pipeline, and the two main fermentation tanks simultaneously carry out secondary fermentation. After fermentation for 20 hours, the two fermentation tanks in the same fermentation operation cycle expand to the same expansion tank, and 25% of the fermentation liquid in the main fermentation tank is pressed to the expansion tank by using the pressure difference. The main fermentation tank and the expansion tank are fermented for another 12 hours. When the tank discharge index is met, the fermentation tank and the expansion tank are discharged to the refining workshop. The other fermentation indexes are the same as those in Example 1, and the volumes of the fermentation tanks are 91.2 tons and 92.1 tons respectively, and the discharge volume of the expansion tank is 71.0 tons.

[0032] Comparative Example 1:

[0033] Heat the culture medium to dissolve, sterilize under high pressure, maintain the temperature at 121°C for 15 minutes, cool the sterilized seed culture medium to 30°C, and add it to the primary fermentation tank. The inoculation amount of the seed liquid is 8% of the volume of the culture medium. The seed liquid is heated at a tank temperature of 36°C, a tank pressure of 0.05Mpa, and a ventilation flow rate of 15M 3 / min, stirring speed of 200rpm, cultured for 20 hours and transferred to the main fermentation tank. The volume of liquid transferred to the main fermentation tank is 110 tons, and the stirring speed in the main fermentation tank is set to 250rpm to ensure that the fermentation liquid is evenly mixed, and the ventilation volume is 60M 3 / min, ensure sufficient oxygen supply, adjust the temperature in the fermentation tank to 36℃, the tank pressure to 0.05Mpa, ferment for 37 hours to meet the tank discharge index, and discharge the fermentation tank and expansion tank to the refining workshop to obtain a main fermentation tank discharge volume of 113.6 tons.

[0034] Comparative Example 2:

[0035] Heat the culture medium to dissolve, sterilize under high pressure, maintain the temperature at 121°C for 15 minutes, cool the sterilized seed culture medium to 30°C, and add it to the primary fermentation tank. The inoculation amount of the seed liquid is 8% of the volume of the culture medium. The seed liquid is heated at a tank temperature of 36°C, a tank pressure of 0.05Mpa, and a ventilation flow rate of 15M 3 / min, stirring speed of 200rpm, cultured for 20 hours and transferred to the main fermentation tank. The volume of liquid transferred to the main fermentation tank is 110 tons, and the stirring speed in the main fermentation tank is set to 250rpm to ensure that the fermentation liquid is evenly mixed, and the ventilation volume is 60M 3 / min, ensure sufficient oxygen supply, adjust the temperature in the fermentation tank to 36℃, the tank pressure to 0.05Mpa, ferment for 36 hours to meet the tank release index, and release the fermentation tank and expansion tank to the refining workshop to obtain a main fermentation tank release volume of 113.1 tons.

[0036] The main fermentation tank in Example 1 is recorded as tank A, the main fermentation tank in Example 2 is recorded as tank B and tank C, the main fermentation tank in Comparative Example 1 is recorded as tank D, and the main fermentation tank in Comparative Example 2 is recorded as tank E. The following table shows the operating data of each fermentation tank:

[0037]

[0038] Tank B and tank C are two tanks with the same cycle. The materials are hydraulically fed into the same expansion tank for cultivation. The volumes of the two main fermentation tanks brought into the expansion tank are basically the same, so the fermentation of the expansion tank is evenly distributed to tanks B and C. It can be seen from the table that in tanks D and E, the potency of the main fermentation tank is lower than that of tanks A, B and C. In tanks A, B and C, the fermentation of bacitracin is carried out by expansion. The fermentation of the ...

[0039] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0040] While the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the invention.

Claims

1. A method for improving the fermentation level of bacitracin, characterized in that, The following steps are involved: Step 1: preparing a plurality of sterile fermentation tanks, including a main fermentation tank, at least one expansion tank and a primary fermentation tank, wherein the main fermentation tank is connected to the expansion tank through a pipeline; Step 2: Inoculate the seed liquid containing bacitracin-producing bacteria into the primary fermentation tank. After the primary fermentation, inoculate the seed liquid in which the bacteria enter the logarithmic growth phase into the pre-sterilized main fermentation tank to start the fermentation process and control the fermentation conditions; Step 3: After the main fermentation tank has been running for 16-20 hours, the sterilized sterile feed liquid is added through the sterile pipeline at a feed rate of 0.5-1 ton / hour; Step 4: After the expansion tank and the pipeline connecting the expansion tank and the main fermentation tank are sterilized, the valve of the main fermentation tank and the expansion tank are opened, and the partial fermentation fluid in the main fermentation tank is hydraulically pressed into the expansion tank by the pressure difference; Step 5: After the material is hydraulically pumped into the expansion tank and the corresponding valve is closed, the expansion tank starts to operate and expand the culture. The flow rate, tank pressure, tank temperature and stirring process are the same as those of the main fermentation tank. After the tank discharge indicators meet the standards, it is discharged to the refining workshop in the same way as the main fermentation tank.

2. The method for improving the fermentation level of bacitracin according to claim 1, characterized in that: The specific process of the primary fermentation is as follows: preparing a seed culture medium suitable for bacitracin-producing bacteria, heating the culture medium to dissolve, high-pressure sterilization, maintaining the temperature at 121°C for 15 minutes, cooling the sterilized seed culture medium to 30°C, and adding it to the primary fermentation tank, the seed liquid is heated at a tank temperature of 34-38°C, a tank pressure of 0.03-0.06Mpa, and a ventilation flow rate of 8-30M 3 / min, and the stirring speed is 200rpm. Cultivate for 15-30 hours and then transfer to the main fermentation tank.

3. The method for improving the fermentation level of bacitracin according to claim 2, characterized in that: The inoculation amount of the seed liquid is 5%-10% of the volume of the culture medium.

4. The method for improving the fermentation level of bacitracin according to claim 3, characterized in that: The fermentation conditions of the main fermentation tank are as follows: turn on the agitator, set the stirring speed to 250 rpm, turn on the air compressor, and set the ventilation volume to 50-150 M 3 / min, adjust the temperature in the fermentation tank to 34-38℃ and the tank pressure to 0.02-0.05Mpa.

5. The method for improving the fermentation level of bacitracin according to claim 4, characterized in that: The main fermentation tank is cultured for 25-50 hours and then discharged.

6. The method for improving the fermentation level of bacitracin according to claim 1, characterized in that: The sterile liquid added in step three is sterilized by high pressure, and the sterile liquid includes a carbon source and a nitrogen source, the carbon source includes glucose and maltose, and the nitrogen source includes peptone and yeast extract.

7. The method for improving the fermentation level of bacitracin according to claim 1, characterized in that: In the step 4, the fermentation liquid entering the expansion tank from the main fermentation tank accounts for 10%-30% of the fermentation liquid in the main fermentation tank, and the tank pressure of the main fermentation tank during the material transfer process does not exceed 0.06Mpa.

8. The method for improving the fermentation level of bacitracin according to claim 1, characterized in that: The sterilization method of the main fermentation tank and the expansion tank is as follows: after the main fermentation tank or the expansion tank is cleaned, the main fermentation tank or the expansion tank is sterilized with steam, the tank temperature is maintained at 121-125° C. for 1-2 hours, and positive pressure is maintained after the sterilization is completed.

9. The method for improving the fermentation level of bacitracin according to claim 1, characterized in that: The pipeline connecting the main fermentation tank and the expansion tank is sterilized with steam at 121-125° C. for 1-2 hours, and the pipeline pressure is maintained at 0.2-0.4Mpa during sterilization.

10. The method for improving the fermentation level of bacitracin according to claim 1, characterized in that: The expansion tank is connected with at least two main fermentation tanks through a pipeline, and the main fermentation tanks expanding into the same expansion tank are in the same fermentation operation cycle.

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