Method for producing tylosin through high-level fermentation

Abstract

The invention discloses a method for producing tylosin through high-level fermentation. The method sequentially comprises slant culture, shake flask seed culture, first-stage seed tank culture, second-stage seed tank culture and fermentation tank culture, in the fermentation tank culture process, polypeptide powder is used as a nitrogen source for tylosin fermentation, the viscosity change of fermentation liquor in the fermentation process is controlled, operation is easy, control is easy, the fermentation unit of tylosin can be greatly increased, and the fermentation cost of tylosin can be greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial industrial fermentation technology, specifically relating to a fermentation method that can significantly increase the fermentation unit of tylosin. Background Technology

[0002] Tylosin is a macrolide antibiotic that was obtained in the United States in 1959 from a culture of Streptomyces freundii. It is a Class III new veterinary drug in China, with a broad antibacterial spectrum and high antibacterial activity against a variety of Gram-positive bacteria and some Gram-negative bacteria. It is the first-line drug for mycoplasma diseases in livestock and poultry.

[0003] Currently, domestic research on the biosynthesis of tylosin using fermentation mainly focuses on improving fermentation levels through strain selection and optimization of fermentation processes. However, process optimization often involves selecting different types of soybean oil and soybean meal, which does not significantly improve potency and results in high raw material costs, making it difficult to apply to large-scale production.

[0004] In 1999, Qi Wei et al., in their study "The Influence of Carbon Source on Tylosin Yield," compared the tylosin production capacity under conditions where starch and vegetable oil were used as carbon sources, finding that using rapeseed oil as a carbon source increased tylosin yield by 79%. In 2002, Peng Haiping, in her study "The Influence of Culture Medium Composition on Tylosin Yield," first used a single-factor method to select the optimal carbon and nitrogen sources, and then used the Plackett-Burman design method to screen out the important factors affecting tylosin yield. Ultimately, process optimization reduced the fermentation cost of tylosin. In 2020, Yang Guangyao, in his study "The Influence of Carbon-Nitrogen Ratio on Tylosin Fermentation," based on the original fermentation culture medium, kept the carbon source (corn flour) content and other components and contents unchanged, and increased the potency of tylosin by 11.2% through the addition of soybean meal. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as high raw material costs and low potency, this invention provides a high-level fermentation method for producing tylosin. This process improves the early potency increase of tylosin by adjusting the culture cycle of the secondary seed tank. At the same time, by optimizing the fermentation medium formula and controlling the fermentation process, the potency of tylosin in the final fermentation broth is increased by more than 30% compared with that before optimization, providing guidance for large-scale production.

[0006] The technical solution of the present invention is as follows: A method for high-level fermentation production of tylosin includes, in sequence, slant culture, shake flask seed culture, primary seed tank culture, secondary seed tank culture and fermenter culture; The secondary seed tank is used for culturing for more than 40 hours before transplanting. During the cultivation in the fermenter, polypeptide powder was used as the nitrogen source of the fermentation medium for cultivation.

[0007] In the present invention, by optimizing the cultivation cycle of the secondary seed tank and simultaneously optimizing the medium formula, the yield of tylosin was significantly increased.

[0008] Furthermore, by controlling the fermentation conditions, the yield of tylosin was further increased. Preferably, after fermentation cultivation, the viscosity of the fermentation broth was measured every 24 hours, and it was more appropriate to maintain the viscosity of the fermentation broth at about 1200 - 1400 mPa·s by timely adding sterile water.

[0009] The present invention also provides a method for high-level fermentation production of tylosin, and the specific steps are as follows: a) Slant culture Prepare the plate medium in proportion, make up the volume with purified water, and dispense it into a triangular flask containing agar powder. Sterilize it with steam at 121.0 ± 2.0 °C for 30 ± 1 minutes. After the plate cools to 70 - 80 °C, pour it into a sterile plate. After the medium coagulates, randomly select a plate or slant, place it in an incubator at 30 ± 2 °C for 2 days, and visually check for colony growth. If there is no abnormal colony growth, it is determined that this batch of medium is qualified.

[0010] b) Shake flask seed culture Take a plate slant, scrape the plate spores, and inoculate them into a triangular flask, and culture at 30 ± 2 °C for 30 - 40 hours for standby.

[0011] c) Primary seed tank culture Dissolve the weighed materials in appropriate amount of water and put them into the seed tank, make up the volume, maintain sterilization at 121.0 ± 2.0 °C for 30 ± 1 minutes. After the temperature stabilizes at 30 ± 2 °C, aseptically inoculate the well-cultured seed liquid in step b, and culture at 30 ± 2 °C with the dissolved oxygen concentration controlled above 45% for 30 - 40 hours and then transfer the seeds.

[0012] d) Secondary seed tank culture Dissolve the weighed materials in appropriate amount of water and put them into the seed tank, make up the volume, sterilize at 121.0 ± 2.0 °C for 30 ± 1 minutes. After the temperature cools down, transfer the primary seed liquid into the secondary seed tank by aseptic operation, and culture at 30 ± 2 °C with the dissolved oxygen concentration controlled above 45% for 30 - 40 hours and then transfer the seeds.

[0013] e) Fermenter culture Dissolve the weighed materials in appropriate amount of water and put them into the seed tank, make up the volume, maintain sterilization at 121.0 ± 2.0 °C for 30 ± 1 minutes. After the temperature cools down, transfer the secondary seed liquid into the fermenter by aseptic operation, and control the dissolved oxygen above 45% by adjusting the rotation speed and ventilation volume at 30 ± 2 °C.

[0014] Preferably, in step a, the slant culture medium composition (by mass percentage) is: 1.0-3.0% soluble starch, 0.05-2.0% beef extract, 0.01-0.08% dipotassium hydrogen phosphate, 0.01-0.08% sodium chloride, 0.01-0.08% magnesium sulfate, 0.001-0.002% ferrous sulfate, 0.05-0.2% potassium nitrate, 1.8-2.0% agar, with the remainder being water. More preferably, the slant culture medium composition (by mass percentage) is: 2.0% soluble starch, 0.1% beef extract, 0.05% dipotassium hydrogen phosphate, 0.05% sodium chloride, 0.05% magnesium sulfate, 0.001% ferrous sulfate, 0.1% potassium nitrate, 1.8-2.0% agar, with the remainder being water. After inoculation of the quick-frozen spore solution onto the slant culture medium, the mycelial growth on the slant is thick and abundant, with a uniform color, turning grayish-white when mature.

[0015] Preferably, in step b, the shake flask culture medium comprises (by mass percentage): 1.0-3.0% corn steep liquor, 0.1-2.0% soybean meal, 0.1-2.0% yeast extract, 0.1-1.0% soybean oil, 0.1-1.0% calcium carbonate, and the remainder being water; more preferably, the shake flask culture medium comprises (by mass percentage): 1.5% corn steep liquor, 0.5% soybean meal, 0.5% yeast extract, 0.4% soybean oil, 0.3% light calcium carbonate, and the remainder being water.

[0016] Preferably, in step c, the composition (by mass percentage) of the primary seed tank culture medium is: 0.5-1.5% soybean meal, 0.5-1.5% corn flour, 1.0-5.0% corn steep liquor, 1.0-5.0% soybean oil, 0.1-1.0% calcium carbonate, and the remainder is water; more preferably, the composition (by mass percentage) of the primary seed tank culture medium is: 0.6% soybean meal, 0.5% corn flour, 1.5% corn steep liquor, 1.0% soybean oil, 0.6% calcium carbonate, 0.04% defoamer, and the remainder is water.

[0017] As a preferred option, in step c, the culture temperature is maintained at 30±2℃, and the dissolved oxygen is controlled to be above 45% by adjusting the stirring speed and air volume. After culturing for 30-40 hours, when the bacterial concentration is ≥18% and no contaminants are found under a microscope, an aseptic transfer operation is performed to transfer the primary seed liquid into the secondary seed tank.

[0018] Preferably, in step d, the composition (by mass percentage) of the secondary seed tank culture medium is: 0.1-2.0% low-temperature soybean meal, 1.0-5.0% corn flour, 0.1-1.5% corn steep liquor, 1.0-5.0% soybean oil, 0.1-2.0% calcium carbonate, 0.5-3.0% corn gluten meal, 1.0-5.0% cottonseed meal, 0.1-0.8% fish meal, 0.1-1.0% yeast powder, 0.01-0.3% diammonium hydrogen phosphate, and the remainder being water; more preferably, the composition (by mass percentage) of the secondary seed tank culture medium is: 0.4% low-temperature soybean meal, 1.8% corn flour, 0.6% corn steep liquor, 2.0% soybean oil, 0.4% calcium carbonate, 0.7% corn gluten meal, 1.2% cottonseed meal, 0.3% fish meal, 0.2% yeast powder, 0.04% diammonium hydrogen phosphate, and the remainder being water.

[0019] Preferably, in step d, the substrate volume in the 15L seed tank is 10L, the cultivation temperature is maintained at 30±1℃, and the aeration ratio is 1.0m. 3 At a speed of 200-650 rpm and a tank pressure of 0.05-0.1 MPa, dissolved oxygen is controlled to be above 45% by adjusting the stirring speed and air volume. After culturing for 30-40 hours, when the bacterial concentration is ≥28% and no contaminants are found under microscopic examination, aseptic transfer is performed to transfer the secondary seed liquid into the fermenter.

[0020] Preferably, in step e, the fermentation medium composition, by mass percentage, is: corn flour 1.0-8.0%, soybean oil 1.0-5.0%, calcium carbonate 0.1-1.0%, corn gluten meal 1.0-6.0%, polypeptide powder 0.1-1.5%, betaine hydrochloride 0.01-0.1%, cottonseed gluten meal 0.1-0.8%, potassium chloride 0.1-0.8%, cobalt chloride 0.001-0.08%, nickel sulfate 0.001-0.01%, with the remainder being water; as a further preferred embodiment, the fermenter medium composition (by mass percentage) is: corn flour 4.0%, soybean oil 1.5%, calcium carbonate 0.8%, corn gluten meal 4.0%, polypeptide powder 1.5%, betaine hydrochloride 0.08%, cottonseed gluten meal 0.8%, potassium chloride 0.5%, cobalt chloride 0.005%, nickel sulfate 0.001%, with the remainder being water.

[0021] Preferably, in step e, the culture temperature is maintained at 30±2℃, and the dissolved oxygen is controlled at above 45% by adjusting the stirring speed and air volume, and the fermentation culture is carried out for 160h-170h.

[0022] Preferably, in step e, after fermentation, the viscosity of the fermentation broth is measured every 24 hours, and sterile water is added in a timely manner to maintain the viscosity of the fermentation broth at 1200-1400 mPa·s.

[0023] Preferably, after inoculation, the pH, cell concentration, viscosity of the fermentation broth are detected at regular intervals, and the tylosin liquid-phase titer and components are detected.

[0024] Compared with the prior art, the advantages of the present invention are as follows: In the method for producing tylosin of the present invention, by adjusting the culture cycle of the secondary seed tank, optimizing the fermentation medium formula, and controlling the viscosity of the fermentation broth during the fermentation process, the final discharging titer of tylosin has been significantly improved. Detailed implementation manners

[0025] Fermentation method of tylosin: a) Slant culture Prepare the plate medium according to the ratio, make up the volume with purified water, and sub-pack into a triangular flask containing agar powder. Sterilize with steam at 121.0 ± 2.0 °C for 30 ± 1 minute. After the plate cools to 70 - 80 °C, pour it into a sterile plate. After the medium coagulates, randomly select a plate or slant, place it in an incubator at 30 ± 2 °C for 2 days, and visually check for colony growth. If there is no abnormal colony growth, it is determined that this batch of medium is qualified.

[0026] b) Shake flask seed culture Take a plate slant, scoop about plate spores, inoculate into a triangular flask, and culture at 30 ± 2 °C for 30 - 40 h for standby.

[0027] c) Primary seed tank culture Dissolve the weighed materials in appropriate amount of water and put them into the seed tank, make up the volume, sterilize at 121.0 ± 2.0 °C for 30 ± 1 minute. After the temperature stabilizes at 30 ± 2 °C, aseptically inoculate the well-cultured seed liquid in step b, and culture at 30 ± 2 °C with the dissolved oxygen concentration controlled above 45% for 30 - 40 hours and then transfer the seeds.

[0028] d) Secondary seed tank culture Dissolve the weighed materials in appropriate amount of water and put them into the seed tank, make up the volume, sterilize at 121.0 ± 2.0 °C for 30 ± 1 minute. After the temperature cools down, aseptically transfer the primary seed liquid into the secondary seed tank at a ratio of 5%, and culture at 30 ± 2 °C with the dissolved oxygen concentration controlled above 45% for 30 - 40 hours and then transfer the seeds.

[0029] e) Fermentation tank culture Dissolve the weighed materials in appropriate amount of water and put them into the fermentation tank, make up the volume, sterilize at 121.0 ± 2.0 °C for 30 ± 1 minute. After the temperature cools down, aseptically transfer the secondary seed liquid into the fermentation tank, and culture at 30 ± 2 °C with the dissolved oxygen concentration controlled above 45% by controlling the rotation speed and aeration volume. <000008​​

[0031] The components of the culture medium for each stage are as follows: The shake flask culture medium consisted of (by weight percentage): 1.5% corn steep liquor, 0.5% soybean meal, 0.5% yeast extract, 0.4% soybean oil, 0.3% calcium carbonate, and the remainder was water.

[0032] The composition (by weight) of the primary seed tank culture medium is as follows: 0.6% soybean meal, 0.5% corn flour, 1.5% corn steep liquor, 1.0% soybean oil, 0.6% calcium carbonate, 0.04% defoamer, and the remainder is water.

[0033] The composition (by weight) of the culture medium in the secondary seed tank was as follows: 0.4% low-temperature soybean meal, 1.8% corn flour, 0.6% corn steep liquor, 2.0% soybean oil, 0.4% calcium carbonate, 0.7% corn gluten meal, 1.2% cottonseed meal, 0.3% fish meal, 0.2% yeast powder, 0.04% diammonium hydrogen phosphate, and the remainder was water.

[0034] The fermentation medium components (by mass percentage) are: corn flour 4.0%, soybean oil 1.5%, calcium carbonate 0.8%, corn gluten meal 4.0%, fish meal 1.5%, betaine hydrochloride 0.08%, cottonseed gluten meal 0.8%, potassium chloride 0.5%, cobalt chloride 0.005%, nickel sulfate 0.001%, and the remainder is water.

[0035] The present invention will be further described below through specific embodiments, and the specific results are as follows.

[0036] Example 1: Experiment on the Periodic Adjustment of the Secondary Seed Tank 1. Shake-flask seed culture: The shake-flask seed culture medium composition (by weight percentage) is: corn steep liquor 1.5%, soybean meal 0.5%, yeast extract 0.5%, soybean oil 0.4%, light calcium carbonate 0.3%, and the remainder is water. Sterilize before use.

[0037] Slant spores were inoculated into sterilized shake flask seed culture medium and cultured at 30°C for 30 hours to obtain shake flask seed solution.

[0038] 2. Primary seed culture: The primary seed culture medium consists of: 0.6% soybean meal, 0.5% corn flour, 1.5% corn steep liquor, 1.0% soybean oil, 0.6% calcium carbonate, 0.04% defoamer, and the remainder is water. Sterilize and use as needed.

[0039] The shake flask seed culture was inoculated into sterilized primary seed culture medium, and cultured at 30°C, dissolved oxygen 30%, and pressure 0.03 MPa for 30 hours with stirring to obtain primary seed culture.

[0040] 3. Secondary seed culture: The secondary seed culture medium consisted of: 0.4% low-temperature soybean meal, 1.8% corn flour, 0.6% corn steep liquor, 2.0% soybean oil, 0.4% calcium carbonate, 0.7% corn gluten meal, 1.2% cottonseed meal, 0.3% fish meal, 0.2% yeast powder, 0.04% diammonium hydrogen phosphate, with the remainder being water. It was sterilized and prepared for use.

[0041] The seed culture obtained in process 2 was inoculated into a sterilized secondary culture medium at an inoculation rate of 20%. The culture temperature was 30℃, dissolved oxygen was 30%, and the pressure was 0.04 MPa. The mixture was stirred and cultured for 45 hours to obtain the secondary seed culture.

[0042] 4. Fermentation culture: The fermentation medium consists of: 4.0% corn flour, 1.5% soybean oil, 0.8% calcium carbonate, 4.0% corn gluten meal, 1.5% fish meal, 0.08% betaine hydrochloride, 0.8% cottonseed gluten meal, 0.5% potassium chloride, 0.005% cobalt chloride, 0.001% nickel sulfate, and the remainder is water. Sterilize and use as is.

[0043] The seed culture obtained in process 3 was inoculated into sterilized fermentation medium at an inoculum rate of 20%. The culture temperature was 30℃, dissolved oxygen was 30%, and the pressure was 0.04 MPa. The mixture was stirred. The initial potency of the fermentation broth was measured after about 30 hours, and fermentation was stopped after 170 hours.

[0044]

[0045] The data above clearly show that in the small-scale test, the initial titer of the secondary seed cultured for 40-50 hours was higher than that of the cultured for 34 hours, and the titer after transfer to the fermentation tank was also higher. To further verify whether the shorter culture period of the secondary seed was the reason for the lower initial titer of fermentation, we cultured the secondary seed in the fermentation tank for 34 hours and then transferred it. The titer results are as follows:

[0046] As can be seen from the table above, shortening the culture period of the secondary seed tank to 34 hours resulted in a significant decrease in the initial titer of the fermentation broth, similar to the batch cultured for 34 hours. This indicates that insufficient seed age in the secondary tank leads to a low initial titer in the fermenter. Therefore, it was ultimately decided to culture the secondary seed for approximately 40-50 hours before transplanting.

[0047] Example 2: Nitrogen source optimization experiment for fermentation culture medium 1. Shake-flask seed culture: The shake-flask seed culture medium consists of (by weight percentage): 1.5% corn steep liquor, 0.5% soybean meal, 0.5% yeast extract, 0.4% soybean oil, 0.3% light calcium carbonate, and the remainder is water. Sterilize before use.

[0048] Slant spores were inoculated into sterilized shake flask seed culture medium and cultured at 30°C for 30 hours to obtain shake flask seed solution.

[0049] 2. Primary seed culture: The primary seed culture medium consists of: 0.6% soybean meal, 0.5% corn flour, 1.5% corn steep liquor, 1.0% soybean oil, 0.6% calcium carbonate, 0.04% defoamer, and the remainder is water. Sterilize and use as needed.

[0050] The shake flask seed culture was inoculated into sterilized primary seed culture medium, and cultured at 30°C, dissolved oxygen 30%, and pressure 0.03 MPa. After stirring, the culture was carried out for 30 hours to obtain the primary seed culture.

[0051] 3. Secondary seed culture: The secondary seed culture medium consists of: 0.4% low-temperature soybean meal, 1.8% corn flour, 0.6% corn steep liquor, 2.0% soybean oil, 0.4% calcium carbonate, 0.7% corn gluten meal, 1.2% cottonseed meal, 0.3% fish meal, 0.2% yeast powder, 0.04% diammonium hydrogen phosphate, and the remainder is water. Sterilize and use as needed.

[0052] The seed culture obtained in process 2 was inoculated into a sterilized secondary culture medium at an inoculation rate of 20%. The culture temperature was 30℃, dissolved oxygen was 30%, and the pressure was 0.04 MPa. The mixture was stirred and cultured for 45 hours to obtain the secondary seed culture.

[0053] 4. Fermentation culture: The fermentation medium consists of: 4.0% corn flour, 1.5% soybean oil, 0.8% calcium carbonate, 4.0% corn gluten meal, 1.5% polypeptide powder, 0.08% betaine hydrochloride, 0.8% cottonseed gluten meal, 0.5% potassium chloride, 0.005% cobalt chloride, 0.001% nickel sulfate, and the remainder is water. Sterilize and use as is.

[0054] The seed culture obtained in process 3 was inoculated into sterilized fermentation medium at a 20% inoculum rate. The culture was carried out at 30°C, dissolved oxygen 30%, and pressure 0.04 MPa with stirring. The initial potency of the fermentation broth was measured after approximately 30 hours, and fermentation was stopped after 170 hours.

[0055] After 45 hours of cultivation in the secondary seed tank, the secondary seed liquid was transferred to the fermenter. The fermenter used polypeptide powder as the nitrogen source for fermentation. The relevant data are shown in the table below.

[0056]

[0057] The data above clearly shows that using polypeptide powder as the nitrogen source for tylosin fermentation increases the fermentation broth potency by about 10% compared to the original formula.

[0058] Example 3: Fermentation Broth Viscosity Control Experiment 1. Shake-flask seed culture: The shake-flask seed culture medium consists of (by weight percentage): 1.5% corn steep liquor, 0.5% soybean meal, 0.5% yeast extract, 0.4% soybean oil, 0.3% light calcium carbonate, and the remainder is water. Sterilize before use.

[0059] Slant spores were inoculated into sterilized shake flask seed culture medium and cultured at 30°C for 30 hours to obtain shake flask seed solution.

[0060] 2. Primary seed culture: The primary seed culture medium consists of: 0.6% soybean meal, 0.5% corn flour, 1.5% corn steep liquor, 1.0% soybean oil, 0.6% calcium carbonate, 0.04% defoamer, and the remainder is water. Sterilize and use as needed.

[0061] The shake flask seed culture was inoculated into sterilized primary seed culture medium, and cultured at 30°C, dissolved oxygen 30%, and pressure 0.03 MPa. After stirring, the culture was carried out for 30 hours to obtain the primary seed culture.

[0062] 3. Secondary seed culture: The secondary seed culture medium consists of: 0.4% low-temperature soybean meal, 1.8% corn flour, 0.6% corn steep liquor, 2.0% soybean oil, 0.4% calcium carbonate, 0.7% corn gluten meal, 1.2% cottonseed meal, 0.3% fish meal, 0.2% yeast powder, 0.04% diammonium hydrogen phosphate, and the remainder is water. Sterilize and use as needed.

[0063] The seed culture obtained in process 2 was inoculated into a sterilized secondary culture medium at an inoculation rate of 20%. The culture temperature was 30℃, dissolved oxygen was 30%, and the pressure was 0.04 MPa. The mixture was stirred and cultured for 45 hours to obtain the secondary seed culture.

[0064] 4. Fermentation culture: The fermentation medium consists of: 4.0% corn flour, 1.5% soybean oil, 0.8% calcium carbonate, 4.0% corn gluten meal, 1.5% fish meal, 0.08% betaine hydrochloride, 0.8% cottonseed gluten meal, 0.5% potassium chloride, 0.005% cobalt chloride, 0.001% nickel sulfate, and the remainder is water. Sterilize and use as is.

[0065] The seed culture obtained in step 3 was inoculated into sterilized fermentation medium at a 20% inoculation rate. The culture was carried out at 30°C, dissolved oxygen 30%, and pressure 0.04 MPa with stirring. The initial potency of the fermentation broth was measured after approximately 30 hours, and the viscosity of the fermentation broth was measured every 24 hours. The viscosity of the fermentation broth was maintained at 1200-1400 mPa·s by adding sterile water in a timely manner. Fermentation was stopped after 170 hours.

[0066]

[0067] The data above shows that maintaining the viscosity of the fermentation broth at around 1200-1400 mPa·s in the later stages of fermentation significantly improves the fermentation broth's potency, increasing it by about 12% compared to the original process.

[0068] Example 4 compares the optimized process with the original process. The optimized secondary seed tank culture conditions, optimized culture medium formulation, viscosity control, and conventional fermentation conditions of Examples 1-3 were compared.

[0069] 1. Shake-flask seed culture: The shake-flask seed culture medium composition (by weight percentage) is: corn steep liquor 1.5%, soybean meal 0.5%, yeast extract 0.5%, soybean oil 0.4%, light calcium carbonate 0.3%, and the remainder is water. Sterilize before use.

[0070] Slant spores were inoculated into sterilized shake flask seed culture medium and cultured at 30°C for 30 hours to obtain shake flask seed solution.

[0071] 2. Primary seed culture: The primary seed culture medium consists of: 0.6% soybean meal, 0.5% corn flour, 1.5% corn steep liquor, 1.0% soybean oil, 0.6% calcium carbonate, 0.04% defoamer, and the remainder is water. Sterilize and use as needed.

[0072] The shake flask seed culture was inoculated into sterilized primary seed culture medium, and cultured at 30°C, dissolved oxygen 30%, and pressure 0.03 MPa. After stirring, the culture was carried out for 30 hours to obtain the primary seed culture.

[0073] 3. Secondary seed culture: The secondary seed culture medium consists of: 0.4% low-temperature soybean meal, 1.8% corn flour, 0.6% corn steep liquor, 2.0% soybean oil, 0.4% calcium carbonate, 0.7% corn gluten meal, 1.2% cottonseed meal, 0.3% fish meal, 0.2% yeast powder, 0.04% diammonium hydrogen phosphate, and the remainder is water. Sterilize and use as needed.

[0074] The seed culture obtained in process 2 was inoculated into a sterilized secondary culture medium at an inoculation rate of 20%. The culture temperature was 30℃, dissolved oxygen was 30%, and the pressure was 0.04 MPa. The mixture was stirred and cultured for 45 hours to obtain the secondary seed culture.

[0075] 4. Fermentation culture: The fermentation medium consists of: 4.0% corn flour, 1.5% soybean oil, 0.8% calcium carbonate, 4.0% corn gluten meal, 1.5% polypeptide powder, 0.08% betaine hydrochloride, 0.8% cottonseed gluten meal, 0.5% potassium chloride, 0.005% cobalt chloride, 0.001% nickel sulfate, and the remainder is water. Sterilize and use as is.

[0076] The seed culture obtained in step 3 was inoculated into sterilized fermentation medium at a 20% inoculation rate. The culture was carried out at 30°C, dissolved oxygen 30%, and pressure 0.04 MPa with stirring. The initial potency of the fermentation broth was measured after approximately 30 hours, and the viscosity of the fermentation broth was measured every 24 hours. The viscosity of the fermentation broth was maintained at 1200-1400 mPa·s by adding sterile water in a timely manner. Fermentation was stopped after 170 hours.

[0077]

[0078] The data from the above embodiments show that the potency of tylosin is increased by about 30% under the optimized process conditions compared with the traditional process.

Claims

1. A method for high-level fermentation production of tylosin, characterized in that, The process includes, in sequence, slant culture, shake flask seed culture, primary seed tank culture, secondary seed tank culture, and fermenter culture; The secondary seed tank is used for culturing for more than 40 hours before transplanting. During the fermentation process, polypeptide powder was used as the nitrogen source for the fermentation medium.

2. The method for high-level fermentation production of tylosin according to claim 1, characterized in that, The amount of polypeptide powder added to the fermentation medium is 0.5-5.0 wt%.

3. The method for high-level fermentation production of tylosin according to claim 1, characterized in that, During the fermentation process, sterile water is added to reduce the viscosity of the fermentation broth.

4. The method for high-level fermentation production of tylosin according to claim 3, characterized in that, The viscosity of the fermentation broth was maintained at 1200-1400 mPa·s.

5. The method for high-level fermentation production of tylosin according to any one of claims 1 to 4, characterized in that, The specific steps are as follows: a) Slant culture Prepare plate culture medium according to the specified ratio, dilute with purified water, and dispense into Erlenmeyer flasks containing agar powder. First, sterilize by steam, then cool the plates to 70-80℃ and pour them into sterile plates. After the culture medium solidifies, randomly select a plate or slant and incubate it in an incubator at 30±2℃ for 1-3 days. Visually inspect for colony growth. If there is no abnormal colony growth, the batch of culture medium is considered qualified. b) Shake-flask seed culture Take a flat, sloping surface, scoop out a portion of the material and place it into an Erlenmeyer flask for shake-flask seed culture; c) Primary seed tank culture Prepare the culture medium in the primary seed tank, and after the temperature stabilizes at 30±2℃, aseptically inoculate the seed liquid prepared in step b and carry out primary seed tank culture. d) Secondary seed tank culture After the culture medium is prepared in the secondary seed tank and cooled, the primary seed solution is transferred into the secondary seed tank under aseptic conditions for secondary seed tank culture. e) Fermenter culture The culture medium is prepared in the fermenter. After the temperature is cooled, the secondary seed liquid is transferred into the fermenter under aseptic conditions for fermentation culture.

6. The method for high-level fermentation production of tylosin according to claim 5, characterized in that, In step e, maintain the culture temperature at 30±2℃, and during the culture process, adjust the stirring speed and aeration rate to keep the dissolved oxygen concentration above 45% for 160-170 hours of fermentation.

7. The method for high-level fermentation production of tylosin according to claim 5, characterized in that, The fermentation medium consists of the following components by mass percentage: corn flour 1.0-8.0%, soybean oil 1.0-5.0%, calcium carbonate 0.1-1.0%, corn gluten meal 1.0-6.0%, polypeptide powder 0.1-1.5%, betaine hydrochloride 0.01-0.1%, cottonseed gluten meal 0.1-0.8%, potassium chloride 0.1-0.8%, cobalt chloride 0.001-0.08%, nickel sulfate 0.001-0.01%, with the remainder being water.

8. The method for high-level fermentation production of tylosin according to claim 7, characterized in that, The fermentation medium components, by mass percentage, are as follows: corn flour 4.0%, soybean oil 1.5%, calcium carbonate 0.8%, corn gluten meal 4.0%, polypeptide powder 1.5%, betaine hydrochloride 0.08%, cottonseed gluten meal 0.8%, potassium chloride 0.5%, cobalt chloride 0.005%, nickel sulfate 0.001%, and the remainder is water.

9. The method for high-level fermentation production of tylosin according to claim 5, characterized in that, Transplanting is performed after 40-50 hours of cultivation in a secondary seed tank.

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