Culture medium and culture method for improving fermentation titer of lincomycin

Abstract

The invention discloses a culture medium for improving the fermentation titer of lincomycin and a culture method, and relates to the field of microbial fermentation. Lincomycin streptomycete is taken as a starting strain, biological nitrogen is added into seeds and a fermentation culture medium basic material, and biological nitrogen and a trace element TE are added into a supplementary material, so that an obvious regulating effect on promoting carbon and nitrogen source metabolism in a fermentation process is achieved, and proper conditions are provided for thallus growth and element production; by adjusting the proportion of biological nitrogen, soybean meal, ammonium sulfate and corn steep liquor supplemented into the mixed nitrogen material in the process, the maintainability of thalli is further improved, and fermentation production of lincomycin is facilitated. The method is suitable for stably and efficiently producing lincomycin on a large scale, and has the advantages of simplicity in operation, remarkable optimization effect and easiness in popularization.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a culture medium and a culture method for improving the fermentation titer of lincomycin. Background Art

[0002] Lincomycin is a lincosamide antibiotic fermented by Streptomyces lincomycin. It is commonly used to treat infectious diseases caused by Gram-positive bacteria. Its structural analogues mainly include lincomycin A, B, C, D, E, and K. Among them, lincomycin A has the best activity. Currently, the research on lincomycin is mainly focused on the strain of Streptomyces lincomycin, culture medium and fermentation process optimization. The fermentation yield of lincomycin has been greatly improved, but due to the long fermentation cycle of lincomycin, the level still needs to be further improved to meet market demand.

[0003] The main production method of lincomycin is microbial fermentation. Although the research on improving the fermentation titer of lincomycin has been going on for many years at home and abroad, there is still no culture method in the prior art that can effectively and significantly improve the fermentation titer of lincomycin. Summary of the invention

[0004] In view of the above defects or deficiencies in the prior art, the present invention provides a culture medium and a culture method for improving the fermentation titer of lincomycin in order to solve the above problems in the prior art.

[0005] Its main technical solutions are: A culture medium for improving the fermentation titer of lincomycin, the culture medium comprising the following components in mass fractions: 0-4% corn starch, 2.5-5.5% glucose, 1-8% soybean powder, 0-8% biological nitrogen, 0.4-10% corn steep liquor, 0.1-5% ammonium sulfate, 0-5% trace elements (TE solution), 0.02-0.1% defoamer, and the balance is water.

[0006] Preferably, the culture medium further comprises the following components in mass fractions: Ammonium nitrate 0.4-1.0%, sodium nitrate 0.25-0.45%, potassium dihydrogen phosphate 0.015-0.025%, calcium carbonate 0.45-0.75%, sodium chloride 0.45-0.75%, soybean oil 0.02-0.15%, defoamer content is 0.02-0.1%, the balance is water, pH 7.0-7.2. Further preferably, the content of the trace elements (TE solution) is: MnSO4·H2O 31-33 mg / L, MgSO4·7H2O 44-46 mg / L, CuSO4·5H2O 0.2-0.3 mg / L, ZnCl2 0.2-0.3 mg / L, CoCl2·6H2O 0.2-0.3 mg / L, and biotin 0.012-0.02 mg / L.

[0007] More preferably, the content of the trace elements (TE solution) is: MnSO4·H2O 32.4 mg / L, MgSO4·7H2O 45.2 mg / L, CuSO4·5H2O 0.25 mg / L, ZnCl2 0.25 mg / L, CoCl2·6H2O 0.22 mg / L, and biotin 0.015 mg / L.

[0008] The fermentation method using the culture medium for improving the fermentation titer of lincomycin has a pH of 6.2-6.8 and a fermentation temperature of 30-32°C during the fermentation process.

[0009] Preferably, the fermentation process adopts the feeding method for feeding, and the mixed material of the feeding includes the following components by mass fraction: corn starch 0-10%, soybean powder 2.0-8.0%, corn steep liquor 3.0-10.0%, biological nitrogen 0-6.0%, ammonium sulfate 1-6%, trace element TE 0-5.0%, defoamer 0.02-0.1%; feeding is started when the total sugar drops to 2.0-3.5g / 100mL, and the total sugar is controlled at 2.0-4.0% by feeding and liquidizing sugar. When the amino nitrogen level drops below 0.5 mg / ml, the mixed material is supplemented, and the amino nitrogen level is controlled at 0.6-0.8 mg / mL by adjusting the supplementation rate of the mixed material, and the pH level is controlled at 6.6-6.8 by adding ammonia water; during the fermentation process, the bacterial concentration is maintained at 38-46%, and the culture period is 264-284 hours; Further preferably, the feed medium also includes the following components in mass fraction: the formula of trace element TE mother solution is: MnSO4·H2O 32.4 mg / L, MgSO4·7H2O 45.2 mg / L, CuSO4·5H2O 0.25 mg / L, ZnCl2 0.25 mg / L, CoCl2 0.22 mg / L, biotin 0.015 mg / L.

[0010] More preferably, during the fermentation process, the temperature is controlled at 30-34°C and the pH is controlled at 6.5-6.9 from 0 to 50 hours; the temperature is controlled at 30-33°C and the pH is controlled at 6.4-6.8 from 51 to 150 hours; and the temperature is controlled at 28-32°C and the pH is controlled at 6.2-6.6 from 151 hours to tank release; Still more preferably, from 0 to 50 h, the temperature is controlled at 32° C. and the pH is controlled at 6.8; from 51 to 150 h, the temperature is controlled at 31° C. and the pH is controlled at 6.6; from 151 h to release the tank, the temperature is controlled at 30° C. and the pH is controlled at 6.4.

[0011] A culture medium and a culture method for improving the fermentation titer of lincomycin, comprising the following steps: (1) Primary seed culture: The cultured lincomycin Streptomyces fermentation seed liquid is inoculated into the primary seed culture medium at an inoculum rate of 0.1-0.2% for cultivation. The culture conditions are: tank pressure 0.05±0.005 Mpa, culture temperature 31±1°C, ventilation ratio 0.8-1.2vvm, dissolved oxygen controlled at 30-60%. The seed tank transplanting standard is: when the bacterial concentration reaches more than 30%, the pH value drops below 6.4, and the culture time is 48-64h, the seed tank is transplanted; (2) Secondary seed culture: Inoculate the cultured lincomycin first-level seed liquid at an inoculum of 10-20% into the first-level seed culture medium for culture. The culture conditions are: tank pressure 0.04±0.005 Mpa, culture temperature 31±1°C, ventilation ratio 0.8-1.5vvm, dissolved oxygen controlled at 30-60%. The seed tank transplanting standard is: when the bacterial concentration reaches more than 40%, the pH value drops below 6.4, and the culture time is 48-64h, transplant the seeds; (3) Fermentation culture: The cultured secondary seed liquid is inoculated into the fermentation medium at an inoculum of 20-30%, and the culture conditions are: tank pressure 0.035±0.005Mpa, culture temperature 31±1°C, ventilation ratio 0.6-1.2vvm, dissolved oxygen range 30%-60%; inorganic nitrogen level is controlled at 0.6-0.8mg / mL by adjusting the feed rate of the mixture, and pH level is controlled at 6.6-6.8 by feeding ammonia water; during the fermentation process, sugar is fed to control the total sugar content at 2.0-4.0%. During the fermentation process, the bacterial concentration is maintained at 38-46%, and the culture period is 280±24h. Furthermore, the mass percentage of each component of the primary seed culture medium is: corn starch 0.5-1.0%, glucose 2.5-4.5%, soybean powder 1.3-2.6%, biological nitrogen 0.1-0.5%, corn steep liquor 0.6-1.4%, ammonium sulfate 0.2-0.4%, ammonium nitrate 0.4-1.0%, sodium nitrate 0.25-0.45%, potassium dihydrogen phosphate 0.015-0.025%, calcium carbonate 0.45-0.75%, sodium chloride 0.45-0.75%, soybean oil 0.02-0.05%, defoaming agent 0.02-0.08%, and the remainder water, pH 7.0-7.2. Furthermore, the mass percentage of each component of the secondary seed culture medium is: corn starch 0.5-2.0%, glucose 2.5-5.5%, soybean powder 1.3-2.6%, biological nitrogen 0.2-0.5%, corn steep liquor 1.0-2.0%, ammonium sulfate 0.2-0.4%, ammonium nitrate 0.4-1.0%, sodium nitrate 0.25-0.45%, potassium dihydrogen phosphate 0.015-0.025%, calcium carbonate 0.45-0.75%, sodium chloride 0.45-0.75%, soybean oil 0.02-0.05%, defoaming agent 0.02-0.08%, and the remainder water, pH 7.0-7.2. Furthermore, the mass percentage of each component of the fermentation medium is: corn starch 0.5-3.0%, glucose 2.5-5.5%, soybean powder 1.3-2.6%, corn steep liquor 1.0-2.5%, biological nitrogen 0.3-0.8%, ammonium sulfate 0.2-0.4%, ammonium nitrate 0.4-1.0%, sodium nitrate 0.25-0.45%, potassium dihydrogen phosphate 0.015-0.025%, calcium carbonate 0.45-0.75%, sodium chloride 0.45-0.75%, soybean oil 0.02-0.1%, defoaming agent 0.02-0.08%, and the remainder water, pH 7.0-7.2. Furthermore, the quality requirements of the first-level seed culture medium after sterilization are: amino nitrogen 60-120 mg / 100 mL, total sugar 3.0-7.0 g / 100 mL, pH 6.4-7.0.

[0012] Furthermore, the quality requirements of the secondary seed culture medium after sterilization are: amino nitrogen 80-160 mg / 100 mL, total sugar 4.0-8.0 g / 100 mL, pH 6.4-7.0.

[0013] Furthermore, the quality requirements of the fermentation medium after sterilization are: amino nitrogen 80-160 mg / 100 mL, total sugar 4.0-8.0 g / 100 mL, pH 6.4-7.0. Furthermore, the fermentation process adopts the feeding method for feeding, and the feed medium composition is: 0-10% starch, 2.0-8.0% soybean powder, 3.0-10.0% corn steep liquor, 0-6.0% biological nitrogen, 1-6% ammonium sulfate, 0-5.0% TE, and 0.02-0.1% defoamer. Feeding starts when the total sugar drops to 2.0-3.5g / 100mL, and the feeding amount is 0.2-1% of the volume of the fermentation liquid.

[0014] Furthermore, the formula of the fermentation feed trace element TE mother solution is: MnSO4·H2O 32.4 mg / L, MgSO4·7H2O 45.2 mg / L, CuSO4·5H2O 0.25 mg / L, ZnCl2 0.25 mg / L, CoCl2 0.22 mg / L, biotin 0.015 mg / L.

[0015] Furthermore, during the fermentation process, the temperature was controlled at 32°C and the pH was controlled at 6.8 from 0 to 50 hours; the temperature was controlled at 31°C and the pH was controlled at 6.6 from 51 to 150 hours; and the temperature was controlled at 30°C and the pH was controlled at 6.4 from 151 hours to tank release.

[0016] The beneficial effect of the present invention is to provide suitable nutrients and metabolic environment for the fermentation production of lincomycin. With respect to the prior art, the method of the present invention has the following advantages: 1. The present invention promotes bacterial growth and anabolism by adding biological nitrogen as a nitrogen source to seeds and fermentation medium, which is beneficial to improving the fermentation starting titer and promoting the accumulation of lincomycin.

[0017] 2. The present invention increases bacterial maintenance and improves the efficiency of lincomycin production by adding biological nitrogen and trace element TE to the fermentation feed solution, which is beneficial to the fermentation production of lincomycin. 3. The present invention promotes the secretion of the secondary metabolite lincomycin by stepwise adjusting the culture temperature and sequentially controlling the pH during the fermentation culture process, thereby providing better conditions for the accumulation of lincomycin. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Comparison of process biomass between Example 1 and Comparative Examples 1-7; Figure 2 Comparison of process biomass between Example 3 and Comparative Example 8; Figure 3 Comparison of process potency between Example 1 and Comparative Examples 1-7; Figure 4 Comparison of process potency of Example 3 and Comparative Example 8. DETAILED DESCRIPTION

[0018] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments. The quality indicators of the biological nitrogen used in the embodiments and comparative examples are: mainly including: protein 49.5%, moisture 9.8%, ash 8.5%, and fineness 60 mesh passing 95%.

[0019] Example 1 Primary seed culture: culture medium volume 9L. Primary seed culture medium: corn starch 0.9%, glucose 4.5, soybean powder 2.5%, biological nitrogen 0.2%, corn steep liquor 1.4%, ammonium sulfate 0.3%, ammonium nitrate 0.6%, sodium nitrate 0.4%, calcium carbonate 0.7%, sodium chloride 0.5%, defoamer 0.08%, soybean oil 0.2%, balance water, pH 7.0. Prepare the first-level seed culture medium in proportion, sterilize at 121-125℃ for 30min, then cool to room temperature and maintain pressure with sterile air. The mass of the first-level seed culture medium after sterilization is: amino nitrogen 105mg / 100mL, total sugar 6.5g / 100mL, pH 6.66. Inoculate the cultured lincomycin Streptomyces seed bottle fermentation liquid at an inoculation rate of 0.2% into the first-level seed culture medium for cultivation. The seed culture conditions are: tank pressure 0.05Mpa, culture temperature 32℃, ventilation ratio 0.6vvm, dissolved oxygen controlled at 30-60%. When the bacterial concentration reaches 30% and the pH value drops to 6.40, transplant the culture, and the culture time is 49h. Secondary seed culture: culture medium volume 15L. Secondary seed culture medium: corn starch 1.0%, glucose 5.5, soybean powder 2.5%, biological nitrogen 0.4%, corn steep liquor 1.6%, ammonium sulfate 0.3%, ammonium nitrate 0.6, sodium nitrate 0.45, calcium carbonate 0.68%, sodium chloride 0.6%, defoamer 0.05%, soybean oil 0.3%, balance water, pH 7.2. Prepare the secondary seed culture medium in proportion, sterilize at 121-125℃ for 30min, then cool to room temperature, and maintain pressure with sterile air. The quality of the secondary seed culture medium after sterilization is as follows: amino nitrogen 112mg / 100mL, total sugar 6.6g / 100mL, pH 6.54. Inoculate the cultured lincomycin primary seed liquid at an inoculum of 15% into the secondary seed culture medium for cultivation. The seed culture conditions are as follows: tank pressure 0.04Mpa, culture temperature 31℃, ventilation ratio 0.8vvm, dissolved oxygen controlled at 30-60%. When the bacterial concentration reaches 40% and the pH value drops to 6.38, transplant the culture, and the culture time is 50h. Fermentation culture: medium volume 25L. Fermentation medium: corn starch 1.0%, glucose 5.5, soybean powder 2.5%, biological nitrogen 0.8%, corn steep liquor 1.8%, ammonium sulfate 0.3%, ammonium nitrate 1.0, sodium nitrate 0.4, calcium carbonate 0.6%, sodium chloride 0.5%, defoamer 0.07%, soybean oil 0.3%, balance water, pH 7.2. Prepare the fermentation medium according to the proportion, sterilize at 121-125℃ for 30min, then cool to room temperature and maintain the pressure with sterile air. The mass of the fermentation medium after sterilization is as follows: amino nitrogen 125mg / 100mL, total sugar 6.5g / 100mL, pH 6.60. The cultured secondary seed liquid is inoculated into a fermentation tank filled with a fermentation medium, the inoculation amount is 30%, the bacterial concentration is maintained at 38-46% during the fermentation process, the culture cycle is 268-284h, and the fermentation is completed to obtain lincomycin fermentation liquid; The fermentation culture conditions are: tank pressure 0.04Mpa, ventilation ratio 0.6-1.2vvm, dissolved oxygen range 40-60%. 0-50h culture temperature 32°C, flow ammonia water to control pH 6.8; 51-150h culture temperature 31°C, flow ammonia water to control pH 6.6; 151h-tank release culture temperature 30°C, flow ammonia water to control pH 6.4. Feeding process control: Sugar supplementation began when the total sugar dropped to 3.0g / 100mL, with a flow rate of 0.8% of the fermentation liquid volume and a sugar concentration of 40% by mass. Mixture supplementation began when the amino nitrogen dropped below 0.5 mg / ml, and the amino nitrogen level was controlled at 0.6mg / mL by adjusting the mixture supplementation acceleration rate, and the pH level was controlled at 6.7 by flow addition of ammonia water. The composition of the mixture culture medium was: 0.5% starch, 3.0% soybean powder, 0.4% biological nitrogen, 2.5% ammonium sulfate, 8.0% corn steep liquor, and 3.0% trace element TE.

[0020] At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation cycle was 274 h, and the lincomycin titer was detected to be 18102 ug / mL. Example 2 Primary seed culture: culture medium volume 9L. Primary seed culture medium: corn starch 0.8%, glucose 4.5%, soybean powder 2.5%, biological nitrogen 0.1%, corn steep liquor 1.5%, ammonium sulfate 0.3%, ammonium nitrate 0.6%, sodium nitrate 0.4%, calcium carbonate 0.7%, sodium chloride 0.5%, defoamer 0.08%, soybean oil 0.2%, balance water, pH 7.0. Prepare the first-level seed culture medium in proportion, sterilize at 121-125℃ for 30min, cool to room temperature, and maintain pressure with sterile air. The mass of the first-level seed culture medium after sterilization is: amino nitrogen 102mg / 100mL, total sugar 6.3g / 100mL, pH 6.58. Inoculate the cultured lincomycin Streptomyces seed bottle fermentation liquid at an inoculum rate of 0.2% into the first-level seed culture medium for cultivation. The seed culture conditions are: tank pressure 0.05Mpa, culture temperature 32℃, ventilation ratio 0.6vvm, dissolved oxygen controlled at 30-60%. When the bacterial concentration reaches 30% and the pH value drops to 6.39, transplant the culture, and the culture time is 52h. Secondary seed culture: culture medium volume 15L. Secondary seed culture medium: corn starch 1.0%, glucose 5.5%, soybean powder 2.5%, biological nitrogen 0.3%, corn steep liquor 1.7%, ammonium sulfate 0.3%, ammonium nitrate 0.6%, sodium nitrate 0.45%, calcium carbonate 0.65%, sodium chloride 0.6%, defoamer 0.05%, soybean oil 0.3%, balance water, pH 7.2. Prepare the secondary seed culture medium in proportion, sterilize at 121-125℃ for 30min, cool to room temperature, and maintain pressure with sterile air. The quality of the secondary seed culture medium after sterilization is: amino nitrogen 115mg / 100mL, total sugar 6.8g / 100mL, pH 6.50. Inoculate the cultured lincomycin primary seed liquid at an inoculum of 15% into the secondary seed culture medium for cultivation. The seed culture conditions are: tank pressure 0.04Mpa, culture temperature 31℃, ventilation ratio 0.8vvm, dissolved oxygen controlled at 30-60%. When the bacterial concentration reaches 40% and the pH value drops to 6.40, transplant the seeds, and the culture time is 49h. Fermentation culture: medium volume 25L. Fermentation medium: corn starch 1.0%, glucose 5.5%, soybean powder 2.5%, biological nitrogen 0.8%, corn steep liquor 1.8%, ammonium sulfate 0.3%, ammonium nitrate 1.0%, sodium nitrate 0.4%, calcium carbonate 0.65%, sodium chloride 0.5%, defoamer 0.07%, soybean oil 0.3%, balance water, pH 7.2. The fermentation medium was prepared in proportion, sterilized at 121-125°C for 30 min, then cooled to room temperature and pressurized with sterile air. The mass of the fermentation medium after sterilization was: amino nitrogen 130 mg / 100 mL, total sugar 6.9 g / 100 mL, pH 6.62. The cultured secondary seed liquid is inoculated into a fermentation tank filled with a fermentation medium, the inoculation amount is 30%, the bacterial concentration is maintained at 38-46% during the fermentation process, the culture cycle is 268-284h, and the fermentation is completed to obtain lincomycin fermentation liquid; The fermentation culture conditions are: tank pressure 0.04Mpa, ventilation ratio 0.6-1.2vvm, dissolved oxygen range 40-60%. 0-50h culture temperature 32°C, flow ammonia water to control pH 6.8; 51-150h culture temperature 31°C, flow ammonia water to control pH 6.6; 151h-tank release culture temperature 30°C, flow ammonia water to control pH 6.4. Feeding process control: Sugar supplementation started when the total sugar dropped to 3.0 g / 100 mL, with the feed rate being 0.8% of the fermentation liquid volume and the sugar concentration being 40% by mass. When the amino nitrogen level drops below 0.5 mg / ml, the feed mixture is supplemented. The amino nitrogen level is controlled at 0.7 mg / mL by adjusting the feed mixture supplementation rate, and the pH level is controlled at 6.6 by adding ammonia water. The composition of the mixed culture medium is: 0.5% starch, 3.0% soybean powder, 0.4% biological nitrogen, 3.0% ammonium sulfate, 8.0% corn steep liquor, and 4.0% trace element TE.

[0021] At the end of the fermentation culture, the bacterial concentration was 46%, the fermentation cycle was 268 h, and the lincomycin titer was detected to be 18386 ug / mL.

[0022] Example 3 Primary seed culture: culture medium volume 9L. Primary seed culture medium: corn starch 0.8%, glucose 4.6%, soybean powder 2.5%, biological nitrogen 0.2%, corn steep liquor 1.4%, ammonium sulfate 0.3%, ammonium nitrate 0.6%, sodium nitrate 0.4%, calcium carbonate 0.7%, sodium chloride 0.6%, defoamer 0.08%, soybean oil 0.2%, balance water, pH 7.0. Prepare the first-level seed culture medium in proportion, sterilize at 121-125℃ for 30min, then cool to room temperature and maintain pressure with sterile air. The mass of the first-level seed culture medium after sterilization is: amino nitrogen 108mg / 100mL, total sugar 6.2g / 100mL, pH 6.52. Inoculate the cultured lincomycin Streptomyces seed bottle fermentation liquid at an inoculation rate of 0.2% into the first-level seed culture medium for cultivation. The seed culture conditions are: tank pressure 0.05Mpa, culture temperature 32℃, ventilation ratio 0.6vvm, dissolved oxygen controlled at 30-60%. When the bacterial concentration reaches 30% and the pH value drops to 6.35, transplant the culture, and the culture time is 48h. Secondary seed culture: culture medium volume 15L. Secondary seed culture medium: corn starch 1.0%, glucose 5.5, soybean powder 2.5%, biological nitrogen 0.5%, corn steep liquor 1.7%, ammonium sulfate 0.3%, ammonium nitrate 0.6%, sodium nitrate 0.45%, calcium carbonate 0.65%, sodium chloride 0.6%, defoamer 0.05%, soybean oil 0.3%, balance water, pH 7.2. Prepare the secondary seed culture medium according to the proportion, sterilize it at 121-125℃ for 30 minutes, then cool it to room temperature and maintain the pressure with sterile air. The mass of the secondary seed culture medium after sterilization is as follows: amino nitrogen 125mg / 100mL, total sugar 6.8g / 100mL, pH 6.51.

[0023] The cultured lincomycin first-level seed liquid was inoculated into the second-level seed culture medium at an inoculation rate of 15% for cultivation. The seed culture conditions were: tank pressure 0.04Mpa, culture temperature 31°C, ventilation ratio 0.8vvm, dissolved oxygen controlled at 30-60%. When the bacterial concentration reached 40% and the pH value dropped to 6.40, the seeds were transplanted and the culture time was 49h. Fermentation culture: medium volume 25L. Fermentation medium: corn starch 1.0%, glucose 5.5, soybean powder 2.5%, biological nitrogen 0.8%, corn steep liquor 1.8%, ammonium sulfate 0.3%, ammonium nitrate 1.0, sodium nitrate 0.4, calcium carbonate 0.65%, sodium chloride 0.5%, defoamer 0.07%, soybean oil 0.3%, balance water, pH 7.2. The fermentation medium was prepared in proportion, sterilized at 121-125°C for 30 min, then cooled to room temperature and pressurized with sterile air. The mass of the fermentation medium after sterilization was: amino nitrogen 130 mg / 100 mL, total sugar 6.9 g / 100 mL, pH 6.62. The cultured secondary seed liquid is inoculated into a fermentation tank filled with a fermentation medium, the inoculation amount is 30%, the bacterial concentration is maintained at 38-46% during the fermentation process, the culture cycle is 268-284h, and the fermentation is completed to obtain lincomycin fermentation liquid; The fermentation culture conditions are: tank pressure 0.04Mpa, ventilation ratio 0.6-1.2vvm, dissolved oxygen range 30-60%. 0-50h culture temperature 32°C, flow ammonia water to control pH 6.8; 51-150h culture temperature 31°C, flow ammonia water to control pH 6.6; 151h-tank release culture temperature 30°C, flow ammonia water to control pH 6.4. Feeding process control: Sugar supplementation started when the total sugar dropped to 3.0g / 100mL, with a feed rate of 0.8% of the fermentation broth volume and a sugar concentration of 40% by mass. When the amino nitrogen dropped below 0.5 mg / 100ml, the mixed material was supplemented, and the amino nitrogen level was controlled at 0.7mg / mL by adjusting the feed rate, and the pH level was controlled at 6.8 by adding ammonia water. The composition of the mixed culture medium is: 0.5% starch, 3.0% soybean powder, 0.5% biological nitrogen, 3.0% ammonium sulfate, 6.0% corn steep liquor, and 5.0% trace element TE.

[0024] At the end of the fermentation culture, the bacterial concentration was 46%, the fermentation cycle was 275 h, and the lincomycin titer was detected to be 19772 ug / mL.

[0025] As can be seen from the examples, the culture medium and culture method for improving the fermentation titer of lincomycin described in the present invention have a higher lincomycin fermentation yield. The inventors conducted comparative tests to determine the degree of improvement.

[0026] Comparative Example 1 Based on Example 1, biological nitrogen and trace element TE were not added to the primary seed culture medium, secondary seed culture medium, fermentation medium, and feed mixture, and the rest was the same as Example 1. At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation cycle was 274h, and the lincomycin titer was 14525 ug / mL.

[0027] No biological nitrogen and trace element TE were added to the seed tank, fermentation tank and feed mixture. The anabolism level in the fermentation tank was low, and the tank discharge level was 14525ug / ml.

[0028] Comparative Example 2 Based on Example 1, the pH value was controlled at 6.8 during the whole fermentation process, and the rest was the same as Example 1.

[0029] At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation cycle was 284 h, and the lincomycin titer was detected to be 14804 ug / mL.

[0030] Comparative Example 3 Based on Example 1, the culture temperature from 0 to 50 h is 29° C.; the culture temperature from 51 to 150 h is 30° C.; the culture temperature from 151 h to tank release is 29° C., and the rest is the same as in Example 1.

[0031] At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation period was 284 h, and the lincomycin titer was 16063 ug / mL. The comparison results are shown in Table 1.

[0032] Comparative Example 4 Based on Example 1, the temperature was controlled at 30° C. throughout the fermentation process, and the rest was the same as Example 1.

[0033] At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation period was 284 hours, and the lincomycin titer was 16418 ug / mL. The comparison results are shown in Table 1.

[0034] Comparative Example 5 Based on Example 1, the culture temperature is 35°C for 0-50h, 31°C for 51-150h, and 32°C for 151h-canning, and the rest is the same as Example 1.

[0035] At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation period was 273 hours, and the lincomycin titer was 15049 ug / mL. The comparison results are shown in Table 1.

[0036] Comparative Example 6 Based on Example 1, biological nitrogen and trace element TE were not added to the feed mixture, and the rest was the same as Example 1.

[0037] At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation period was 279 hours, and the lincomycin titer was 15115 ug / mL. The comparison results are shown in Table 1.

[0038] Comparative Example 7 Based on Example 1, no trace element TE is added to the supplementary feed mixture, and the rest is the same as Example 1.

[0039] At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation period was 275 hours, and the lincomycin titer was 15720 ug / mL. The comparison results are shown in Table 1. Table 1

[0040] From the comparison between Example 1 and Comparative Example 1, it can be seen that the first-level seed culture medium, the second-level seed culture medium, the fermentation culture medium, and the feed mixture do not add biological nitrogen and trace element TE, which has a greater impact on the final canning level; from the comparison between Example 1 and Comparative Example 2, it can be seen that the pH is controlled at 6.8 throughout the fermentation process, and the canning level is greatly reduced compared with the step-by-step pH adjustment in Example 1; from Example 1 and Comparative Example 3, it can be seen that the 0-50h culture temperature is 29°C; the 51-150h culture temperature is 30°C; the 151h-canning culture temperature is 29°C, and the canning level is reduced compared with the step-by-step temperature adjustment scheme in Example 1; from the comparison between Example 1 and Comparative Example 4, it can be seen that the fermentation process The temperature is controlled at 30°C throughout the whole process. Compared with the step-by-step temperature adjustment scheme of Example 1, the tank discharge level is reduced, but it is not much different from Comparative Example 3. By comparing Example 1 with Comparative Example 5, it can be seen that the 0-50h culture temperature is 35°C; the 51-150h culture temperature is 31°C; the 151h-tank discharge culture temperature is 32°C, which is significantly reduced compared with the step-by-step temperature adjustment scheme of Example 1. By comparing Example 1 with Comparative Example 6, it can be seen that no biological nitrogen and trace element TE are added to the fermentation tank feed mixture, which has a greater impact on the final tank discharge level. By comparing Example 1 with Comparative Example 7, it can be seen that no trace element TE is added to the fermentation tank feed mixture, and the tank discharge level is reduced.

[0041] It can be seen from Example 1 and Comparative Examples 1-7 that high nitrogen production efficiency can be obtained by adding biological nitrogen to the base materials of the seed tank and the fermentation tank, adding biological nitrogen and trace elements TE to the supplementary feed mixture of the fermentation tank, and dynamically adjusting the temperature and pH during the fermentation process.

[0042] Comparative Example 8 Based on Example 3, the feeding process control was changed, and the rest was the same as Example 3. Comparative Example 8: Sugar supplementation was started when the total sugar dropped to 3.0 g / 100 mL, the flow addition rate was 0.8% of the fermentation broth volume, and the sugar concentration mass percentage was 40%; the mixed material was supplemented when the amino nitrogen dropped below 0.5 mg / ml, and the amino nitrogen level was controlled at 0.4 mg / mL by adjusting the mixed material supplementation acceleration rate.

[0043] At the end of the fermentation culture, the bacterial concentration was 44%, the fermentation cycle was 275 h, and the lincomycin titer was 15462 ug / mL.

[0044] Table 2

[0045] It can be seen from Example 3 and Comparative Example 8 that as the feed rate increases, the process-controlled amino nitrogen range is adjusted upward, the accumulation of lincomycin in the process increases, and the production efficiency is positively correlated with the amino nitrogen level.

[0046] The culture medium and culture method for improving the fermentation titer of lincomycin disclosed in the embodiment can significantly improve the fermentation titer of lincomycin compared with the comparative example, reaching more than 18000ug / mL. In summary: the present invention discloses a culture medium and a culture method for improving the fermentation titer of lincomycin. By adding biological nitrogen as a nitrogen source to the seed culture medium and the fermentation culture medium, the growth and anabolism of the bacteria are promoted, which is beneficial to improving the fermentation starting titer and promoting the accumulation of lincomycin; by adding biological nitrogen and trace elements TE to the fermentation feed liquid, the maintenance of the bacteria is increased and the efficiency of the production of lincomycin is improved, which is beneficial to the fermentation production of lincomycin; by step-by-step adjusting the culture temperature and time-controlled pH during the fermentation culture process, the secretion of the secondary metabolite lincomycin is promoted, providing better conditions for the accumulation of lincomycin; by increasing the amino nitrogen control level and then adjusting the mixed material feeding rate, the bacteria are continuously provided with a rich organic nitrogen source, the maintenance of the bacteria for the production of lincomycin is enhanced, and the titer growth is maintained and the final level is improved. The present invention is suitable for large-scale stable and efficient production of lincomycin. The technical solution of the present invention is explained through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above specific embodiments to be implemented. Any improvements made by technicians in the relevant field based on the present invention, or equivalent replacement of the materials selected by the present invention, etc., fall within the scope of protection of the patent.

Claims

1. A culture medium for improving the fermentation titer of lincomycin, characterized in that: The culture medium comprises the following components in mass fraction: 0-4% corn starch, 2.5-5.5% glucose, 1-8% soybean powder, 0-8% biological nitrogen, 0.4-10% corn steep liquor, 0.1-5% ammonium sulfate, 0-5% trace elements, 0.02-0.1% defoamer, and the balance is water.

2. The culture medium for improving the fermentation titer of lincomycin according to claim 1, characterized in that: The culture medium also includes the following components in mass fractions: Ammonium nitrate 0.4-1.0%, sodium nitrate 0.25-0.45%, potassium dihydrogen phosphate 0.015-0.025%, calcium carbonate 0.45-0.75%, sodium chloride 0.45-0.75%, soybean oil 0.02-0.15%, defoamer content is 0.02-0.1%, the balance is water, pH 7.0-7.

2.

3. The culture medium for improving the fermentation titer of lincomycin according to claim 1, characterized in that: The contents of the trace elements are: MnSO4·H2O 31-33 mg / L, MgSO4·7H2O 44-46 mg / L, CuSO4·5H2O 0.2-0.3 mg / L, ZnCl2 0.2-0.3 mg / L, CoCl2·6H2O 0.2-0.3 mg / L, and biotin 0.012-0.02 mg / L.

4. The culture medium for improving the fermentation titer of lincomycin according to claim 3, characterized in that: The contents of the trace elements are: MnSO4·H2O 32.4 mg / L, MgSO4·7H2O 45.2 mg / L, CuSO4·5H2O 0.25 mg / L, ZnCl2 0.25 mg / L, CoCl2·6H2O 0.22 mg / L, and biotin 0.015 mg / L.

5. A fermentation method using the culture medium for improving the fermentation titer of lincomycin according to any one of claims 1 to 4, characterized in that: The pH of the fermentation process is 6.2-6.8, and the fermentation temperature is 30-32°C.

6. The fermentation method of the culture medium for improving the fermentation titer of lincomycin according to claim 5, characterized in that: The fermentation process adopts a feeding method to feed, and the mixed material of the feeding includes the following components by mass fraction: 0-10% corn starch, 2.0-8.0% soybean powder, 3.0-10.0% corn steep liquor, 0-6.0% biological nitrogen, 1-6% ammonium sulfate, 0-5.0% trace elements, and 0.02-0.1% defoamer; feeding is started when the total sugar drops to 2.0-3.5g / 100mL, and the total sugar is controlled at 2.0-4.0% by feeding liquid sugar. When the amino nitrogen level drops below 0.5 mg / ml, the mixed material is supplemented, and the amino nitrogen level is controlled at 0.6-0.8 mg / mL by adjusting the supplementation rate of the mixed material, and the pH level is controlled at 6.6-6.8 by adding ammonia water; during the fermentation process, the bacterial concentration is maintained at 38-46%, and the culture period is 264-284 hours; Trace elements in the materials: MnSO4·H2O 31-33 mg / L, MgSO4·7H2O 44-46 mg / L, CuSO4·5H2O0.2-0.3 mg / L, ZnCl2 0.2-0.3 mg / L, CoCl2·6H2O 0.2-0.3 mg / L, biotin 0.012-0.02 mg / L; Preferably, the trace element formula is: MnSO4·H2O 32.4 mg / L, MgSO4·7H2O 45.2 mg / L, CuSO4·5H2O0.25 mg / L, ZnCl2 0.25 mg / L, CoCl20.22 mg / L, biotin 0.015 mg / L.

7. The fermentation method of the culture medium for improving the fermentation titer of lincomycin according to claim 5, characterized in that: During the fermentation process, the temperature is controlled at 30-34°C and the pH is controlled at 6.5-6.9 for 0-50h; the temperature is controlled at 30-33°C and the pH is controlled at 6.4-6.8 for 51-150h; the temperature is controlled at 28-32°C and the pH is controlled at 6.2-6.6 for 151h-tank release; Preferably, from 0 to 50 hours, the temperature is controlled at 32°C and the pH is controlled at 6.8; from 51 to 150 hours, the temperature is controlled at 31°C and the pH is controlled at 6.6; from 151 hours to release the tank, the temperature is controlled at 30°C and the pH is controlled at 6.

4.

8. The fermentation method of the culture medium for improving the fermentation titer of lincomycin according to claim 5, characterized in that: The fermentation method comprises the following steps: (1) Primary seed culture: The cultured lincomycin Streptomyces fermentation seed liquid is inoculated into the primary seed culture medium at an inoculum rate of 0.1-0.2% for cultivation. The culture conditions are: tank pressure 0.05±0.005 Mpa, culture temperature 31±1°C, ventilation ratio 0.8-1.2vvm, dissolved oxygen controlled at 30-60%; seed tank transplanting standard: when the bacterial concentration reaches more than 30%, the pH value drops below 6.4, and the culture time is 48-64h, transplanting is performed; (2) Secondary seed culture: Inoculate the cultured lincomycin first-level seed liquid at an inoculum rate of 10-20% into the first-level seed culture medium for culture. The culture conditions are: tank pressure 0.04±0.005 Mpa, culture temperature 31±1°C, ventilation ratio 0.8-1.5vvm, dissolved oxygen controlled at 30-60%; seed tank transplanting standard: when the bacterial concentration reaches more than 40%, the pH value drops below 6.4, and the culture time is 48-64h, transplant; (3) Fermentation culture: The cultured secondary seed liquid is inoculated into the fermentation medium at an inoculation rate of 20-30% and the culture conditions are as follows: tank pressure 0.035±0.005Mpa, culture temperature 31±1°C, ventilation ratio 0.6-1.2vvm, dissolved oxygen range 30%-60%; the amino nitrogen level is controlled at 0.6-0.8mg / mL by adjusting the feed rate of the mixture, and the pH level is controlled at 6.6-6.8 by feeding ammonia water; during the fermentation process, the total sugar is controlled at 2.0-4.0% by feeding liquid sugar; during the fermentation process, the bacterial concentration is maintained at 38-46%, and the culture period is 264-284h.

9. The fermentation method of the culture medium for improving the fermentation titer of lincomycin according to claim 8, characterized in that: The mass percentage of each component of the primary seed culture medium is: corn starch 0.5-1.0%, glucose 2.5-4.5%, soybean powder 1.3-2.6%, biological nitrogen 0.1-0.5%, corn steep liquor 0.6-1.4%, ammonium sulfate 0.2-0.4%, ammonium nitrate 0.4-1.0%, sodium nitrate 0.25-0.45%, potassium dihydrogen phosphate 0.015-0.025%, calcium carbonate 0.45-0.75%, sodium chloride 0.45-0.75%, soybean oil 0.02-0.05%, defoamer 0.02-0.08%, balance water, pH 7.0-7.2; The mass percentage of each component of the secondary seed culture medium is: corn starch 0.5-2.0%, glucose 2.5-5.5%, soybean powder 1.3-2.6%, biological nitrogen 0.2-0.5%, corn steep liquor 1.0-2.0%, ammonium sulfate 0.2-0.4%, ammonium nitrate 0.4-1.0%, sodium nitrate 0.25-0.45%, potassium dihydrogen phosphate 0.015-0.025%, calcium carbonate 0.45-0.75%, sodium chloride 0.45-0.75%, soybean oil 0.02-0.05%, defoamer 0.02-0.08%, balance water, pH 7.0-7.2; The mass percentage of each component of the fermentation medium is: corn starch 0.5-3.0%, glucose 2.5-5.5%, soybean powder 1.3-2.6%, corn steep liquor 1.0-2.5%, biological nitrogen 0.3-0.8%, ammonium sulfate 0.2-0.4%, ammonium nitrate 0.4-1.0%, sodium nitrate 0.25-0.45%, potassium dihydrogen phosphate 0.015-0.025%, calcium carbonate 0.45-0.75%, sodium chloride 0.45-0.75%, soybean oil 0.02-0.1%, defoamer 0.02-0.08%, balance water, pH 7.0-7.

2.

10. The fermentation method of the culture medium for improving the fermentation titer of lincomycin according to claim 9, characterized in that: The quality requirements of the first-level seed culture medium after sterilization are: amino nitrogen 60-120 mg / 100 mL, total sugar 3.0-7.0 g / 100 mL, pH 6.4-7.0; the quality requirements of the second-level seed culture medium after sterilization are: amino nitrogen 80-160 mg / 100 mL, total sugar 4.0-8.0 g / 100 mL, pH 6.4-7.0; the quality requirements of the fermentation medium after sterilization are: amino nitrogen 80-160 mg / 100 mL, total sugar 4.0 -8.0 g / 100 mL, pH 6.4-7.0.

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