A fermentation method for increasing the growth rate of aureomycin titer after 90 hours of fermentation and application thereof

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to a fermentation method for increasing the titer growth rate of aureomycin after 90 hours of fermentation and application thereof. The present application achieves the effect of comprehensively regulating the fermentation process of aureomycin by regulating the seed inoculation amount of Streptomyces aureofaciens, the total sugar concentration after seed tank depletion, the seed age and pH value after seed transfer, the ratio of slow-acting organic nitrogen to readily available organic nitrogen in the fermentation medium, the total sugar concentration after fermentation tank depletion and the total sugar concentration in the fermentation process, thereby increasing the titer growth rate of aureomycin after 90 hours of fermentation. Further, the method can control the amount of sugar supplement in the fermentation process to a certain extent without being too high, which is conducive to reducing material consumption, while avoiding premature autolysis of mycelium in the fermentation process, which is conducive to reducing the difficulty and cost of sewage treatment. In addition, the method is simple to operate, has strong popularization, has no side effects on fermentation production and product quality, and achieves good economic and ecological benefits.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to a fermentation method for increasing the titer growth rate of aureomycin after 90 hours of fermentation and application thereof. BACKGROUND

[0002] Aureomycin is a broad-spectrum antibiotic and is widely used in the livestock breeding industry. It has broad antibacterial activity against most gram-positive and gram-negative bacteria, rickettsia, mycoplasma (mycoplasma), spirochetes, large viruses, etc. In addition, it also has relatively significant therapeutic effects on chicken lamina dorsi disease, chronic respiratory disease, infectious synovitis, coccidiosis, swine leptospirosis, atrophic rhinitis, bacterial enteritis, and pig dysentery.

[0003] At present, aureomycin premix is one of the important product forms of aureomycin, and the global production capacity is about 110,000 tons. In July 2020, the management mode of aureomycin varieties changed from "veterinary drug letter" to "veterinary drug letter", and the market competition further increased. Reducing production costs and improving production efficiency are important aspects of improving product market competitiveness.

[0004] During the fermentation of aureomycin, as the fermentation proceeds, the consumption of nutrients, the accumulation of harmful metabolites, and the adverse effects of improper process control in the early and middle stages of fermentation gradually appear. The mycelium is severely fragmented, and the titer grows slowly, which seriously restricts the final fermentation efficiency of aureomycin. That is, fluctuations in strains, fermentation raw materials, process control, and other aspects will cause a decrease in the late titer growth rate, resulting in high unit production costs of aureomycin. For example, when the age of Aureobasidium is too short, the proliferation time is longer, and as the proliferation number increases, the strain's ability to produce resistance will decrease. When the age of Aureobasidium is too long, the strain's activity decreases, and premature autolysis is not conducive to the growth of the titer in the later stage. For example, when the total nitrogen content of the fast-acting organic nitrogen source in the fermentation medium is high, the early bacterial concentration is often too high, the dissolved oxygen is low, and the viscosity is high, causing abnormal metabolism. When the total nitrogen content of the slow-acting organic nitrogen source is low, the late bacterial concentration often cannot be maintained at an appropriate level, resulting in a decrease in the amount of ammonia and sugar supplementation, and an increase in dissolved oxygen, which is not conducive to the growth of the titer in the later stage. In addition, if the total sugar after fermentation tank consumption is too low, the sugar supplementation time will be too early, and if the total sugar after fermentation tank consumption is too high, it means that the volume after fermentation tank consumption is large. Because when the nutrient substance is below a certain limit, Aureobasidium cannot be utilized. The final efficiency of aureomycin fermentation is greatly affected by the growth rate of the titer in the later stage. A low growth rate of the titer in the later stage not only affects the fermentation level, but also indicates that the mycelium autolyzes prematurely, aureomycin is released from the cell into the fermentation broth, and aureomycin is lost in the extraction section. At the same time, it increases the difficulty of treating the discharged wastewater. SUMMARY

[0005] The present application is directed to a fermentation method for increasing the titer growth rate of aureomycin after 90 hours of fermentation and its application, which can significantly increase the titer growth rate of aureomycin after 90 hours of fermentation, avoid premature autolysis of mycelium in the fermentation process, and reduce material consumption.

[0006] The present application provides a fermentation method for increasing the titer growth rate of aureomycin after 90 hours of fermentation, comprising the following steps:

[0007] Inoculating aureomycin spores into a seed culture solution for seed culture to obtain a seed solution;

[0008] Transferring the seed solution into a fermentation medium for fermentation culture to obtain a fermentation culture solution containing aureomycin;

[0009] The inoculation amount of the aureomycin spores is (2.0-2.3)×10 8 The total sugar concentration after consumption of the seed culture solution is 3.8-4.3%;

[0010] The seed age of the transfer is 22.0-24.0 h; and the pH value of the seed solution is 5.70-5.85;

[0011] The ratio of delayed effective organic nitrogen to readily available organic nitrogen in the fermentation medium is 1.90-2.10; the total sugar concentration after consumption of the fermentation medium is 6.8-7.3%; the total sugar concentration at the 25th hour of fermentation is ≤3.5%, and the total sugar concentration before 90 hours is ≥2.0%.

[0012] Preferably, the fermentation temperature at the 17th-19th hour of fermentation is reduced from 31-33℃ to 28.5-29.5℃.

[0013] Preferably, the seed culture solution comprises the following components in mass percentage: soybean meal 2.7-2.9%, corn starch 4.3-4.7%, sodium chloride 0.08-0.12%, ammonium sulfate 0.28-0.32%, calcium carbonate 0.38-0.42%, yeast powder 0.9-1.1%, magnesium sulfate 0.03-0.05%, potassium dihydrogen phosphate 0.05-0.07%, and the rest is water.

[0014] Preferably, the fermentation medium comprises the following components in mass percentage: peanut cake powder 1.9-2.0%, soybean cake powder 0.9-1.3%, corn starch 5.8-6.2%, amylase 0.02-0.03%, sodium chloride 0.2-0.4%, ammonium sulfate 0.5-0.6%, calcium carbonate 0.60-0.65%, yeast powder 0.85-0.95%, corn syrup 1.3-1.4%, magnesium sulfate 0.02-0.03%, potassium dihydrogen phosphate 0.01-0.02%, diquat 0.020-0.025%, and the balance of water.

[0015] Preferably, the first time of the fermentation culture is 76-90h, and the first time is ≤5min.

[0016] Preferably, the exhaust amount of the fermentation tank during the first time is 200-300m 3 / h.

[0017] Preferably, the temperature of the fermentation culture is 28.5-33℃.

[0018] Preferably, the cycle of the fermentation culture is 105-115h.

[0019] The application also provides the application of the method in the production of aureomycin related products.

[0020] Preferably, the aureomycin related products comprise aureomycin premix.

[0021] Beneficial effects:

[0022] The application provides a fermentation method for improving the titer growth rate of aureomycin after 90h of fermentation, comprising the following steps: inoculating aureomycin spores into a seed culture solution for seed culture to obtain a seed solution; and inoculating the seed solution into a fermentation medium for fermentation culture to obtain a fermentation culture solution containing aureomycin; the inoculation amount of the aureomycin spores is (2.0-2.3)×10 8The inoculum size is 1 / L; the total sugar concentration after sterilization in the seed tank is 3.8–4.3%; the seed age after transplanting is 22.0–24.0 h; the pH of the seed solution is 5.70–5.85; the ratio of slow-release organic nitrogen to readily available organic nitrogen in the fermentation medium is 1.90–2.10; the total sugar concentration after sterilization in the fermentation tank is 6.8–7.3%; the total sugar concentration at 25 h of fermentation is ≤3.5%, and the total sugar concentration before 90 h is ≥2.0%. This invention achieves comprehensive control of the chlortetracycline fermentation process by regulating the seed inoculum size, the total sugar concentration after sterilization in the seed tank, the transplanting age and pH, the ratio of slow-release organic nitrogen to readily available organic nitrogen in the fermentation medium, the total sugar concentration after sterilization in the fermentation tank, and the total sugar concentration during fermentation. This significantly improves the potency rate of chlortetracycline after 90 hours of fermentation.

[0023] The method described in this invention can control the amount of sugar added during fermentation to a certain extent, preventing excessive levels and reducing material consumption. Furthermore, by controlling the amount of inoculated spores, their age, and pH value, the method prevents the inoculum from becoming too old in the seed tank. By controlling the ratio of fast-acting to slow-acting organic nitrogen sources and the total sugar concentration after sterilization and 25 hours, the inoculum concentration is kept within a suitable range, avoiding excessive nutrient consumption. Insufficient nutrients in the later stages of fermentation can prevent premature mycelial autolysis, thus reducing the difficulty and cost of wastewater treatment. In addition, the method is simple to operate, widely applicable, and has no side effects on fermentation production or product quality, achieving good economic and ecological benefits. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0025] Figure 1 This is the standard curve graph from Test Example 1. Detailed Implementation

[0026] This invention provides a fermentation method for increasing the potency rate of chlortetracycline after 90 hours of fermentation, comprising the following steps:

[0027] The spores of Streptomyces aureus were inoculated into the seed culture medium for seed culture to obtain the seed culture.

[0028] The seed culture was transferred to a fermentation medium for fermentation culture to obtain a fermentation culture broth containing chlortetracycline;

[0029] The inoculation amount of *Streptomyces aureus* spores was (2.0–2.3) × 10⁻⁶. 8 The total sugar concentration of the seeds cultured was 3.8–4.3% after sterilization in the seed tank;

[0030] The seed age of the seed culture is 22.0-24.0h; the pH value of the seed liquid is 5.70-5.85;

[0031] The ratio of the delayed organic nitrogen and the readily available organic nitrogen in the fermentation medium is 1.90-2.10; the total sugar concentration after the seed tank is consumed in the fermentation culture is 6.8-7.3%; the total sugar concentration at the 25th hour of the fermentation culture is ≤3.5%, and the total sugar concentration before 90h is ≥2.0%.

[0032] The present application inoculates the spores of Streptomyces aureofaciens into a seed culture solution to perform seed culture, and obtains a seed liquid. The inoculation amount of the spores of Streptomyces aureofaciens in the present application is (2.0-2.3)×10 8 / mL, preferably 2.26×10 8 / mL. The temperature of the seed culture in the present application is preferably 31-33℃, preferably 32℃; the total sugar concentration after the seed tank is consumed in the seed culture is 3.8-4.3%, preferably 4.13%. The strain type of the Streptomyces aureofaciens in the present application is not specially limited, and the conventional Streptomyces aureofaciens in the art can be used, for example, the Streptomyces aureofaciens with the strain number M14-2 is used in the examples of the present application.

[0033] The seed culture solution in the present application preferably comprises the following components with the mass percentage: soybean cake powder 2.7-2.9%, corn starch 4.3-4.7%, sodium chloride 0.08-0.12%, ammonium sulfate 0.28-0.32%, calcium carbonate 0.38-0.42%, yeast powder 0.9-1.1%, magnesium sulfate 0.03-0.05%, potassium dihydrogen phosphate 0.05-0.07%, and the rest is water.

[0034] The seed culture solution in the present application preferably comprises 2.7-2.9% of soybean cake powder, more preferably 2.8%, in terms of mass percentage.

[0035] The seed culture solution in the present application preferably comprises 4.3-4.7% of corn starch, further preferably 4.4-4.6%, more preferably 4.6%.

[0036] The seed culture solution in the present application preferably comprises 0.08-0.12% of sodium chloride, further preferably 0.09-0.11%, more preferably 0.1%.

[0037] The seed culture solution in the present application preferably comprises 0.28-0.32% of ammonium sulfate, further preferably 0.29-0.31%, more preferably 0.29%.

[0038] The seed culture solution in the present application preferably comprises 0.38-0.42% of calcium carbonate, further preferably 0.39-0.41%, more preferably 0.40%.

[0039] The seed culture solution preferably comprises 0.9-1.1% of yeast powder, more preferably 1.0%.

[0040] The seed culture solution preferably comprises 0.03-0.05% of magnesium sulfate, further preferably 0.03%.

[0041] The seed culture solution preferably comprises 0.05-0.07% of potassium dihydrogen phosphate, further preferably 0.05-0.06%, more preferably 0.05%.

[0042] The seed culture solution preferably comprises 0.07-0.08%, further preferably 0.07%.

[0043] In addition to the above-mentioned components in percentage mass, other components in percentage mass are supplemented with water.

[0044] In the seed culture process, in order to utilize the soybean meal and corn starch in the seed culture solution, Aureomycin Streptomyces secretes enzymes utilizing soybean meal and corn starch, which can be moved into the fermenter together with the seed solution during the seed moving process, which is beneficial to shorten the delay period after entering the fermenter.

[0045] The pH value of the seed culture solution is the natural pH value, without special adjustment.

[0046] The present application does not have special limitations on other operations in the seed culture process, and the conventional seed culture process in the art can be used for operation.

[0047] After obtaining the seed solution, the seed solution is moved to the fermentation medium for fermentation culture to obtain a fermentation culture solution containing aureomycin.

[0048] The seed age of the seed moving is 22.0-24.0h, preferably 22.5-23h, more preferably 22.5h, that is, the seed solution of seed culture for 22.0-24.0h is inoculated into the fermentation medium for fermentation. The pH value of the seed culture solution during the seed moving is 5.70-5.85, preferably 5.75-5.80, more preferably 5.76.

[0049] The ratio of the slow-acting organic nitrogen to the readily available organic nitrogen in the fermentation medium is 1.90-2.10, preferably 2.00-2.10, more preferably 2.07. By controlling the ratio of the slow-acting organic nitrogen to the readily available organic nitrogen, the concentration of the Aureomycin Streptomyces in the late fermentation period can be maintained at a suitable level, and the titer growth rate in the late Aureomycin fermentation period can be improved.

[0050] The fermentation medium of the present application preferably comprises the following components in the mass percentage: peanut cake powder 1.9 to 2.0%, soybean cake powder 0.9 to 1.3%, corn starch 5.8 to 6.2%, amylase 0.02 to 0.03%, sodium chloride 0.2 to 0.4%, ammonium sulfate 0.5 to 0.6%, calcium carbonate 0.60 to 0.65%, yeast powder 0.85 to 0.95%, corn steep liquor 1.3 to 1.4%, magnesium sulfate 0.02 to 0.03%, potassium dihydrogen phosphate 0.01 to 0.02%, and the balance water.

[0051] The fermentation medium of the present application preferably comprises 1.9 to 2.0% of peanut cake powder, further preferably 1.95 to 2.0%, and more preferably 2.0%. The peanut cake powder of the present application is preferably a source of the slow release organic nitrogen.

[0052] The fermentation medium of the present application preferably comprises 0.9 to 1.3% of soybean cake powder, further preferably 0.95 to 1.25%, and more preferably 1.0 to 1.20%, and most preferably 1.0%. The soybean cake powder of the present application is preferably a source of the slow release organic nitrogen.

[0053] The fermentation medium of the present application preferably comprises 5.8 to 6.2% of corn starch, further preferably 5.9 to 6.1%, and more preferably 6.0%.

[0054] The fermentation medium of the present application preferably comprises 0.02 to 0.03% of amylase, further preferably 0.02 to 0.025%, and more preferably 0.0.2%.

[0055] The fermentation medium of the present application preferably comprises 0.2 to 0.4% of sodium chloride, further preferably 0.2 to 0.3%, and more preferably 0.2%.

[0056] The fermentation medium of the present application preferably comprises 0.5 to 0.6% of ammonium sulfate, further preferably 0.5 to 0.55%, and more preferably 0.5%.

[0057] The fermentation medium of the present application preferably comprises 0.60 to 0.65% of ammonium carbonate, further preferably 0.60%.

[0058] The fermentation medium of the present application preferably comprises 0.85 to 0.95% of yeast powder, further preferably 0.85 to 0.90%, and more preferably 0.85%. The yeast powder of the present application is preferably a source of the readily available organic nitrogen.

[0059] The fermentation medium of the present application preferably comprises 1.3 to 1.4% of corn steep liquor, further preferably 1.3%. The corn steep liquor of the present application is preferably a source of the readily available organic nitrogen.

[0060] The fermentation medium of the present application preferably comprises 0.02-0.03% of magnesium sulfate, more preferably 0.02%.

[0061] The fermentation medium of the present application preferably comprises 0.01-0.02% of potassium dihydrogen phosphate, further preferably 0.01-0.015%, more preferably 0.01%.

[0062] The fermentation medium of the present application preferably comprises 0.020-0.025% of surfactant, further preferably 0.020%.

[0063] In addition to the above-mentioned components in the percentage mass, other percentage components of the fermentation medium of the present application are preferably supplemented with water.

[0064] The pH value of the fermentation medium of the present application is preferably the natural pH value, without special adjustment.

[0065] The temperature of the fermentation culture of the present application is preferably 28.5-33℃. The fermentation temperature of the fermentation culture of the present application for 0-17h is preferably 31-33℃, and the fermentation temperature after 17h is preferably 28.5-29.5℃. The temperature of 31-33℃ of the present application is conducive to the growth and reproduction of Streptomyces aureus, and the temperature of 28.5-29.5℃ is conducive to the production of aureomycin by Streptomyces aureus. The total sugar concentration of the fermentation tank of the fermentation culture of the present application is 6.8-7.3%, preferably 6.9-7.28%, more preferably 7.26%. The total sugar concentration of the fermentation culture of the present application at the 25th hour is ≤3.5%, preferably 2.8-3.5, more preferably 3.05%, and the total sugar concentration before the 90th hour is ≥2.0%, preferably 2.0-2.2.

[0066] The fermentation temperature of the fermentation culture of the present application at the 17.0-19.0th hour is preferably reduced from 31-33℃ to 28.5-29.5℃, and the dissolved oxygen content during the temperature reduction is preferably 25-35%, more preferably 32%.

[0067] The fermentation culture of the present application preferably comprises 1-3 times of aeration, more preferably 1 time. The first aeration is at the 76-90th hour, further preferably at the 80-85th hour, more preferably at the 83th hour. The duration of the first aeration is preferably ≤5min, more preferably 3min. During the aeration, the exhaust valve of the fermentation tank is closed and then reopened for one quarter of a week, which has the effect of avoiding tank stoving. The cycle of the fermentation culture of the present application is preferably 105-115h, further preferably 110-112h, more preferably 112h.

[0068] The fermentation culture of the present application is not particularly limited in other conditions, and can be adjusted according to the conventional fermentation culture conditions in the art.

[0069] The method of the present application achieves the effect of comprehensive control of the chlortetracycline fermentation process by regulating the seed inoculation amount, total sugar concentration after seed tank depletion, seed age and pH value, ratio of slow-acting organic nitrogen to readily available organic nitrogen in the fermentation medium, total sugar concentration after fermentation tank depletion and total sugar concentration during fermentation, each condition influences each other and is indispensable to improve the potency growth rate of chlortetracycline after 90 hours of fermentation, and the effect is remarkable.

[0070] Based on the above advantages, the present application also provides the application of the method in the chlortetracycline related product. The chlortetracycline related product preferably includes a chlortetracycline premix.

[0071] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0072] Example 1

[0073] A fermentation method for improving the potency growth rate of chlortetracycline after 90 hours of fermentation, the steps are as follows:

[0074] In a Chinese-style workshop, the 114 tank is used as the fermentation tank, the total volume of the 114 tank is 3m 3 , the tank body is provided with a feeding device and an online monitoring and automatic control system of pH, dissolved oxygen and temperature;

[0075] The 116 tank is used as the seed tank, the total volume is 1m 3 , and the temperature online monitoring and automatic control system is configured, and the seed tank corresponding to the 114 tank fermentation tank is the 116 tank.

[0076] Seed culture stage: the seed culture solution in the 116 tank is composed of the following components with mass percentage (%): soybean meal 2.8, corn starch 4.6, sodium chloride 0.1, ammonium sulfate 0.29, calcium carbonate 0.4, yeast powder 1.0, magnesium sulfate 0.03, potassium dihydrogen phosphate 0.05, bubble enemy 0.07 and water 90.66, and the pH is natural;

[0077] According to the constant volume of 180 liters of the 116 seed tank, the ingredients are prepared, after the end of the material beating and constant volume, the material beating valve and the tank cover are closed, then the steam valves of the air inlet steam, the material outlet steam, the sight glass steam and the sampling steam and the small exhaust valves are opened, the temperature is raised, the intermediate check is made on whether the steam flows through the pipelines, when the temperature rises to 118℃ and the tank pressure is greater than 0.1Mpa, the pressure holding timing is started, the pressure is held at 118-125℃ for 30 minutes, then the small exhaust valves and the steam valves are closed, the temperature is lowered, and when the tank pressure is reduced to below 0.08Mpa, the sterile air can be introduced into the tank;

[0078] When the temperature of the tank is below 50℃, sample and detect the pH, total sugar and amino nitrogen of the medium in the 116 tank; the pH of the medium in the 116 tank is 6.11, the total sugar is 4.13%, and the amino nitrogen is 110 mg / dL; when the temperature of the tank is lowered to 32-33℃, inoculate;

[0079] The seed tank 116 uses the strain M14-2, and the spore concentration is 18.5 billion / mL; 2.2 mL of the spore solution is taken by a sterile syringe in the clean room super-clean workbench, and the syringe with 2.2 mL of the spore solution is connected to the 116 tank from the inoculation port, so that the inoculation amount of the corresponding seed tank of the test tank is 2.26 x 10 8 8 spores per liter; cultivate at 32+1℃, and sample and detect the pH and observe the mycelium every half an hour after 19 hours.

[0080] In the fermentation and cultivation stage, the fermentation medium in the 114 tank contains the following components by mass percentage (%): peanut cake powder 2.0, soybean cake powder 1.0, corn starch 6.0, amylase 0.02, sodium chloride 0.2, ammonium sulfate 0.5, calcium carbonate 0.60, yeast powder 0.85, corn syrup 1.3, magnesium sulfate 0.02, potassium dihydrogen phosphate 0.01, bubble enemy 0.020 and water 87.48, and the pH value after sterilization is natural; the ratio of the slow-acting and quick-acting organic nitrogen sources in the fermentation tank 114 is 2.07;

[0081] The fermentation tank 114 is prepared according to the constant volume of 1.5 tons, and after the material is punched and the constant volume is completed, the material punching valve and the tank cover are closed, and then the steam valves of the air inlet, the material outlet, the sight glass and the sampling are opened, the temperature is raised, and the steam flow of each pipeline is checked in the middle; when the temperature is raised to 118℃ and the tank pressure is greater than 0.1 Mpa, the pressure holding is started, and the pressure is maintained at 118-125℃ for 30 minutes; then the small exhaust and steam valves are closed, and the temperature is lowered; when the tank pressure is lowered to below 0.08 Mpa, sterile air can be introduced into the tank;

[0082] When the temperature of the tank is below 50℃, sample and detect the pH, total sugar and amino nitrogen of the medium in the 114 tank; the pH of the medium in the 114 tank is 6.08, the total sugar is 7.26%, and the amino nitrogen is 222 mg / dL; when the temperature of the tank is lowered to 32-33℃, the seed solution reaches the inoculation standard, and the inoculation operation is performed through the sterilized inoculation pipeline;

[0083] The seed tank 116 corresponds to the seed tank transplanting seed age of 22.5h, the transplanting pH value is 5.76, and the seed liquid is transplanted into the 114 fermentation tanks. During the process of transplanting the seed liquid into the fermentation tank, the dissolved oxygen electrode is calibrated. After the transplanting is completed, the temperature and dissolved oxygen start the automatic control. The temperature is controlled according to 31-33℃. When the fermentation pH value is reduced to 5.95, the pH automatic control is started. When the total sugar concentration is reduced to about 4.3%, the total sugar concentration is supplemented, and the automatic control of the sugar supplementing system is started.

[0084] At the 18th hour of the 114 tank fermentation culture, the dissolved oxygen is 32%. On the temperature control system, the set value is set to 29℃ from 32℃. At the 25th hour, the total sugar concentration is 3.05%. At the 90th hour, the total sugar concentration is 2.12%. The first time of the 114 tank fermentation process is 83 hours. The exhaust valve flow is controlled at 200-300m 3 / h during the 3-minute blowing process. The 114 tank fermentation process is only blown once. The fermentation cycle of the 114 tank is 110h.

[0085] Comparative Example 1

[0086] A fermentation method for improving the titer growth rate of chlortetracycline after 90 hours of fermentation, the steps are as follows:

[0087] In a Chinese-style workshop, 115 tanks are used as control fermentation tanks. The total volume of the 115 tank is 3m 3 . The tank body is provided with a feeding device and an online monitoring and automatic control system of pH, dissolved oxygen and temperature.

[0088] The 117 tank is a control seed tank with a total volume of 1m 3 . The temperature online monitoring and automatic control system is configured. The seed tank corresponding to the 115 tank fermentation tank is the 117 tank.

[0089] Seed culture stage: the seed culture solution in the 117 tank contains the following components (% by mass): soybean meal 2.8, corn starch 4.6, sodium chloride 0.1, ammonium sulfate 0.29, calcium carbonate 0.4, yeast powder 1.0, magnesium sulfate 0.03, potassium dihydrogen phosphate 0.05, bubble enemy 0.07 and water 90.66, and the pH is natural. The seed culture solution used in the 117 control seed tank is the same batch as the seed culture solution in the 116 tank in Example 1, and both meet the company's quality standards after quality inspection.

[0090] According to 117 seed tank constant volume 180 liters to carry out the ingredients, beat the material, constant volume, after closing the beat material valve, tank cover, then open the inlet steam, discharge steam, sight glass steam, sampling steam, etc. Steam valve and each small exhaust valve, temperature, intermediate check whether the pipeline is steam flow, when the temperature rises to 118 ℃ and tank pressure greater than 0.1 Mpa, start pressure timing, 118-125 ℃ pressure 30 minutes, then close each small exhaust and steam valve, cooling, when the tank pressure decreases to 0.08 Mpa or less, can be sterile air into the tank;

[0091] When the tank temperature is lower than 50 ℃, sample 117 tank medium pH, total sugar, amino nitrogen index; 117 tank medium pH is 6.08, total sugar is 4.35%, amino nitrogen is 114 mg / dL, when the tank temperature drops to 32-33 ℃, inoculation;

[0092] 117 seed tank using the strain number M14-2, the spore concentration is 18.5 billion / mL, using 5 mL sterile syringe in clean room super clean bench to take 1.8 mL spore liquid, the syringe with 1.8 mL spore liquid from the inoculation port into 117 tank, then the test tank corresponding to the seed tank inoculation amount is 1.85 x 10 8 Individual spore per liter; 32+1 ℃ culture, 19 h, every half hour sample detection pH and mycelium.

[0093] Fermentation culture stage: the fermentation medium in 115 tank is as follows: peanut cake powder 2.0, soybean meal 1.2, corn starch 6.0, amylase 0.02, sodium chloride 0.2, ammonium sulfate 0.5, calcium carbonate 0.60, yeast powder 0.85, corn syrup 1.3, magnesium sulfate 0.02, potassium dihydrogen phosphate 0.01, bubble enemy 0.020 and water 87.28, after consumption, pH value is natural; The ratio of slow-acting and quick-acting organic nitrogen source in fermentation tank 115 is 2.21;

[0094] Fermentation tank 115 according to constant volume 1.5 tons to prepare medium, beat the material, constant volume, after closing the beat material valve, tank cover, then open the inlet steam, discharge steam, sight glass steam, sampling steam, etc. Steam valve and each small exhaust valve, temperature, intermediate check whether the pipeline is steam flow, when the temperature rises to 118 ℃ and tank pressure greater than 0.1 Mpa, start pressure timing, 118-125 ℃ pressure 30 minutes, then close each small exhaust and steam valve, cooling, when the tank pressure decreases to 0.08 Mpa or less, can be sterile air into the tank;

[0095] When the temperature of the tank is below 50℃, the pH, total sugar and amino nitrogen of the medium in the 115 tank are detected; the pH of the medium in the 115 tank is 6.06, the total sugar is 7.35%, and the amino nitrogen is 226 mg / dL; the temperature of the fermentation tank is reduced to 32-33℃, and the seed liquid is waited to reach the seed transfer standard, and the seed transfer operation is performed through the sterilized seed transfer pipeline;

[0096] 117 The seed tank corresponding to the seed tank seed transfer seed age is 24.5h, the pH value of the seed transfer is 5.60, and the seed liquid is transferred to the 115 fermentation tank. During the process of transferring the seed liquid into the fermentation tank, the dissolved oxygen electrode is calibrated. After the seed transfer is completed, the temperature and dissolved oxygen start automatic control. The temperature is controlled at 32+1℃. When the fermentation pH is reduced to 5.95, the pH starts automatic control. When the total sugar concentration is reduced to about 4.3%, the total sugar concentration is supplemented according to the total sugar concentration. The total sugar concentration starts automatic control.

[0097] At the 16th hour of the 115 tank fermentation culture, the dissolved oxygen is 39%, and on the temperature control system, the set value is set from 32℃ to 29℃. At the 25th hour, the total sugar concentration is 3.80%, and at the 90th hour, the total sugar concentration is 1.90%. The first time of the 115 tank fermentation process is 93h, the aeration process is 6 minutes, and the aeration process exhaust valve flow is 0-50m 3 / h. The fermentation period of the 114 tank is 110h. The 115 tank fermentation process is only aeration once.

[0098] Test Example 1

[0099] The titer of chlortetracycline in the fermentation broth at the 90th hour and the 110th hour of fermentation in Example 1 and Comparative Example 1 is determined, and the determination method is as follows:

[0100] 1. 75mL of fermentation broth is taken in a 150mL triangular flask, an appropriate amount of oxalic acid is added, and then it is shaken for about 1 minute, and then it is placed in a multi-tank water bath for 5 minutes, taken out, and then shaken for about 1 minute. The pH is tested with pH paper to be acidified to pH 1.5-1.8, and then filtered with filter paper into a dry test tube.

[0101] 2. 5mL of the acidified filtrate is taken into a triangular flask containing dilution water (the dilution water is determined according to the dilution multiple and distilled water, and the dilution multiple is determined according to the titer of about 1000μ / mL after dilution).

[0102] 3. 1mL of the diluted solution is taken into two 50mL volumetric flasks, and 5mL of 2mol / L hydrochloric acid is added to each of them. One of them is immediately diluted to the mark with distilled water and shaken well, and the other one is placed in a 100℃ water bath for heating for 5 minutes, and then cooled and diluted to the mark with distilled water.

[0103] 4. The spectrophotometer is turned on and preheated for 20-30 minutes, the wavelength is adjusted to 440nm, and the solutions in the above volumetric flasks are compared with a 1cm cuvette, and the absorbance is read.

[0104] 5. Calculation:

[0105] 5.1 Read the standard curve table of absorbance (The standard curve is prepared by using chlortetracycline hydrochloride standard to prepare solutions with different concentrations, then 1 mL of each solution with the same concentration is taken into two 50 mL volumetric flasks, 5 mL of 2 mol / L hydrochloric acid is added to each, one of which is used as a blank and immediately diluted to the mark with distilled water and shaken, the other is heated in a 100°C water bath for 5 minutes and then cooled and diluted to the mark with distilled water. The spectrophotometer is turned on and preheated for 20-30 minutes, the wavelength is set to 440 nm, and 1 cm cuvettes are used to compare the solutions in the above volumetric flasks, and the absorbance is read), the corresponding unit value x is found according to the absorbance value and the standard curve, and the standard curve is established according to the concentration of the standard and the absorbance value, wherein the standard curve table is shown in Table 1, and the standard curve graph is shown in Figure 1, in which the horizontal axis represents the absorbance value, and the vertical axis represents the titer (μ / mL). Figure 1 Figure 1

[0106] Table 1 Standard curve table

[0107]

[0108] 5.2 Titer (μ / mL) in the sample = n x x (n: dilution multiple).

[0109] The determination results show that the titers of the fermentation broth (dilution multiple is 20 times) of Example 1 and Comparative Example 1 at the 90th hour of fermentation are 20106 μ / mL and 19839 μ / mL, respectively; at the 110th hour of fermentation, the titers of the fermentation broth (dilution multiple is 20 times) of Example 1 and Comparative Example 1 are 23728 μ / mL and 22925 μ / mL, respectively; the titer growth rate of Example 1 after 90 hours of fermentation is 181.1 μ / mL / h, and the titer growth rate of Comparative Example 1 after 90 hours of fermentation is 154.3 μ / mL / h, and the titer growth rate of Example 1 is increased by 17.4% compared with that of Comparative Example 1.

[0110] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.​​

Claims

1. A fermentation method for increasing the potency rate of chlortetracycline after 90 hours of fermentation, characterized in that, Includes the following steps: The spores of Streptomyces aureus were inoculated into the seed culture medium for seed culture to obtain the seed culture. The seed culture was transferred to a fermentation medium for fermentation to obtain a fermentation broth containing chlortetracycline; the inoculum size of *Streptomyces aureus* spores was 2.26 × 10⁻⁶. 8 The total sugar concentration of the seed culture medium after sterilization was 4.13%; The seedlings were transplanted at an age of 22.5 hours; the pH of the seed solution was 5.

76. The ratio of slow-release organic nitrogen to readily available organic nitrogen in the fermentation medium was 2.07; the total sugar concentration after sterilization in the fermentation medium was 7.26%; the total sugar concentration at 25 hours of fermentation was 3.05%, and the total sugar concentration before 90 hours was 2.12%. The fermentation temperature was reduced from 32℃ to 29℃ during the 17th to 19th hour of fermentation culture. The seed culture medium comprises the following components by weight percentage: 2.8% soybean meal, 4.6% corn starch, 0.1% sodium chloride, 0.29% ammonium sulfate, 0.4% calcium carbonate, 1.0% yeast powder, 0.03% magnesium sulfate, 0.05% potassium dihydrogen phosphate, 0.07% bubbly precipitant, and the balance being water. The fermentation medium comprises the following components in weight percentage: 2.0% peanut meal, 1.0% soybean meal, 6.0% corn starch, 0.02% amylase, 0.2% sodium chloride, 0.5% ammonium sulfate, 0.60% calcium carbonate, 0.85% yeast powder, 1.3% corn steep liquor, 0.02% magnesium sulfate, 0.01% potassium dihydrogen phosphate, 0.020% foaming agent, and the balance being water; The first release of the fermentation culture was at the 83rd hour, and the duration of the first release was 3 minutes. During the first carryover process, the exhaust volume of the fermenter was 200-300 m³ / h. 3 / h; The fermentation culture temperature is 29~32℃; The fermentation culture period is 110 hours.

2. The application of the fermentation method according to claim 1 in the production of chlortetracycline-related products.

3. The application according to claim 2, characterized in that, The chlortetracycline-related products include chlortetracycline premixes.

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