A fermentation method of aureomycin and application thereof

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to a fermentation method of aureomycin and application thereof. The present application reduces the mass of slow-acting nitrogen substances in fermentation raw materials, and adds papain in the fermentation raw materials, thereby improving the utilization rate of nitrogen substances in the fermentation raw materials to meet the fermentation demand of streptomyces aureofaciens, and reducing the use amount of nitrogen substances; meanwhile, the residual sugar concentration in the fermentation broth is reduced, thereby achieving the effect of reducing the COD of wastewater discharged in the front end of aureomycin fermentation. Meanwhile, the fermentation method is simple to operate and easy to popularize, which greatly saves the raw material cost and environmental protection cost, and greatly reduces the preparation cost of aureomycin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to a fermentation method of aureomycin and application thereof. BACKGROUND

[0002] Aureomycin is a broad-spectrum antibiotic, which has the effects of preventing and treating diseases and improving feed efficiency. At present, the global production capacity of aureomycin premix is about 110,000 tons, and new enterprises are continuously joining in, while the original production enterprises are also increasing the production capacity. With the development of the livestock and poultry breeding industry, the market of aureomycin will further grow.

[0003] With the continuous improvement of the national discharge standard of fermentation industrial wastewater, the proportion of environmental protection cost in the production cost of enterprises is becoming higher and higher. At the same time, the wastewater pollutant discharge standard of aureomycin is strictly regulated in the “Fermentation Pharmaceutical Industry Water Pollutant Discharge Standard GB21903-2008”, and the unit product drainage base is 500 cubic / t, and the chemical oxygen demand (COD) (COD refers to the amount of reducing substances that need to be oxidized in the water sample measured by chemical method) is not higher than 120 (100, applicable to the combined production enterprises producing fermentation raw materials and mixed preparation at the same time) mg / L. In order to reduce the environmental protection cost of aureomycin fermentation, it is one of the important links to reduce the COD of aureomycin fermentation wastewater.

[0004] In the aureomycin fermentation process, the factors affecting the COD of the filtrate after tank discharge mainly include two aspects, one is the dosage of raw materials in the medium formula, and the other is the utilization rate of the raw materials by the aureus streptomyces. At present, although there are reports on the improvement of wastewater treatment process to reduce the COD of discharged aureomycin wastewater, how to reduce the COD of discharged wastewater from the front end of aureomycin fermentation by process optimization and improving the utilization rate of raw materials has been a difficult problem in the field. SUMMARY

[0005] The purpose of the present application is to provide a fermentation method of aureomycin and application thereof, which can reduce the COD of aureomycin fermentation wastewater by improving the utilization rate of fermentation raw materials and reducing the residual material of fermentation culture solution, thereby achieving the technical effect of reducing the environmental protection cost of aureomycin from the front end of aureomycin fermentation.

[0006] The present application provides a fermentation method of aureomycin, which comprises the following steps:

[0007] The aureus streptomyces seed liquid is inoculated into the fermentation medium for fermentation culture, and the fermentation culture solution containing aureomycin is obtained, which can be discharged into the tank.

[0008] The mass percentage of the slow-acting nitrogen substance in the fermentation medium is 2.8-3.0%;

[0009] The fermentation medium comprises papain, and the mass of the papain is 0.1-0.3% of the total mass of the delayed nitrogen substances in the fermentation medium.

[0010] The residual sugar concentration of the fermentation medium after tanking is 0.8-1.2%.

[0011] Preferably, the delayed nitrogen substances comprise peanut cake powder and soybean cake powder.

[0012] Preferably, the fermentation medium for chlortetracycline comprises the following components in the following mass percentages: the total amount of peanut cake powder and soybean cake powder is 2.8-3.0%, 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%, and the rest is water.

[0013] Preferably, the activity of the papain is ≥200,000 units per gram.

[0014] Preferably, the total sugar concentration in the fermentation medium is ≤2.0% at the 90th hour of the fermentation.

[0015] Preferably, the fermentation process is accompanied by feeding, and the feeding medium preferably comprises the following components in the following mass percentages: corn starch 35-40%, amylase 0.06-0.08%, calcium carbonate 0.3-0.4%, sodium chloride 0.11-0.12%, ammonium sulfate 0.3-0.4%, magnesium sulfate 0.02-0.03%, calcium chloride 0.2-0.3%, and the rest is water.

[0016] Preferably, the amount of the feeding medium supplied per hour is 0.0-0.4% of the volume of the fermentation medium at 90h-2h before tanking.

[0017] Preferably, the total sugar concentration of the feeding medium after consumption is 31-35%.

[0018] The application also provides the use of the method described in the above technical solution in the production of chlortetracycline-related products.

[0019] Preferably, the chlortetracycline-related products comprise chlortetracycline premix.

[0020] Advantages:

[0021] The application provides a fermentation method of aureomycin, which comprises the following steps: inoculating a seed solution of Streptomyces aureofaciens into a fermentation medium to perform fermentation culture, obtaining a fermentation culture solution containing aureomycin, and discharging the fermentation culture solution; the total mass percentage of the delayed nitrogen substances in the fermentation medium is 10% lower than that in an aureomycin fermentation medium; the fermentation medium comprises papain, and the mass of the papain is 0.1-0.3% of the total mass of the delayed nitrogen substances in the fermentation medium; and the residual sugar concentration of the fermentation culture solution discharged is 0.8-1.2%. The application reduces the mass of the delayed nitrogen substances in the fermentation raw material, adds papain in the fermentation raw material, improves the utilization rate of the nitrogen substances in the fermentation raw material while meeting the fermentation demand of the Streptomyces aureofaciens, reduces the residual sugar concentration in the fermentation culture solution, and reduces the COD of the sewage discharged from the front end of the aureomycin fermentation.

[0022] Meanwhile, the fermentation method is simple to operate, easy to popularize, greatly saves the raw material cost and the environmental protection cost, and greatly reduces the fermentation cost of aureomycin. DETAILED DESCRIPTION

[0023] The application provides a method for reducing the COD of the filter solution of aureomycin fermentation, which comprises the following steps:

[0024] Inoculating a seed solution of Streptomyces aureofaciens into a fermentation medium to perform fermentation culture, obtaining a fermentation culture solution containing aureomycin, and discharging the fermentation culture solution; the mass percentage of the delayed nitrogen substances in the fermentation medium is 2.8-3.0%; the fermentation medium comprises papain, and the mass of the papain is 0.1-0.3% of the total mass of the delayed nitrogen substances in the fermentation medium; and the residual sugar concentration of the fermentation culture solution discharged is 0.8-1.2%.

[0025] The application preferably prepares a seed solution of Streptomyces aureofaciens, and the preparation method of the seed solution of Streptomyces aureofaciens preferably comprises the following steps: inoculating spores of Streptomyces aureofaciens into a seed culture solution to perform seed culture, and obtaining the seed solution of Streptomyces aureofaciens.

[0026] The inoculation amount of the spores of Streptomyces aureofaciens is preferably (2.0-2.3) x 10 8 / L, and more preferably 2.14 x 10 8 / L. The application does not have special limitations on the strain type of the Streptomyces aureofaciens, and the conventional Streptomyces aureofaciens in the field can be used, for example, the Streptomyces aureofaciens with the strain number M14-2 is used in the examples of the application.

[0027] The seed culture solution of the present application preferably comprises the following components in the 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 balance of water.

[0028] The seed culture solution of the present application preferably comprises 2.7-2.9% of soybean meal in the mass percentage, more preferably 2.8%.

[0029] The seed culture solution of the present application preferably comprises 4.3-4.7% of corn starch, further preferably 4.4-4.6%, more preferably 4.6%.

[0030] The seed culture solution of the present application preferably comprises 0.08-0.12% of sodium chloride, further preferably 0.09-0.11%, more preferably 0.1%.

[0031] The seed culture solution of the present application preferably comprises 0.28-0.32% of ammonium sulfate, further preferably 0.29-0.31%, more preferably 0.29%.

[0032] The seed culture solution of the present application preferably comprises 0.38-0.42% of calcium carbonate, further preferably 0.39-0.41%, more preferably 0.40%.

[0033] The seed culture solution of the present application preferably comprises 0.9-1.1% of yeast powder, more preferably 1.0%.

[0034] The seed culture solution of the present application preferably comprises 0.03-0.05% of magnesium sulfate, further preferably 0.03%.

[0035] The seed culture solution of the present application preferably comprises 0.05-0.07% of potassium dihydrogen phosphate, further preferably 0.05-0.06%, more preferably 0.05%.

[0036] The seed culture solution of the present application preferably comprises 0.07-0.08%, further preferably 0.07%.

[0037] The seed culture solution of the present application comprises the above mass percentage components, and other percentage components are supplemented with water.

[0038] The pH value of the seed culture solution of the present application is the natural pH value, without special adjustment.

[0039] The pH value of the seed culture solution during the inoculation of the present application is preferably 6.0-6.2; the total sugar concentration is preferably 3.8-4.2%; and the amino nitrogen content is preferably 100-120 mg / dL.

[0040] 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.

[0041] After obtaining the Streptomyces aureofaciens seed solution, the present application moves the Streptomyces aureofaciens seed solution to a fermentation medium for fermentation culture to obtain a fermentation culture solution containing chlortetracycline, and then is discharged.

[0042] The fermentation medium of the present application includes papain, and the mass of the papain is 0.1-0.3% of the mass of the delayed nitrogen substance in the fermentation medium, preferably 0.2%. The activity unit of the papain of the present application is preferably ≥200,000 units per gram, more preferably 200,000 units per gram.

[0043] The fermentation medium of the present application preferably includes the following components with the mass percentage: the total amount of peanut cake powder and soybean cake powder is 2.8-3.0%, corn starch is 5.8-6.2%, amylase is 0.02-0.03%, sodium chloride is 0.2-0.4%, ammonium sulfate is 0.5-0.6%, calcium carbonate is 0.60-0.65%, yeast powder is 0.85-0.95%, corn syrup is 1.3-1.4%, magnesium sulfate is 0.02-0.03%, potassium dihydrogen phosphate is 0.01-0.02%, and the rest is water.

[0044] The fermentation medium of the present application preferably includes 2.8-3.0% of peanut cake powder and soybean cake powder, and further preferably 2.8%. The peanut cake powder and soybean cake powder of the present application are preferably the source of the delayed nitrogen substance. The mass ratio of the peanut cake powder and soybean cake powder of the present application is preferably 1.9:0.9.

[0045] The fermentation medium of the present application preferably includes 5.8-6.2% of corn starch, further preferably 5.9-6.1%, and more preferably 6.0%.

[0046] The fermentation medium of the present application preferably includes 0.02-0.03% of amylase, further preferably 0.02-0.025%, and more preferably 0.0.2%.

[0047] The fermentation medium of the present application preferably includes 0.2-0.4% of sodium chloride, further preferably 0.2-0.3%, and more preferably 0.2%.

[0048] The fermentation medium preferably comprises 0.5-0.6% of ammonium sulfate, further preferably 0.5-0.55%, and more preferably 0.5%.

[0049] The fermentation medium preferably comprises 0.60-0.65% of ammonium carbonate, further preferably 0.60%.

[0050] The fermentation medium preferably comprises 0.85-0.95% of yeast powder, further preferably 0.85-0.90%, and more preferably 0.85%.

[0051] The fermentation medium preferably comprises 1.3-1.4% of corn syrup, further preferably 1.3%.

[0052] The fermentation medium preferably comprises 0.02-0.03% of magnesium sulfate, and more preferably 0.02%.

[0053] The fermentation medium preferably comprises 0.01-0.02% of potassium dihydrogen phosphate, further preferably 0.01-0.015%, and more preferably 0.01%.

[0054] The fermentation medium preferably comprises 0.020-0.025% of Tween 80, further preferably 0.020%.

[0055] The fermentation medium comprises the above-mentioned components in the percentage of mass, and other components are supplemented with water.

[0056] The seed age for inoculation is preferably 22.0-24.0h, and more preferably 23.0h, that is, the seed liquid of seed culture for 22.0-24.0h is inoculated into the fermentation medium for fermentation.

[0057] The pH value of the fermentation medium for inoculation is preferably 6.0-6.2, the total sugar concentration is preferably 6.7-7.2%, and the content of amino nitrogen is preferably 200-230mg / dL. The fermentation culture temperature is preferably 32-33℃. The fermentation is preferably supplemented according to the total sugar concentration in the fermentation broth, so that the total sugar concentration in the fermentation culture is 2.8-3.5% at 20-30h, 2.0-2.5% at 30-90h, 1.5-2.0% at 90-100h, and 0.8%≤total sugar in fermentation tank≤1.2%.

[0058] The feed medium of the present application preferably comprises the following components in mass percentage: corn starch 35-40%, amylase 0.06-0.08%, calcium carbonate 0.3-0.4%, sodium chloride 0.11-0.12%, ammonium sulfate 0.3-0.4%, magnesium sulfate 0.02-0.03%, calcium chloride 0.2-0.3%, and the balance water.

[0059] The feed medium of the present application preferably comprises corn starch in mass percentage of 35-40%, further preferably 37-40%, and more preferably 40%.

[0060] The feed medium of the present application preferably comprises amylase in mass percentage of 0.06-0.08%, and further preferably 0.07%.

[0061] The feed medium of the present application preferably comprises calcium carbonate in mass percentage of 0.3-0.4%, and further preferably 0.35%.

[0062] The feed medium of the present application preferably comprises sodium chloride in mass percentage of 0.11-0.12%, and further preferably 0.11%.

[0063] The feed medium of the present application preferably comprises ammonium sulfate in mass percentage of 0.3-0.4%, and further preferably 0.3%.

[0064] The feed medium of the present application preferably comprises magnesium sulfate in mass percentage of 0.02-0.03%, and further preferably 0.02%.

[0065] The feed medium of the present application preferably comprises calcium chloride in mass percentage of 0.3-0.4%, and further preferably 0.3%.

[0066] The feed medium of the present application comprises the above-mentioned components in mass percentage, and other components are supplemented with water. The feed medium of the present application has the effects of maintaining the concentration of Streptomyces aureofaciens and promoting the production of aureomycin by Streptomyces aureofaciens.

[0067] The total sugar concentration in the fermentation broth of the fermentation culture of the present application is preferably ≤2.0% at the 90th hour. The amount of the feed medium supplemented per hour is 0.0-0.4% of the volume of the fermentation broth at 90h-2h before the tank is discharged. The above-mentioned feed frequency and feed amount can control the residual sugar concentration at 0.8-1.2% when the tank is discharged, and preferably 1.17%. The present application preferably stops feeding at 2h before the tank is discharged, and the total sugar concentration after the consumption of the feed medium is preferably 31-35%, and more preferably 32-34%.

[0068] The period of the fermentation culture of the present application is preferably 105-115h, and more preferably 110h.

[0069] The fermentation culture has no special limitation in other conditions, and the conventional fermentation culture condition in the art can be used.

[0070] The fermentation method reduces the mass of the delayed effect nitrogen substances in the fermentation raw material, and adds papain in the fermentation raw material, so as to meet the fermentation requirement of Streptomyces aureofaciens and improve the utilization rate of the nitrogen substances in the fermentation raw material; and the residual sugar concentration in the fermentation culture solution is reduced, so as to reduce the COD of the sewage discharged in the fermentation of aureomycin from the front end.

[0071] Based on the above technical advantages, the application also provides the application of the method in the production of aureomycin related products. The aureomycin related products preferably include aureomycin premix. The specification of the aureomycin premix has no special limitation, and the conventional specification in the art can be used.

[0072] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0073] Experimental Example 1

[0074] The selection test of the protease is as follows:

[0075] 500 mL of peanut cake powder solution with a mass fraction of 2% and soybean meal solution with a mass fraction of 1% are configured, and the pH of the solution is adjusted to 6.3 by using hydrochloric acid, then 0.3% of the substrate mass of different proteases (the enzyme activity is 200,000 units / g) are added, and the hydrolysis is carried out for 30 min, 60 min and 120 min respectively, then the change of amino nitrogen before and after the hydrolysis is detected by using the formaldehyde titration method, so as to verify the hydrolysis effect of different proteases, and the results are shown in Table 1:

[0076] Table 1 Hydrolysis effect of different proteases on peanut cake powder and soybean meal

[0077]

[0078]

[0079] It can be obtained from Table 1 that the hydrolysis effect of papain on peanut cake powder and soybean meal is the best, and then the papain is selected to hydrolyze the peanut cake powder and soybean meal.

[0080] Experimental Example 2

[0081] The optimization of the addition amount of papain is as follows:

[0082] The peanut cake powder and 1% soybean cake powder solution 500 mL with a quality score of 2% was configured, and the solution pH was adjusted to 6.3 with hydrochloric acid, then the papain (enzyme activity was 200,000 units / g) was added in the proportion of 0.1%, 0.2%, 0.3%, 0.4%, 0.5% of the mass of peanut cake powder and soybean cake powder, three repeats for each proportion, respectively, hydrolyzed for 60 min, and the hydrolysis effect of different proportions was verified, and the results are shown in Table 2:

[0083] Table 2 Effect of different papain addition ratios on hydrolysis of peanut cake powder and soybean cake powder

[0084]

[0085] From Table 2, it can be concluded that as the addition of papain ratio increases, the amino nitrogen level gradually increases, and when the addition ratio reaches 0.3%, the amino nitrogen level increases limitedly, and then the subsequent test is carried out with 0.3% papain of the total mass of peanut cake powder and soybean cake powder.

[0086] Example 1

[0087] A fermentation method of aureomycin, the steps are as follows:

[0088] In the pilot plant, 114 tanks are used as fermentation tanks, and the total volume of 114 tanks is 3m 3 The tank is configured with a feeding device and an online monitoring and control system of pH, dissolved oxygen and temperature;

[0089] 116 tanks are used as seed tanks, and the total volume is 1m 3 The corresponding seed tank 116 of the 114 tank fermentation tank is configured with a temperature online monitoring and control system;

[0090] 201 tanks are used as feeding tanks, and the total volume is 3m 3 The 201 tank is used to feed the 114 tank.

[0091] Seed culture stage: the seed culture solution in the 116 tank is composed of the following components with a mass percentage of (%): soybean cake powder 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, pH is natural;

[0092] According to the formula of seed culture solution, the ingredients are prepared according to 116 seed tank constant volume 180 liters, after the end of the constant volume, the valve is closed, the tank cover, then open the inlet steam, discharge steam, sight glass steam, sampling steam, etc. Steam valve and each small exhaust valve, temperature rise, 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, the sterile air into the tank can be passed;

[0093] When the tank temperature is lower than 50 ℃, the pH, total sugar and amino nitrogen of the culture medium in the 116 tank are detected; the pH of the culture medium in the 116 tank is 6.09, the total sugar is 4.01%, and the amino nitrogen is 108 mg / dL; when the tank temperature drops to 32-33 ℃, inoculation is carried out;

[0094] The strain number used in the 116 seed tank is M14-2, and the spore concentration is 19.3 billion / mL. 2.0 mL of spore solution is taken in a sterile syringe in a clean room super-clean workbench, the syringe is connected to the 116 tank through the inoculation port, and then the temperature automatic control system is started; 31-33 ℃ culture, 19h, every half hour, pH and mycelium are detected.

[0095] In the fermentation culture stage, the fermentation medium in the 114 tank is as follows: peanut cake powder 1.9, soybean cake powder 0.9, 200,000 units / g of papain 0.0084, 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 87.6716, pH value is natural;

[0096] The fermentation tank 114 is prepared according to the constant volume 1500 liters, after the end of the constant volume, the valve is closed, the tank cover, then open the inlet steam, discharge steam, sight glass steam, sampling steam, etc. Steam valve and each small exhaust valve, temperature rise, 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, the sterile air into the tank can be passed;

[0097] 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 6.12, the total sugar is 6.93%, and the amino nitrogen is 215 mg / dL; the temperature of the fermentation tank is reduced to 32-33℃, and the seed liquid is waited to reach the inoculation standard; the inoculation operation is performed through the sterilized inoculation pipeline;

[0098] The feed medium in the 201 tank comprises the following components in percentage by mass (%): corn starch 40, amylase 0.07, calcium carbonate 0.35, sodium chloride 0.11, ammonium sulfate 0.3, magnesium sulfate 0.02, calcium chloride 0.3 and water 58.85, and the pH value is natural.

[0099] The constant volume of the 201 tank is 1000 liters; after the material is punched and the constant volume is reached, 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; when the temperature is raised to 118℃ and the tank pressure is greater than 0.1 Mpa, the pressure holding is started, the temperature is kept at 118-125℃ for 30 minutes, then the small exhaust valves and the steam valves are closed, and the temperature is reduced; when the tank pressure is reduced to below 0.08 Mpa, the sterile air can be introduced into the tank;

[0100] When the temperature of the tank is below 50℃, sample and detect the pH, total sugar, reducing sugar and amino nitrogen of the feed medium in the 201 tank; the indexes of the feed medium in the 201 tank are as follows: the pH value is 5.33, the total sugar is 33.4%, the reducing sugar is 5.5%, and the amino nitrogen is 118 mg / dL;

[0101] The 201 tank is kept at 20-50℃ for standby;

[0102] The seed tank of the 116 tank corresponds to a seed tank inoculation seed age of 23.0 h, and the inoculation pH value is 5.66; the seed liquid is moved into the 114 fermentation tank; during the process of moving the seed liquid into the fermentation tank, the dissolved oxygen electrode is calibrated; after the inoculation is completed, the temperature and the dissolved oxygen start the automatic control; the temperature is controlled at 31-33℃; when the fermentation pH value is reduced to 5.95, the pH value starts the automatic control; when the total sugar concentration is reduced to about 4.3%, the total sugar is supplemented according to the total sugar concentration; the total sugar supplement system starts the automatic control;

[0103] When the dissolved oxygen is 32% at the 17.5th hour of the fermentation culture in the 114 tank, the set value is set from 32℃ to 29℃ on the temperature automatic control system;

[0104] The total sugar in the 114 tank at the 92th hour of the fermentation is 1.83%;

[0105] After the fermentation culture for 90 h, the feed rate is 0.1-0.4% of the volume of the fermentation liquid per hour according to the total sugar concentration and the consumption rate of the fermentation liquid; the feed is stopped two hours before the tank is discharged;

[0106] Total sugar in tank 1.17%

[0107] At 110 hours of fermentation, the fermentation was stopped, and 114 tank of fermentation broth was pumped into two small storage tanks, 60 kg of light calcium carbonate was added to each tank, and stirred for 40 minutes;

[0108] The fermentation broth after adding calcium carbonate and stirring was pumped into a plate frame, the filtration pressure was 0.58 MPa, the squeezing pressure was 0.85 MPa, 150 mL of filtrate was collected at 10 minutes after the fermentation broth entered the plate frame, 40 minutes after the fermentation broth entered the plate frame, and after the fermentation broth entered the plate frame, respectively, then stirred in a 1000 mL beaker for 1 minute, mixed uniformly, then the COD of the filtrate was determined by potassium dichromate method, and the titer of the fermentation broth in 201 tank was 23824 μ / mL; the COD of the fermentation broth filtrate was 24569 mg / L.

[0109] Comparative Example 1

[0110] A fermentation method of aureomycin, the steps are as follows:

[0111] In the pilot plant, 115 tank was used as a control fermentation tank, the total volume of 115 tank was 3m 3 , the tank was equipped with a feeding device and an online monitoring and automatic control system of pH, dissolved oxygen and temperature;

[0112] 117 tank was a control seed tank, the total volume was 1m 3 , equipped with an online monitoring and automatic control system of temperature, and the seed tank corresponding to the 115 tank fermentation tank was 117 tank;

[0113] 202 tank was a feeding tank, the total volume was 3m 3 , and 202 tank was used to feed 115 tank.

[0114] Seed culture stage: the seed culture solution in 117 tank was 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, pH was natural; the seed culture solution used in 117 control seed tank was the same batch as the seed culture solution in 116 tank in Example 1, and both met the company's quality standards after quality inspection.

[0115] According to 117 seed tank constant volume 180 liters to carry out the ingredients, beat the material, constant volume after the end, close 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 rise, 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 below, can pass into the tank sterile air;

[0116] When the tank temperature is lower than 50℃, sample 117 tank medium pH, total sugar, amino nitrogen index; 117 tank medium pH is 6.10, total sugar is 4.01%, amino nitrogen is 108 mg / dL, when the tank temperature drops to 32-33℃, inoculation;

[0117] 117 seed tank using the strain number M14-2, the spore concentration is 19.3 billion / mL, using 5 mL sterile syringe in clean room super clean bench to take 2.0 mL spore liquid, the syringe from the inoculation port into 117 tank, then start temperature control system, 31-33℃ culture, 19h after, every half hour sample detection pH and microscopic mycelium.

[0118] Fermentation culture stage: the fermentation medium in 115 tank is as follows: peanut cake powder 2.1, soybean meal 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 87.38, pH value is natural;

[0119] Fermentation tank 115 according to constant volume 1500 liters to prepare medium, beat the material, constant volume after the end, close 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 rise, 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 below, can pass into the tank sterile air;

[0120] When the tank temperature is lower than 50℃, sample 115 tank medium pH, total sugar, amino nitrogen index; 115 tank medium pH is 6.08, total sugar is 6.85%, amino nitrogen is 213 mg / dL, fermentation tank temperature drops to 32-33℃, wait for seed liquid to reach the seed standard, through the sterilized seed transfer pipeline to carry out seed transfer operation;

[0121] 202 tank feed medium is the following mass percentage of components (%): corn starch 40, amylase 0.07, calcium carbonate 0.35, sodium chloride 0.11, ammonium sulfate 0.3, magnesium sulfate 0.02, calcium chloride 0.3 and water 58.85, pH value is natural.

[0122] The constant volume of the 202 tank is 1200 liters. After the material is beaten and the constant volume is completed, the beating valve and the tank cover are closed, and then the steam valves of the inlet air, the discharge, the sight glass and the sampling are opened, and the exhaust valves are opened. Temperature is raised, and the steam flow of each pipeline is checked. When the temperature is raised to 118℃ and the tank pressure is greater than 0.1Mpa, the pressure holding is started, and the pressure is kept for 30 minutes at 118-125℃. Then the exhaust valves and the steam valves are closed, and the temperature is lowered. When the tank pressure is reduced to 0.08Mpa or less, sterile air can be introduced into the tank;

[0123] When the tank temperature is lower than 50℃, the pH value, total sugar, reducing sugar and amino nitrogen of the 202 tank feed medium are detected. The pH value of the 202 tank feed medium is 5.38, the total sugar is 30.2%, the reducing sugar is 5.2%, and the amino nitrogen is 110mg / dL;

[0124] The 202 tank is kept at 20-50℃ for standby;

[0125] The seed tank of 117 corresponds to the seed tank of 23.0h. The pH value of the seed tank is 5.62, and the seed liquid is moved into the 115 fermentation tank. During the process of moving the seed liquid into the fermentation tank, the dissolved oxygen electrode is calibrated. After the seed is moved, the temperature and dissolved oxygen start automatic control. The temperature is controlled at 31-33℃. When the fermentation pH value is reduced to 5.95, the pH value starts automatic control. When the total sugar concentration is reduced to about 4.3%, the total sugar concentration is supplemented. The total sugar concentration starts automatic control.

[0126] At the 17.5h of the 115 tank fermentation, the dissolved oxygen is 31%. On the temperature control system, the set value is set from 32℃ to 29℃.

[0127] At the 92h of the 115 tank fermentation, the total sugar is 2.10%.

[0128] After 90h of fermentation culture, according to the total sugar concentration and consumption rate of the fermentation broth, the feeding rate is 0.1-0.4% of the fermentation broth volume per hour. Two hours before the tank is discharged, the feeding is stopped.

[0129] The total sugar of the 115 tank is 1.35%.

[0130] At the 110h of fermentation culture, the fermentation is stopped, the 115 tank fermentation broth is discharged into two small storage tanks, and 60 kilograms of light calcium carbonate is added to each tank, and stirred for 40 minutes.

[0131] The fermentation liquor after stirring evenly with calcium carbonate was poured into a plate frame, the filtration pressure was 0.58 MPa, the squeezing pressure was 0.85 MPa, 150 mL of filtrate was connected respectively at 10 minutes after the fermentation liquor entered the plate frame, 40 minutes after the fermentation liquor entered the plate frame and after the fermentation liquor entered the plate frame completely, then the mixture was stirred in a 1000 mL beaker for 1 minute, and then the COD of the filtrate was determined by the potassium dichromate method, so that the titer of the fermentation liquor of the 202th tank was 23552 μ / mL; the COD of the filtrate of the fermentation liquor was 25768 mg / L.

[0132] It can be concluded from Example 1 and Comparative Example 1 that, compared with Comparative Example 1, the fermentation of aureomycin is carried out by the fermentation method described in Example 1, the COD in the fermentation filtrate is reduced by 1199 mg / L on the basis of ensuring the titer of the fermentation liquor.

[0133] Example 2

[0134] The method in Example 1 was used for aureomycin fermentation, except that the total volume of the fermentation tank was 50 L, and the fermentation medium was composed of the following components with mass percentage (%): peanut cake powder 2.0, soybean cake powder 1.0, 200,000 units / g of papain 0.009, corn starch 6.0, amylase 0.02, sodium chloride 0.2, ammonium sulfate 0.5, calcium carbonate 0.60, yeast powder 0.85, corn slurry 1.3, magnesium sulfate 0.02, potassium dihydrogen phosphate 0.01, bubble enemy 0.020 and water 87.471, and the pH value was natural; the fermentation period was 111 h, the total sugar after fermentation was 1.14%, the titer of the fermentation liquor was 23745 μ / mL, and the COD of the fermentation liquor was 24155 mg / L.

[0135] Comparative Example 2

[0136] The method in Example 1 was used for aureomycin fermentation, except that the fermentation medium was composed of the following components with mass percentage (%): peanut cake powder 1.7, soybean cake powder 0.85, 200,000 units / g of papain 0.00855, corn starch 6.0, amylase 0.02, sodium chloride 0.2, ammonium sulfate 0.5, calcium carbonate 0.60, yeast powder 0.85, corn slurry 1.3, magnesium sulfate 0.02, potassium dihydrogen phosphate 0.01, bubble enemy 0.020 and water 87.92145, and the pH value was natural; the fermentation period was 111.5 h, the total sugar after fermentation was 1.19%, the titer of the fermentation liquor was 23461 μ / mL, and the COD of the fermentation liquor was 23353 mg / L.

[0137] Comparative Example 3

[0138] The method in Example 1 is used for the fermentation of aureomycin, with the difference that the fermentation medium is composed of the following components in mass percentage (%): peanut cake powder 1.60, soybean cake powder 0.8, 200,000 units / g of papain 0.0072, 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 88.0728, with the pH value being natural; the fermentation period is 111.5 h, the total sugar after fermentation is 1.19%, the titer of the fermentation liquor is 21,107 μ / mL, and the COD of the filtrate of the fermentation liquor is 22,845 mg / L.

[0139] From the above examples, it can be concluded that the fermentation method described in the present application can reduce the COD of the wastewater discharged in the fermentation of aureomycin by reducing the nitrogen-containing substances in the fermentation raw materials from the front end of the fermentation and improving the utilization rate of the raw materials on the basis of ensuring the titer of the fermentation liquor.

[0140] 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 by people according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A fermentation method for chlortetracycline, characterized in that, Includes the following steps: The seed culture of Streptomyces aureus was transferred to a fermentation medium for fermentation culture to obtain a fermentation broth containing chlortetracycline, which was then placed in a tank. The fermentation medium contains 2.8-3.0% by mass of delayed-release nitrogen compounds. The fermentation medium includes papain, and the mass of the papain is 0.1-0.3% of the total mass of delayed-release nitrogenous substances in the fermentation medium. The residual sugar concentration of the fermentation broth discharged into the tank is 0.8-1.2%; The delayed-release nitrogen compounds include peanut meal powder and soybean meal powder; The fermentation medium comprises the following components by weight percentage: 2.8-3.0% total of peanut cake powder and soybean cake powder, 5.8-6.2% corn starch, 0.02-0.03% amylase, 0.2-0.4% sodium chloride, 0.5-0.6% ammonium sulfate, 0.60-0.65% calcium carbonate, 0.85-0.95% yeast powder, 1.3-1.4% corn steep liquor, 0.02-0.03% magnesium sulfate, 0.01-0.02% potassium dihydrogen phosphate, 0.020-0.025% foaming agent, and the balance being water; The papain activity is ≥200,000 units per gram; The fermentation process is accompanied by feeding, and the feeding medium includes the following components in weight percentage: corn starch 35-40%, amylase 0.06-0.08%, calcium carbonate 0.3-0.4%, sodium chloride 0.11-0.12%, ammonium sulfate 0.3-0.4%, magnesium sulfate 0.02-0.03%, calcium chloride 0.2-0.3%, and the balance being water; During the 90 hours of fermentation culture to 2 hours before discharge, the amount of the feeding medium added per hour is 0.1 to 0.4% of the fermentation liquid volume.

2. The fermentation method according to claim 1, characterized in that, At the 90th hour of fermentation, the total sugar concentration in the fermentation broth was ≤2.0%.

3. The fermentation method according to claim 1, characterized in that, The total sugar concentration of the supplemental culture medium after sterilization is 31-35%.

4. The application of the fermentation method according to any one of claims 1 to 3 in the production of chlortetracycline-related products.

5. The application according to claim 4, characterized in that, The chlortetracycline-related products include chlortetracycline premixes.

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