CN109182411B - Process for producing lincomycin by feed fermentation - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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- General Health & Medical Sciences (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention relates to a process for producing lincomycin by feed fermentation, which comprises the following steps: seed culture, shake flask culture, secondary tank culture and fermentation tank fermentation culture. In the fermentation culture process, small materials consisting of 8-12 parts of soybean cake powder, 15-20 parts of corn steep liquor, 4-5 parts of calcium carbonate, 2-3 parts of magnesium sulfate, 0.2-0.4 part of leucine, 0.3-0.5 part of simethicone, 0.3-0.5 part of soybean oil and 200 parts of water are added; supplementing ammonium sulfate solution or ammonia water as required to regulate and control the pH of the fermentation liquor; sugar solution is supplemented as required to ensure that the content of reducing sugar in the fermentation liquor is in a proper range. The titer of the lincomycin obtained by fermentation production by adopting the process provided by the invention is over 10000U/mL, and the content of the component B in the separated and purified lincomycin is lower than 0.5%.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a process for producing lincomycin by fed-batch fermentation.
Background
Lincomycin belongs to lincomamine antibiotics, is a narrow-spectrum antibiotic, acts on 50S subunit of sensitive sclerotium to prevent the extension of peptide chain, thereby inhibiting the protein synthesis of bacterial cells, has better effect on gram-positive cocci, and particularly has high-efficiency bacteriostatic action on anaerobic bacteria, staphylococcus aureus and pneumococcus. The action mechanism of the compound is similar to that of erythromycin, belongs to bacteriostatic agents and is mainly used for various infections caused by sensitive bacteria clinically, such as pneumonia, meningitis, endocarditis, cellulitis, tonsillitis, erysipelas, furuncle, urinary system infection and the like. The product can enter bone tissue and has special affinity with bone, so it is especially suitable for infection caused by anaerobic bacteria and Staphylococcus aureus osteomyelitis. Lincomycin is generally lincomycin A, while lincomycin B has low antibacterial activity and high toxicity and can be regarded as impurities in lincomycin.
Lincomycin is produced in china in the seventies of the last century and is already a very mature product. However, for a long time, fermentation product quality, yield and cycle time have seemed to have reached limits due to limitations in process development. Chinese patent CN101220385 discloses a method for regulating and controlling a lincomycin fermentation process by using ammonium ions so as to improve the lincomycin yield, which specifically comprises the following steps: adjusting the initial ammonium ion concentration in the fermentation medium to 57 +/-5 mmoL/L, supplementing ammonium ions (ammonium sulfate or ammonium nitrate) when the ammonium ion concentration is 0.2-1.5mmoL/L, and then controlling the ammonium ion concentration to be 2-5mmoL/L until the end of fermentation. The strain cultured by fermentation is streptomyces lincolnensis L-427, the yield of the lincomycin obtained by fermentation is improved, and the biological value reaches 7000U/mL.
The fermentation culture process of the streptomyces lincolnensis is singly regulated and controlled by regulating and controlling the ammonium ion concentration, although the yield of the lincomycin is improved, the content of the lincomycin B in the product cannot be determined, and the yield of the lincomycin is still to be improved. In the prior art, a feeding mode is adopted, so that the culture process cannot be accurately controlled, the local reducing sugar and amino nitrogen content in feed liquid before and after feeding are high, inhibition effect on growth and metabolism of thalli is possibly generated, the growth difference of the thalli in a fermentation tank is large, and the lincomycin fermentation production is extremely unfavorable. Therefore, the research on a fermentation process with high lincomycin fermentation yield, high titer and low lincomycin B content is a long-term target of lincomycin fermentation production.
Disclosure of Invention
The invention aims to provide a process for producing lincomycin by supplemented fermentation, which is characterized in that small materials mainly comprising soybean cake powder and corn steep liquor are supplemented at regular time, ammonium sulfate solution or ammonia water and sugar solution are supplemented as required in the fermentation process, so that the pH value in fermentation liquor is stable, the environment is stable, the dissolved oxygen limitation in fermentation for 120 hours is avoided, the thallus grows well, the lincomycin titer is high, and the lincomycin B content is low.
Specifically, aiming at the defects of the prior art, the invention provides the following technical scheme:
a process for producing lincomycin by feed fermentation comprises the following steps: seed culture, shake flask culture, second grade jar is cultivateed and fermentation cylinder fermentation culture, carries out the feed supplement at fermentation cylinder fermentation culture in-process, specifically includes:
(1) adding small materials;
(2) regulating and controlling the pH value of the fermentation liquor by supplementing an ammonium sulfate solution or ammonia water;
(3) when the content of reducing sugar in the fermentation liquor is lower than 0.3%, sugar is supplemented;
wherein, the small material comprises the following components in parts by weight: 8-12 parts of soybean cake powder, 15-20 parts of corn steep liquor, 4-5 parts of calcium carbonate, 2-3 parts of magnesium sulfate, 0.2-0.4 part of leucine, 0.3-0.5 part of simethicone, 0.3-0.5 part of soybean oil and 200 parts of water.
According to the method, the small materials are supplemented on time in the fermentation culture process, and the reagent and the sugar solution are supplemented as required, so that the environment of the fermentation liquid is stable, the small materials are rich in nutrition and suitable for the growth requirement of the strain, and the factors act synergistically, so that the lincomycin yield after the fermentation is finished is high, and the content of the component B is low.
Preferably, the conditions of the fermentation culture are: the culture temperature is controlled at 30 +/-1 ℃, the tank pressure is maintained at 0.02-0.04MPa, the stirring is started in the whole process, and the culture period is 180-203 hours after the fermentation tank is inoculated.
Preferably, the rotation speed of stirring is specifically as follows: the rotation speed from 0 to 90 hours is 120-150 rpm, and the rotation speed from 90 hours to the end of fermentation is 80-90 rpm.
Preferably, the pH of the fermentation broth is controlled between 6.0 and 7.0.
Preferably, the method for supplementing the ammonium sulfate solution or the ammonia water comprises the following steps: when the pH value of the fermentation liquor exceeds 7.5, supplementing an ammonium sulfate solution to reduce the pH value to 6.5-7.0; when the pH value of the fermentation liquor is lower than 5.5, adding ammonia water to raise the pH value to 6.0-6.5.
Preferably, the pH value of the ammonium sulfate solution after sterilization is 3.98-4.05, and the concentration of the ammonia water is 20%.
Preferably, the method for supplementing the small materials comprises the following steps: the feed is supplemented once every 40 hours of fermentation culture, then every 24 hours, and the last supplement is supplemented 6 times in total at the 160 th hour of fermentation culture, and the volume of the small feed supplemented each time is 1.5-2.0L.
Preferably, the culture medium for fermentation culture comprises the following components in parts by weight: 10-12 parts of soybean cake powder, 15-18 parts of glucose, 15-20 parts of corn starch, 1.0-2.5 parts of magnesium sulfate, 0.3-0.5 part of leucine, 0.3-0.5 part of tyrosine, 0.5-0.8 part of potassium dihydrogen phosphate, 5-6 parts of calcium carbonate, 1.5-2.5 parts of ammonium nitrate, 2.0-5.0 parts of sodium chloride, 0.8-1.5 parts of simethicone and 120 parts of 100 parts of water.
The fermentation medium provided by the invention meets the nutritional requirements of streptomyces lincolnensis and is suitable for the growth of strains.
Preferably, the sugar supplement refers to glucose solution, sucrose solution or fructose solution with the mass concentration of 15-25%.
Preferably, the reducing sugar content in the fermentation liquor is 0.4-1.0% after sugar supplement.
Compared with the prior art, the invention has the advantages that:
1. the culture medium and culture conditions of each stage provided by the invention are very suitable for the growth of streptomyces lincolnensis. In the fermentation process, small materials mainly comprising soybean cake powder and corn steep liquor are supplemented, namely, an organic nitrogen source is continuously supplemented, so that the environment in the fermentation liquid is stable, the thalli can quickly complete primary metabolism and enter secondary metabolism, the phenomenon of limited dissolved oxygen in 120 hours in the fermentation process is avoided, and the thalli grow well.
2. And according to the pH value of the fermentation liquor, adding ammonium sulfate solution or ammonia water as required in due time to keep the pH value of the fermentation liquor stable, thereby being beneficial to the growth of thalli and the accumulation of lincomycin.
3. According to the content of reducing sugar in the fermentation liquor, sugar is supplemented timely according to needs, so that the nutrient source of the thalli is ensured, and the growth of the thalli and the accumulation of lincomycin are facilitated.
In conclusion, the lincomycin is supplemented in the process of producing lincomycin by fermentation, and the supplement method and the supplement components have synergistic effect, so that the dissolved oxygen in the fermentation process is not limited, the pH is relatively stable, the thallus grows well, the titer of the obtained lincomycin is improved to more than 10000U/mL, the yield is obviously improved, and the content of the lincomycin B component in the product is lower than 0.5%, so that the lincomycin B component meets the requirements of the pharmaceutical field.
Detailed Description
The invention provides a process for producing lincomycin by feed fermentation, which comprises the following steps: seed culture, shake flask culture, secondary tank culture and fermentation tank fermentation culture.
Seed medium (g/L): 25 parts of soybean cake powder, 8 parts of soluble starch, 20 parts of glucose, 20 parts of corn steep liquor, 0.8 part of ammonium sulfate, 0.6 part of magnesium sulfate, 0.4 part of monopotassium phosphate, and sterilizing for 30min at 121 ℃.
Shake flask medium (g/L): 30-40 parts of soybean cake powder, 10-15 parts of soluble starch, 20-30 parts of glucose, 15-25 parts of molasses, 25-35 parts of corn steep liquor, 1.0-1.5 parts of ammonium nitrate, 0.5-0.8 part of calcium carbonate, 0.5-0.8 part of magnesium sulfate, 0.3-0.6 part of dipotassium hydrogen phosphate, and sterilizing at 121 ℃ for 30 min.
Secondary tank medium (g/L): 35 parts of soybean cake powder, 15 parts of soluble starch, 40 parts of glucose, 30 parts of corn steep liquor, 0.5-0.8 part of biotin, 0.5 part of ammonium nitrate, 0.5 part of ammonium sulfate, 1.5 parts of calcium carbonate, 0.5 part of monopotassium phosphate, 1.5 parts of sodium chloride and 3.0mL/L of natural sodium citrate, and sterilizing at 121 ℃ for 30 min.
Fermentation medium of fermentation tank (parts by weight): 10-12 parts of soybean cake powder, 15-18 parts of glucose, 15-20 parts of corn starch, 1.0-2.5 parts of magnesium sulfate, 0.3-0.5 part of leucine, 0.3-0.5 part of tyrosine, 0.5-0.8 part of potassium dihydrogen phosphate, 5-6 parts of calcium carbonate, 1.5-2.5 parts of ammonium nitrate, 2.0-5.0 parts of sodium chloride, 0.8-1.5 parts of simethicone and 120 parts of 100 parts of water; sterilizing at 121 deg.C for 30 min.
The seed culture, shake flask culture and secondary tank culture are all performed at 30 deg.C for 2-3 days, 5-7 days and 2-4 days respectively; the content of the 750mL shake flask is 150mL, and the rotating speed is 200 r/min.
Inoculating the seeds cultured in the second-stage tank into a 50L fermentation tank (FUS-50L multi-parameter full-automatic control fermentation tank) by a differential pressure method, wherein the inoculation amount is 15-20%. The fermentation conditions of the fermentation tank are as follows: the culture temperature is controlled to be 30 +/-1 ℃, the compressed air inlet after filtration and sterilization is opened in the whole process, the tank pressure is maintained to be 0.02-0.04MPa, the stirring is started in the whole process, the rotating speed is 120-150 rpm in 0-90 hours, and the rotating speed is 80-90 rpm from 90 hours to the end of fermentation; the culture period after the fermentation tank is inoculated is 180-203 hours.
Feeding materials as required in the fermentation culture process, which comprises the following steps: adding small materials; adding ammonium sulfate solution or ammonia water, and regulating the pH value of the fermentation liquor to 6.0-7.0; when the content of reducing sugar in the fermentation liquor is lower than 0.3%, adding 15-25% of glucose solution, sucrose solution or fructose solution to make the content of reducing sugar in the fermentation liquor be 0.4-1.0%.
The method for supplementing the small materials comprises the following steps: the feed is supplemented once every 40 hours of fermentation culture, then every 24 hours, and the last supplement is supplemented 6 times in total at the 160 th hour of fermentation culture, and the volume of the small feed supplemented each time is 1.5-2.0L. Specifically, under the protection of flame, small materials to be supplemented are poured in through a feeding port. The supplementary small materials comprise the following components in parts by weight: 8-12 parts of soybean cake powder, 15-20 parts of corn steep liquor, 4-5 parts of calcium carbonate, 2-3 parts of magnesium sulfate, 0.2-0.4 part of leucine, 0.3-0.5 part of simethicone, 0.3-0.5 part of soybean oil and 200 parts of water.
The method for replenishing the ammonium sulfate solution or the ammonia water comprises the following steps: when the pH value of the fermentation liquor exceeds 7.5, supplementing an ammonium sulfate solution to reduce the pH value to 6.5-7.0; when the pH value of the fermentation liquor is lower than 5.5, adding ammonia water to raise the pH value to 6.0-6.5. The pH value of the ammonium sulfate solution after sterilization is 3.98-4.05, and the concentration of ammonia water is 20%.
During the fermentation process, samples are taken every 24 hours for reducing sugar determination, amino nitrogen determination and biological value determination. The reducing sugar is measured by a Fehling reagent method, and the amino nitrogen is measured by a formaldehyde titration method.
The fermentation titer is measured by adopting a tube-disc method: the method comprises the steps of comparing the sizes of inhibition zones generated by a standard product (lincomycin with a calibrated content detected by an HPLC method) and a test product (fermentation liquor) on inoculated test bacteria (micrococcus luteus) by utilizing the diffusion effect of antibiotics in an agar culture medium and adopting the design of a quantitative reaction parallel line principle, and determining the biological potency of the test product.
When the pH value of the fermentation liquor is more than or equal to 6.8, the reducing sugar is less than 0.6%, the amino nitrogen is less than 0.5mg/mL, the bacterial concentration is less than or equal to 55%, the biological value is more than or equal to 10000U/mL, and the fermentation time is within 203 hours, the fermentation is finished, and the fermentation tank is placed.
After the fermentation culture is finished and the tank is placed, the method also comprises the following steps: and (3) separating and purifying lincomycin from the fermentation liquor. Isolation and purification of lincomycin can be carried out by techniques familiar to those skilled in the art, such as three-step purification by ammonium sulfate precipitation, DEAE-Sepharose ion exchange, and Ultrogel AcA34 gel filtration; or purifying by Ni-IDA affinity chromatography.
The invention is further illustrated by the following specific examples. The materials, reagents and equipment related to the invention are all commercially available; wherein,
streptomyces lincolnensis (Streptomyces lincolnensis) with the number of CGMCC4.5969, purchased from China general microbiological culture Collection center;
micrococcus luteus (Micrococcus luteus) with the number of CGMCC1.10554 purchased from China general microbiological culture Collection center;
lincomycin standards were purchased from Sigma;
corn steep liquor was purchased from north China pharmaceutical Kangxin Co., Ltd, lot number 20060701;
soybean cake flour was purchased from beijing soja scott technologies ltd;
molasses (CAS: 68476-78-8) was purchased from Nanjing Banuno Biotech Ltd;
biotin (CAS: 58-85-5) was purchased from Beijing Xinsaiwei chemical science and technology, Inc.;
bubble enemy (CAS: 110-11-1) was purchased from Shanghai Song chemical science and technology, Inc.;
FUS-50L multi-parameter full-automatic control fermentation tank is purchased from Shanghai national intensive biochemical engineering equipment, Inc.
Example 1
A process for producing lincomycin by feed fermentation comprises the following steps: seed culture, shake flask culture, secondary tank culture and fermentation tank fermentation culture.
Seed medium (g/L): 25 parts of soybean cake powder, 8 parts of soluble starch, 20 parts of glucose, 20 parts of corn steep liquor, 0.8 part of ammonium sulfate, 0.6 part of magnesium sulfate, 0.4 part of monopotassium phosphate, and sterilizing for 30min at 121 ℃.
Shake flask medium (g/L): 30 parts of soybean cake powder, 10 parts of soluble starch, 20 parts of glucose, 15 parts of molasses, 25 parts of corn steep liquor, 1.0 part of ammonium nitrate, 0.5 part of calcium carbonate, 0.5 part of magnesium sulfate and 0.3 part of dipotassium hydrogen phosphate, and sterilizing the mixture for 30min at 121 ℃.
Secondary tank medium (g/L): 35 parts of soybean cake powder, 15 parts of soluble starch, 40 parts of glucose, 30 parts of corn steep liquor, 0.5 part of biotin, 0.5 part of ammonium nitrate, 0.5 part of ammonium sulfate, 1.5 parts of calcium carbonate, 0.5 part of monopotassium phosphate, 1.5 parts of sodium chloride and 3.0mL/L of natural killer, and sterilizing at 121 ℃ for 30 min.
Fermentation medium of fermentation tank (parts by weight): 10 parts of soybean cake powder, 15 parts of glucose, 15 parts of corn starch, 1.0 part of magnesium sulfate, 0.3 part of leucine, 0.3 part of tyrosine, 0.5 part of monopotassium phosphate, 5 parts of calcium carbonate, 1.5 parts of ammonium nitrate, 2.0 parts of sodium chloride, 0.8 part of simethicone and 100 parts of water; sterilizing at 121 deg.C for 30 min.
The seed culture, the shake culture and the second-stage tank culture are all carried out at 30 ℃, and the culture periods are respectively 2 days, 5 days and 2 days; the content of the 750mL shake flask is 150mL, and the rotating speed is 200 r/min.
Inoculating the seeds cultured in the second-stage tank into a 50L fermentation tank by adopting a differential pressure method, wherein the inoculation amount is 15%. The fermentation conditions of the fermentation tank are as follows: the culture temperature is controlled to be 30 +/-1 ℃, a compressed air inlet after filtration and sterilization is opened in the whole process, the tank pressure is maintained at 0.02MPa, the stirring is started in the whole process, the rotating speed is 120 r/min for 0-90 hours, and the rotating speed is 80 r/min from 90 hours to the end of fermentation; the culture period after inoculation of the fermenter was 180 hours.
Samples were taken every 24 hours during the fermentation to determine reducing sugars, amino nitrogen and biological potency. Feeding materials according to needs, including feeding small materials; supplementing ammonium sulfate solution or ammonia water; and sugar supplement.
The method for replenishing the small materials comprises the following steps: the feed is supplemented once every 40 hours of fermentation culture, then every 24 hours, and the last supplement is supplemented 6 times in total at the 160 th hour of fermentation culture, and the volume of the small feed supplemented every time is 1.5L. Specifically, under the protection of flame, small materials to be supplemented are poured in through a feeding port. The supplemented small ingredients comprise 8 parts of soybean cake powder, 15 parts of corn steep liquor, 4 parts of calcium carbonate, 2 parts of magnesium sulfate, 0.2 part of leucine, 0.3 part of simethicone, 0.3 part of soybean oil and 200 parts of water.
The method for replenishing the ammonium sulfate solution or the ammonia water comprises the following steps: when the pH value of the fermentation liquor exceeds 7.5, supplementing an ammonium sulfate solution to reduce the pH value to 6.5; when the pH of the fermentation liquor is lower than 5.5, ammonia water is added, and the pH is increased to 6.0. The pH value of the supplemented ammonium sulfate solution after sterilization is 3.98, and the concentration of ammonia water is 20%; the pH value is controlled to be 6.0-7.0 in the whole fermentation process.
When the content of reducing sugar in the fermentation liquor is lower than 0.3%, a glucose solution with the mass concentration of 15% is supplemented, so that the content of reducing sugar in the fermentation liquor is 0.4%.
The measurement of the biological potency was performed by the tube-disc method, and the measurement was performed every 24 hours. And when the fermentation is carried out for 180 hours, the pH value of the fermentation liquid is 6.8, the reducing sugar content is 0.5%, the amino nitrogen content is 0.3mg/mL, the bacteria concentration is 55%, and the biological value is 10600U/mL, and the fermentation is finished and put into a tank. And (3) separating and purifying by adopting a Ni-IDA affinity chromatography one-step method to obtain lincomycin, and determining the lincomycin B content by adopting an HPLC method.
Example 2
A process for producing lincomycin by feed fermentation comprises the following steps: seed culture, shake flask culture, secondary tank culture and fermentation tank fermentation culture.
Seed medium (g/L): 25 parts of soybean cake powder, 8 parts of soluble starch, 20 parts of glucose, 20 parts of corn steep liquor, 0.8 part of ammonium sulfate, 0.6 part of magnesium sulfate, 0.4 part of monopotassium phosphate, and sterilizing for 30min at 121 ℃.
Shake flask medium (g/L): 35 parts of soybean cake powder, 12 parts of soluble starch, 25 parts of glucose, 20 parts of molasses, 30 parts of corn steep liquor, 1.2 parts of ammonium nitrate, 0.7 part of calcium carbonate, 0.6 part of magnesium sulfate and 0.5 part of dipotassium hydrogen phosphate, and sterilizing the mixture for 30min at 121 ℃.
Secondary tank medium (g/L): 35 parts of soybean cake powder, 15 parts of soluble starch, 40 parts of glucose, 30 parts of corn steep liquor, 0.7 part of biotin, 0.5 part of ammonium nitrate, 0.5 part of ammonium sulfate, 1.5 parts of calcium carbonate, 0.5 part of monopotassium phosphate, 1.5 parts of sodium chloride and 3.0mL/L of natural killer, and sterilizing at 121 ℃ for 30 min.
Fermentation medium of fermentation tank (parts by weight): 11 parts of soybean cake powder, 16 parts of glucose, 18 parts of corn starch, 1.5 parts of magnesium sulfate, 0.4 part of leucine, 0.4 part of tyrosine, 0.6 part of monopotassium phosphate, 5.5 parts of calcium carbonate, 2.0 parts of ammonium nitrate, 3.2 parts of sodium chloride, 1.2 parts of simethicone and 110 parts of water; sterilizing at 121 deg.C for 30 min.
The seed culture, the shake culture and the second-stage tank culture are all carried out at 30 ℃, and the culture periods are 3 days, 7 days and 4 days respectively; the content of the 750mL shake flask is 150mL, and the rotating speed is 200 r/min.
Inoculating the seeds cultured in the second-stage tank into a 50L fermentation tank by adopting a differential pressure method, wherein the inoculation amount is 18%. The fermentation conditions of the fermentation tank are as follows: the culture temperature is controlled at 30 +/-1 ℃, a compressed air inlet after filtration and sterilization is opened in the whole process, the tank pressure is maintained at 0.03MPa, the stirring is started in the whole process, the rotating speed is 135 revolutions per minute for 0 to 90 hours, and the rotating speed is 85 revolutions per minute from 90 hours to the end of fermentation; the cultivation period after inoculation of the fermenter was 200 hours.
Samples were taken every 24 hours during the fermentation to determine reducing sugars, amino nitrogen and biological potency. Feeding materials according to needs, including feeding small materials; supplementing ammonium sulfate solution or ammonia water; and sugar supplement.
The method for replenishing the small materials comprises the following steps: the feed is supplemented once every 40 hours of fermentation culture, then every 24 hours, and the last supplement is supplemented 6 times in total at the 160 th hour of fermentation culture, and the volume of the small feed supplemented every time is 1.6L. Specifically, under the protection of flame, small materials to be supplemented are poured in through a feeding port. The supplementary small ingredients comprise 10 parts of soybean cake powder, 16 parts of corn steep liquor, 4.5 parts of calcium carbonate, 2.5 parts of magnesium sulfate, 0.3 part of leucine, 0.4 part of simethicone, 0.4 part of soybean oil and 200 parts of water.
The method for replenishing the ammonium sulfate solution or the ammonia water comprises the following steps: when the pH value of the fermentation liquor exceeds 7.5, supplementing an ammonium sulfate solution to reduce the pH value to 6.8; when the pH of the fermentation liquor is lower than 5.5, ammonia water is added, and the pH is increased to 6.2. The pH value of the supplemented ammonium sulfate solution after sterilization is 4.01, and the concentration of ammonia water is 20%; the pH value is controlled to be 6.0-7.0 in the whole fermentation process.
When the content of reducing sugar in the fermentation liquor is lower than 0.3%, a glucose solution with the mass concentration of 20% is supplemented, so that the content of reducing sugar in the fermentation liquor is 0.6%.
The measurement of the biological potency was performed by the tube-disc method, and the measurement was performed every 24 hours. When the fermentation is carried out for 200 hours, the pH value of the fermentation liquor is 7.0, the reducing sugar content is 0.4%, the amino nitrogen content is 0.2mg/mL, the bacteria concentration is 50%, and the biological potency is 11000U/mL, and the fermentation is finished and put into a tank. And (3) separating and purifying by adopting a Ni-IDA affinity chromatography one-step method to obtain lincomycin, and determining the lincomycin B content by adopting an HPLC method.
Example 3
A process for producing lincomycin by feed fermentation comprises the following steps: seed culture, shake flask culture, secondary tank culture and fermentation tank fermentation culture.
Seed medium (g/L): 25 parts of soybean cake powder, 8 parts of soluble starch, 20 parts of glucose, 20 parts of corn steep liquor, 0.8 part of ammonium sulfate, 0.6 part of magnesium sulfate, 0.4 part of monopotassium phosphate, and sterilizing for 30min at 121 ℃.
Shake flask medium (g/L): 40 parts of soybean cake powder, 15 parts of soluble starch, 30 parts of glucose, 25 parts of molasses, 35 parts of corn steep liquor, 1.5 parts of ammonium nitrate, 0.8 part of calcium carbonate, 0.8 part of magnesium sulfate and 0.6 part of dipotassium hydrogen phosphate, and sterilizing the soybean cake powder for 30min at 121 ℃.
Secondary tank medium (g/L): 35 parts of soybean cake powder, 15 parts of soluble starch, 40 parts of glucose, 30 parts of corn steep liquor, 0.8 part of biotin, 0.5 part of ammonium nitrate, 0.5 part of ammonium sulfate, 1.5 parts of calcium carbonate, 0.5 part of monopotassium phosphate, 1.5 parts of sodium chloride and 3.0mL/L of natural killer, and sterilizing at 121 ℃ for 30 min.
Fermentation medium of fermentation tank (parts by weight): 12 parts of soybean cake powder, 18 parts of glucose, 20 parts of corn starch, 2.5 parts of magnesium sulfate, 0.5 part of leucine, 0.5 part of tyrosine, 0.8 part of monopotassium phosphate, 6 parts of calcium carbonate, 2.5 parts of ammonium nitrate, 5.0 parts of sodium chloride, 1.5 parts of simethicone and 120 parts of water; sterilizing at 121 deg.C for 30 min.
The seed culture, the shake culture and the second-stage tank culture are all carried out at 30 ℃, and the culture periods are 3 days, 7 days and 4 days respectively; the content of the 750mL shake flask is 150mL, and the rotating speed is 200 r/min.
Inoculating the seeds cultured in the second-stage tank into a 50L fermentation tank by adopting a differential pressure method, wherein the inoculation amount is 20%. The fermentation conditions of the fermentation tank are as follows: the culture temperature is controlled to be 30 +/-1 ℃, a compressed air inlet after filtration and sterilization is opened in the whole process, the tank pressure is maintained at 0.04MPa, the stirring is started in the whole process, the rotating speed is 150 revolutions per minute after 0-90 hours, and the rotating speed is 90 revolutions per minute after 90 hours and fermentation is finished; the incubation period after inoculation of the fermenter was 203 hours.
Samples were taken every 24 hours during the fermentation to determine reducing sugars, amino nitrogen and biological potency. Feeding materials according to needs, including feeding small materials; supplementing ammonium sulfate solution or ammonia water; and sugar supplement.
The method for replenishing the small materials comprises the following steps: the feed is supplemented once every 40 hours of fermentation culture, then every 24 hours, and the last supplement is supplemented for 6 times in total at the 160 th hour of fermentation culture, and the volume of the small feed supplemented every time is 2.0L. Specifically, under the protection of flame, small materials to be supplemented are poured in through a feeding port. The supplementary small ingredients comprise 12 parts of soybean cake powder, 20 parts of corn steep liquor, 5 parts of calcium carbonate, 3 parts of magnesium sulfate, 0.4 part of leucine, 0.5 part of simethicone, 0.5 part of soybean oil and 200 parts of water.
The method for replenishing the ammonium sulfate solution or the ammonia water comprises the following steps: when the pH value of the fermentation liquor exceeds 7.5, supplementing an ammonium sulfate solution to reduce the pH value to 7.0; when the pH of the fermentation liquor is lower than 5.5, ammonia water is added, and the pH is increased to 6.5. The pH value of the supplemented ammonium sulfate solution after sterilization is 4.05, and the concentration of ammonia water is 20%; the pH value is controlled to be 6.0-7.0 in the whole fermentation process.
When the content of reducing sugar in the fermentation liquor is lower than 0.3%, a glucose solution with the mass concentration of 25% is supplemented, so that the content of reducing sugar in the fermentation liquor is 1.0%.
The measurement of the biological potency was performed by the tube-disc method, and the measurement was performed every 24 hours. When the fermentation is carried out for 203 hours, the pH value of the fermentation liquid is 6.8, the reducing sugar content is 0.5%, the amino nitrogen content is 0.4mg/mL, the bacterial concentration is 55%, and the biological potency is 10890U/mL, and the fermentation is finished and put into a tank. And (3) separating and purifying by adopting a Ni-IDA affinity chromatography one-step method to obtain lincomycin, and determining the lincomycin B content by adopting an HPLC method.
Example 4
A process for producing lincomycin by feed fermentation comprises the following steps: seed culture, shake flask culture, secondary tank culture and fermentation tank fermentation culture.
The seed culture medium, shake flask culture medium and secondary tank culture medium were the same as in example 2, and the conditions of the seed culture, shake flask culture and secondary tank culture were also the same as in example 2.
Fermentation medium of fermentation tank (parts by weight): 10 parts of soybean cake powder, 18 parts of glucose, 20 parts of corn starch, 1.5 parts of magnesium sulfate, 0.5 part of leucine, 0.3 part of tyrosine, 0.7 part of monopotassium phosphate, 5 parts of calcium carbonate, 2.5 parts of ammonium nitrate, 4.0 parts of sodium chloride, 1.5 parts of simethicone and 120 parts of water; sterilizing at 121 deg.C for 30 min.
Inoculating the seeds cultured in the second-stage tank into a 50L fermentation tank by adopting a differential pressure method, wherein the inoculation amount is 20%. The fermentation conditions of the fermentation tank are as follows: the culture temperature is controlled to be 30 +/-1 ℃, a compressed air inlet after filtration and sterilization is opened in the whole process, the tank pressure is maintained at 0.03MPa, the stirring is started in the whole process, the rotating speed is 150 rpm after 0-90 hours, and the rotating speed is 85 rpm after 90 hours and the fermentation is finished; the cultivation period after inoculation of the fermenter was 200 hours.
Samples were taken every 24 hours during the fermentation to determine reducing sugars, amino nitrogen and biological potency. Feeding materials according to needs, including feeding small materials; supplementing ammonium sulfate solution or ammonia water; and sugar supplement.
The method for replenishing the small materials comprises the following steps: the feed is supplemented once every 40 hours of fermentation culture, then every 24 hours, and the last supplement is supplemented 6 times in total at the 160 th hour of fermentation culture, and the volume of the small feed supplemented every time is 1.6L. Specifically, under the protection of flame, small materials to be supplemented are poured in through a feeding port. The supplementary small ingredients comprise 10 parts of soybean cake powder, 20 parts of corn steep liquor, 5 parts of calcium carbonate, 2.5 parts of magnesium sulfate, 0.4 part of leucine, 0.5 part of simethicone, 0.3 part of soybean oil and 200 parts of water.
The method for replenishing the ammonium sulfate solution or the ammonia water comprises the following steps: when the pH value of the fermentation liquor exceeds 7.5, supplementing an ammonium sulfate solution to reduce the pH value to 7.0; when the pH of the fermentation liquor is lower than 5.5, ammonia water is added, and the pH is increased to 6.0. The pH value of the supplemented ammonium sulfate solution after sterilization is 4.00, and the concentration of ammonia water is 20%; the pH value is controlled to be 6.0-7.0 in the whole fermentation process.
When the content of reducing sugar in the fermentation liquor is lower than 0.3%, adding sucrose solution with the mass concentration of 20% to ensure that the content of reducing sugar in the fermentation liquor is 0.5%.
The measurement of the biological potency was performed by the tube-disc method, and the measurement was performed every 24 hours. When the fermentation is carried out for 200 hours, the pH value of the fermentation liquid is 7.0, the reducing sugar content is 0.3%, the amino nitrogen content is 0.2mg/mL, the bacteria concentration is 51%, and the biological value is 10430U/mL, and the fermentation is finished and the tank is placed. And (3) separating and purifying by adopting a Ni-IDA affinity chromatography one-step method to obtain lincomycin, and determining the lincomycin B content by adopting an HPLC method.
Example 5
A process for producing lincomycin by feed fermentation comprises the following steps: seed culture, shake flask culture, secondary tank culture and fermentation tank fermentation culture.
The seed culture medium, shake flask culture medium and secondary tank culture medium were the same as in example 1, and the conditions of the seed culture, shake flask culture and secondary tank culture were also the same as in example 1.
Fermentation medium of fermentation tank (parts by weight): 12 parts of soybean cake powder, 15 parts of glucose, 15 parts of corn starch, 1.8 parts of magnesium sulfate, 0.5 part of leucine, 0.4 part of tyrosine, 0.8 part of monopotassium phosphate, 5 parts of calcium carbonate, 2.3 parts of ammonium nitrate, 4.0 parts of sodium chloride, 1.5 parts of simethicone and 100 parts of water; sterilizing at 121 deg.C for 30 min.
Inoculating the seeds cultured in the second-stage tank into a 50L fermentation tank by adopting a differential pressure method, wherein the inoculation amount is 20%. The fermentation conditions of the fermentation tank are as follows: the culture temperature is controlled to be 30 +/-1 ℃, a compressed air inlet after filtration and sterilization is opened in the whole process, the tank pressure is maintained at 0.04MPa, the stirring is started in the whole process, the rotating speed is 140 revolutions per minute for 0 to 90 hours, and the rotating speed from 90 hours to the end of fermentation is 80 revolutions per minute; the incubation period after inoculation of the fermenter was 190 hours.
Samples were taken every 24 hours during the fermentation to determine reducing sugars, amino nitrogen and biological potency. Feeding materials according to needs, including feeding small materials; supplementing ammonium sulfate solution or ammonia water; and sugar supplement.
The method for replenishing the small materials comprises the following steps: the feed is supplemented once every 40 hours of fermentation culture, then every 24 hours, and the last supplement is supplemented 6 times in total at the 160 th hour of fermentation culture, and the volume of the small feed supplemented every time is 1.8L. Specifically, under the protection of flame, small materials to be supplemented are poured in through a feeding port. The supplementary small ingredients comprise 10 parts of soybean cake powder, 15 parts of corn steep liquor, 5 parts of calcium carbonate, 2.2 parts of magnesium sulfate, 0.35 part of leucine, 0.42 part of simethicone, 0.5 part of soybean oil and 200 parts of water.
The method for replenishing the ammonium sulfate solution or the ammonia water comprises the following steps: when the pH value of the fermentation liquor exceeds 7.5, supplementing an ammonium sulfate solution to reduce the pH value to 6.7; when the pH of the fermentation liquor is lower than 5.5, ammonia water is added, and the pH is increased to 6.4. The pH value of the supplemented ammonium sulfate solution after sterilization is 3.99, and the concentration of ammonia water is 20%; the pH value is controlled to be 6.0-7.0 in the whole fermentation process.
When the content of reducing sugar in the fermentation liquor is lower than 0.3%, a fructose solution with the mass concentration of 22% is supplemented, so that the content of reducing sugar in the fermentation liquor is 0.9%.
The measurement of the biological potency was performed by the tube-disc method, and the measurement was performed every 24 hours. And when the fermentation is carried out for 190 hours, the pH value of the fermentation liquor is 7.1, the reducing sugar content is 0.3%, the amino nitrogen content is 0.3mg/mL, the bacteria concentration is 50%, and the biological value is 10710U/mL, and the fermentation is finished and put into a tank. And (3) separating and purifying by adopting a Ni-IDA affinity chromatography one-step method to obtain lincomycin, and determining the lincomycin B content by adopting an HPLC method.
Comparative example 1
This comparative example differs from example 1 only in that the addition of small materials was not carried out during the fermentation culture, and the remainder was the same as
Example 1.
Comparative example 2
The comparative example is different from example 2 only in that ammonium sulfate solution or ammonia water is not additionally added in the fermentation culture process to regulate the pH of the fermentation liquor, and the rest is the same as example 2.
Comparative example 3
This comparative example differs from example 2 only in that no sugar supplementation is performed during the fermentation culture, and the rest is the same as example 2.
Comparative example 4
This comparative example is different from example 2 only in that no feeding operation was performed during the fermentation culture, and each stage of culture was performed using the same medium and culture conditions as in example 2.
Comparative example 5
The comparative example differs from example 2 in that the additional ingredients consisted of 15 parts by weight of soybean meal, 6 parts by weight of calcium carbonate, 2 parts by weight of magnesium sulfate, 0.8 part by weight of soybean oil and 100 parts by weight of water. The rest is the same as example 2.
Comparative example 6
The difference between the comparative example and the example 2 is that the method for supplementing the small materials is different, and the method for supplementing the small materials in the comparative example is as follows: the feed is supplemented once in 40 hours of fermentation culture, and then is supplemented once in 52 hours, 70 hours, 100 hours and 150 hours respectively, and the volume of the feed supplemented each time is 3L. The rest is the same as example 2.
Comparative example 7
This comparative example differs from example 2 in that the reducing sugar content in the fermentation broth was not monitored during the fermentation, but glucose solutions with a concentration of 20% were added once at 24 hours, 60 hours, 90 hours and 150 hours of the fermentation, respectively, at a volume of 1L. The rest is the same as example 2.
Test example
And respectively taking fermentation liquor when the fermentation is carried out for 120 hours and the fermentation is finished and putting the fermentation tank, and measuring the biological value of lincomycin by adopting a pipe-disc method, wherein the results are shown in table 1.
As can be seen from the data in Table 1, examples 1-5 produced lincomycin by fed-batch fermentation, the lincomycin titer at the time of tank discharge was over 10000U/mL, and the titer at 120 hours of fermentation was already higher than the tank discharge titer of comparative examples 1-7; the tapping titer of example 2 was highest. Wherein, the comparative example 1 is that small materials are not supplemented in the fermentation process, the comparative example 2 is that a reagent is not supplemented in the fermentation process to control the pH value, the comparative example 3 is that sugar is not supplemented in the fermentation process, the comparative example 4 is that a common fermentation method is adopted and no material is supplemented, the composition of the small materials supplemented in the comparative example 5 is out of the protection range of the invention, the mode of supplementing the small materials in the comparative example 6 is not proper, and the comparative example 7 adopts timed sugar supplement. Therefore, the addition of small materials, the addition of ammonium sulfate solution or ammonia water and the addition of sugar can influence the yield of the lincomycin produced by fermentation, and the composition, the addition mode and the addition mode of the small materials and the addition mode of the sugar solution also influence the yield of the lincomycin.
| Group of | 120 hour titer U/mL | Put jar valence U/mL |
| Example 1 | 7206 | 10600 |
| Example 2 | 7310 | 11000 |
| Example 3 | 7438 | 10890 |
| Example 4 | 6997 | 10430 |
| Example 5 | 7069 | 10710 |
| Comparative example 1 | 3604 | 5532 |
| Comparative example 2 | 4086 | 6538 |
| Comparative example 3 | 3902 | 5998 |
| Comparative example 4 | 2884 | 4795 |
| Comparative example 5 | 5032 | 6984 |
| Comparative example 6 | 4893 | 6487 |
| Comparative example 7 | 5167 | 6841 |
Table 1.
Each example and comparative example was subjected to lincomycin purification under the same process conditions after canning, and then the lincomycin B content in the purified lincomycin was measured by HPLC, and the results are shown in table 2.
The content determination conditions of the lincomycin B are as follows:
measuring with Shimadzu HPT-20A liquid chromatograph, variable wavelength ultraviolet detector, C18 chromatographic column with detection wavelength of 214nm at room temperature.
The mobile phase was methanol-acetonitrile-0.05M phosphate buffer (16: 16:68 by volume) at a flow rate of 1.0 mL/min.
| Group of | Lincomycin B content (%) |
| Example 1 | 0.45 |
| Example 2 | 0.40 |
| Example 3 | 0.43 |
| Example 4 | 0.48 |
| Example 5 | 0.45 |
| Comparative example 1 | 5.42 |
| Comparative example 2 | 3.81 |
| Comparative example 3 | 4.16 |
| Comparative example 4 | 7.69 |
| Comparative example 5 | 2.33 |
| Comparative example 6 | 1.98 |
| Comparative example 7 | 2.67 |
Table 2.
As is apparent from the data in Table 2, the lincomycin B content in the lincomycin prepared in examples 1-5 is less than 0.5%, and the product purity is higher than that of the lincomycin prepared in each comparative example.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (4)
1. A process for producing lincomycin by feed fermentation comprises the following steps: seed culture, shake flask culture, second grade jar is cultivateed and fermentation cylinder fermentation culture, its characterized in that carries out the feed supplement in fermentation cylinder fermentation culture process, specifically includes:
(1) adding small materials;
(2) regulating and controlling the pH value of the fermentation liquor by supplementing an ammonium sulfate solution or ammonia water;
(3) when the content of reducing sugar in the fermentation liquor is lower than 0.3%, sugar is supplemented;
wherein the small materials comprise the following components in parts by weight: 8-12 parts of soybean cake powder, 15-20 parts of corn steep liquor, 4-5 parts of calcium carbonate, 2-3 parts of magnesium sulfate, 0.2-0.4 part of leucine, 0.3-0.5 part of simethicone, 0.3-0.5 part of soybean oil and 200 parts of water;
the method for replenishing the ammonium sulfate solution or the ammonia water comprises the following steps: when the pH value of the fermentation liquor exceeds 7.5, supplementing an ammonium sulfate solution to reduce the pH value to 6.5-7.0; when the pH value of the fermentation liquor is lower than 5.5, adding ammonia water to raise the pH value to 6.0-6.5; controlling the pH value of the fermentation liquor to be 6.0-7.0;
the method for supplementing the small materials comprises the following steps: supplementing once every 40 hours of fermentation culture, then supplementing once every 24 hours, wherein the last supplementing is supplementing 6 times in total in the 160 th hour of fermentation culture, and the volume of each supplemented small material is 1.5-2.0L;
the conditions of fermentation culture are as follows: the culture temperature is controlled at 30 +/-1 ℃, the tank pressure is maintained at 0.02-0.04MPa, the stirring is started in the whole process, and the culture period is 180-203 hours after the fermentation tank is inoculated;
the culture medium for fermentation culture comprises the following components in parts by weight: 10-12 parts of soybean cake powder, 15-18 parts of glucose, 15-20 parts of corn starch, 1.0-2.5 parts of magnesium sulfate, 0.3-0.5 part of leucine, 0.3-0.5 part of tyrosine, 0.5-0.8 part of potassium dihydrogen phosphate, 5-6 parts of calcium carbonate, 1.5-2.5 parts of ammonium nitrate, 2.0-5.0 parts of sodium chloride, 0.8-1.5 parts of simethicone and 120 parts of 100 parts of water;
after sugar supplement, the reducing sugar content in the fermentation liquor is 0.4-1.0%.
2. The process according to claim 1, characterized in that the rotation speed of the stirring is in particular: the rotation speed from 0 to 90 hours is 120-150 rpm, and the rotation speed from 90 hours to the end of fermentation is 80-90 rpm.
3. The process as claimed in claim 1, wherein the ammonium sulfate solution has a pH of 3.98-4.05 after sterilization, and the concentration of the ammonia water is 20%.
4. The process according to any one of claims 1 to 3, wherein the sugar supplement is a glucose solution, a sucrose solution or a fructose solution with a supplemental concentration of 15 to 25% by mass.
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