CN114807275A - Fermentation method for increasing pristinamycin yield - Google Patents
Fermentation method for increasing pristinamycin yield Download PDFInfo
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- CN114807275A CN114807275A CN202210584384.0A CN202210584384A CN114807275A CN 114807275 A CN114807275 A CN 114807275A CN 202210584384 A CN202210584384 A CN 202210584384A CN 114807275 A CN114807275 A CN 114807275A
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- Prior art keywords
- fermentation
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- pristinamycin
- culture
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Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 93
- 230000004151 fermentation Effects 0.000 title claims abstract description 93
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- JOOMGSFOCRDAHL-XKCHLWDXSA-N pristinamycin-IIB Natural products CC(C)[C@@H]1OC(=O)[C@H]2CCCN2C(=O)c3coc(CC(=O)C[C@@H](O)C=C(C)C=CCNC(=O)C=C[C@H]1C)n3 JOOMGSFOCRDAHL-XKCHLWDXSA-N 0.000 title claims abstract description 53
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Classifications
-
- C—CHEMISTRY; METALLURGY
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Abstract
The invention provides a fermentation method for improving pristinamycin yield, which comprises the following steps: in the fermentation culture process, n-propanol is supplemented into the fermentation culture medium. The method realizes the great improvement of the pristinamycin yield, reduces the production cost and is beneficial to industrial production.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a fermentation method for improving pristinamycin yield.
Background
Pristinamycin (privtinamycins) belonging to the class of streptogramins, isolated from Streptomyces pristinaespiralis (Streptomyces pristinaespiralis) in 1955, consisted of about 30% pristinamycin I and 70% pristinamycin II. Wherein pristinamycin I is a cyclic hexapeptide lactone consisting of pristinamycin IA, pristinamycin IB and pristinamycin IC, and pristinamycin II is a polyunsaturated lactone consisting of pristinamycin IIA and pristinamycin IIB. Pristinamycin IA and pristinamycin IIA are the main components of pristinamycin.
Pristinamycin has strong bactericidal activity on gram-positive bacteria and is mainly used for treating infection caused by gram-positive bacteria including drug-resistant bacteria. However, the natural pristinamycin has poor water solubility and is not easily prepared into parenteral preparations, so that clinical application is limited. The problem can be solved by chemically modifying natural pristinamycin IA and IIA to obtain a water-soluble derivative RP59500 (trade name Synercid), which is the first commercially available injection of the streptogramins.
The pristinamycin has the following specific structural formula:
although pristinamycin has a wide range of applications and finished preparations thereof are on the market, the lower fermentation yield and higher production cost of pristinamycin are still major factors limiting the applications and development thereof.
Disclosure of Invention
The invention provides a fermentation method for improving pristinamycin yield, aiming at solving the problems of low pristinamycin yield and high production cost in the prior art, and the method comprises the following steps:
in the fermentation culture process, n-propanol is supplemented into the fermentation culture medium.
Preferably, the time for said supplementation is 10-60h, preferably 10-50h, more preferably 20-50h, most preferably 30-40h after the start of the fermentation culture.
Preferably, the n-propanol is continuously supplemented at a rate of 0.04-0.1%/day, preferably 0.06-0.08%/day, based on the initial volume of the fermentation medium.
Preferably, the fermentation medium comprises: 2-10g/100mL carbon source, 2-5g/100mL nitrogen source, 0.2-1.0g/100mL inorganic salt.
Preferably, the carbon source of the fermentation medium is selected from one of glucose, sucrose, lactose, maltose, maltodextrin, soluble starch, sorbitol, glycerol or a combination of the above, preferably a combination of soluble starch, glucose and sucrose.
Preferably, the nitrogen source of the fermentation medium is selected from one of soybean cake powder, yeast powder, corn gluten meal, cottonseed cake powder, peanut cake powder, soybean meal, corn steep liquor dry powder, yeast extract and peptone or a combination of the above substances, and preferably the combination of the soybean cake powder and the yeast powder.
Preferably, the fermentation medium comprises: 3.0 plus or minus 0.6 of soluble starch, 2.0 plus or minus 0.4 of glucose, 0.5 plus or minus 0.1 of cane sugar, 2.0 plus or minus 0.4 of soybean cake powder, 0.5 plus or minus 0.1 of yeast powder, 0.2 plus or minus 0.04 of ammonium sulfate, 0.1 plus or minus 0.02 of manganese sulfate, 0.01 plus or minus 0.002 of ferrous sulfate, 0.05 plus or minus 0.01 of potassium dihydrogen phosphate, 0.3 plus or minus 0.06 of calcium carbonate, the unit is g/100mL, and the pH value is 7.0 plus or minus 0.2.
Preferably, the time for fermentation culture is 80-160h, preferably 100-160 h.
Preferably, the conditions of the fermentation culture are as follows: the temperature is 27 plus or minus 1.0 ℃, the ventilation volume is 0.5 to 1.0VVM, the tank pressure is 0.045 to 0.065MPa, and the dissolved oxygen is 30 to 100 percent.
The invention controls the glucose content at 0.2-1.0% (the content of glucose in the real-time fermentation medium, unit is g/mL) by feeding glucose solution. The glucose solution has a concentration of 50% (g/mL), is used after being sterilized at 121.0-123.0 deg.C for 30min, and is fed when the glucose content in the culture medium is less than 1.0% during fermentation.
The strain used in the fermentation culture of the invention is any Streptomyces pristinaespiralis capable of producing pristinamycin, such as Streptomyces pristinaespiralis, ATCC 25486 (the preservation number of which in the China general microbiological culture Collection center is CGMCC 4.1352) which can be purchased from China general microbiological culture Collection center.
From the biosynthesis point of view, the starting units for pristinamycin synthesis are amino acids, including phenylalanine, threonine, methionine, lysine, proline, valine, and the like. However, the inventors have found that the addition of amino acids does not significantly increase the production of pristinamycin and therefore presumably the main limiting factor hindering the synthesis of pristinamycin is not the starting amino acid but rather the extension unit of its carbon chain. The formation of the lactone ring of pristinamycin takes acetic acid as a starting unit, the inventor speculates that the extension unit of the carbon chain of pristinamycin may be short-chain fatty acid/short-chain fatty acid sodium, and according to the principle, the addition of sodium formate, sodium acetate, sodium propionate, sodium succinate and the like is respectively tried, but the improvement of the yield of pristinamycin is not obviously influenced.
Normally, n-propanol is toxic to the growth and metabolism of microorganisms, but the inventors have surprisingly found that the addition of n-propanol (especially under certain conditions) can greatly increase the production of pristinamycin. But the addition of ethanol, similar to n-propanol, reduced the production of pristinamycin.
Compared with the prior art, the invention has the following beneficial effects:
according to the method, the n-propanol is supplemented into the fermentation culture medium in the fermentation culture process, so that the yield of pristinamycin can be greatly improved, the fermentation culture medium is simple in component and low in cost, is simple and easy to implement in process control, reduces the production cost of pristinamycin, and is beneficial to industrial production.
Detailed Description
The following examples further illustrate the invention. However, the invention may be embodied in many different forms, such as different media or similar strains, all of which may achieve similar results, and it should not be construed as limited to the embodiments set forth herein, which are provided for the purpose of making the disclosure more complete. In the following examples, all temperatures are in degrees celsius unless otherwise indicated; unless otherwise indicated, various starting materials and reagents were obtained commercially and were used without further purification; unless otherwise indicated, each solvent is a technical grade solvent and is used without further treatment.
The pristinamycin content measured in the embodiment refers to the sum of the contents of two main components of pristinamycin IA and pristinamycin IIA, and the detection conditions are as follows:
a chromatographic column: agilent Zorbax C 18 Column (4.6mm x250mm x 5 μm);
mobile phase A: phosphate buffer (0.03mol/L potassium dihydrogen phosphate solution, pH adjusted to 2.3 with phosphoric acid);
mobile phase B: acetonitrile;
flow rate: 1.0 mL/min;
linear gradient: in 0-22min, phase B is 35-35%, in 22-40 min: the phase B is 35 to 80 percent and is mixed in 40 to 50 min: phase B is 80 to 35 percent, and the phase B is mixed in 50 to 60 min: the phase B is 35 to 35 percent;
column temperature: 30 ℃;
detection wavelength: 210 nm;
sample introduction amount: 10 μ L.
Comparative example: screening of supplementary components in fermentation culture process
(1) Selecting a starting strain: the strain used in the experiment was Streptomyces pristinaespiralis (ATCC 25486).
(2) Slant culture of the strain: a slant strain was obtained by thawing Streptomyces pristinaespiralis (ATCC 25486) in a glycerol tube, inoculating the thawed tube to a slant medium, and culturing at 27. + -. 1.0 ℃ for 165 hours.
The slant culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.1 part of dipotassium hydrogen phosphate, 0.1 part of magnesium sulfate and 2.0 parts of agar, wherein the unit is g/100mL, and the pH value is 7.0.
(3) Strain shake flask culture: digging 2-5cm under aseptic condition 2 And (3) scattering the slant strains prepared in the step (2) by using a homogenizer, inoculating the scattered slant strains into a shake flask culture medium, and culturing the shake flask at the temperature of 27 +/-1.0 ℃ for 28 hours to obtain mature shake flask seed liquid.
The shake flask culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium hydrogen phosphate, 0.2 part of sodium chloride and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
(4) Seed tank culture: 15L of seeds are canned into 10L of seed culture medium, sterilized at 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃. Inoculating the shake flask seed solution obtained in the step (3) into a seed tank culture medium in an inoculation amount of 0.05 percent of volume ratio, controlling the culture temperature to be 27 +/-1.0 ℃, controlling the ventilation rate to be 0.5-1.0VVM, controlling the tank pressure to be 0.045-0.065MPa, controlling the initial stirring rotation speed to be 150RPM, controlling the stirring rotation speed to be 150RPM and 550RPM in the culture process, controlling the dissolved oxygen to be more than 30 percent, and culturing for 35 hours to obtain the seed tank seed solution.
The components and the dosage of the seeding tank culture medium are as follows: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium phosphate, 0.2 part of sodium chloride, 0.3 part of calcium carbonate and 0.05 part of defoaming agent, wherein the unit is g/100mL, and the pH value is 7.0.
(5) Fermentation culture: 50L of fermentation medium is canned for 30L, sterilized at the temperature of 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃, the seed liquid of the seed tank obtained in the step (4) is inoculated into the fermentation medium by the inoculation amount of 5 percent of the volume ratio, the fermentation temperature is controlled to be 27 +/-1.0 ℃, the ventilation amount is controlled to be 0.5-1.0VVM, the tank pressure is 0.045-0.065MPa, the initial stirring rotation speed is 150RPM, the stirring rotation speed is controlled to be 150RPM 550RPM in the culture process, the dissolved oxygen is controlled to be 30-100 percent, and the glucose content is controlled to be 0.2-1.0 percent. After fermentation culture for 30h, water, different short-chain fatty acid sodium salts, ethanol, n-propanol and different amino acids are continuously supplemented into the culture medium respectively, wherein the supplementing rate is 0.06 percent/day of the initial volume of the fermentation culture medium. The experiment supplemented with an equal amount of water was a control experiment. After fermentation culture for 100h, the pristinamycin content in the culture medium is detected, and the experimental results are shown in tables 1 and 2:
table 1: effect of adding different short-chain fatty acid sodium, ethanol and n-propanol on pristinamycin yield
| Supplementary substance | Sodium formate | Sodium acetate | Sodium propionate | Succinic acid sodium salt | Ethanol | N-propanol |
| Pristinamycin yield (mg/L) | 1194.15 | 1246.32 | 1295.89 | 1209.65 | 1045.90 | 1869.97 |
Table 2: effect of different amino acid additions on pristinamycin yield
| Name of amino acid | Phenylalanine (PHE) | Threonine | Methionine | Lysine | Proline | Valine |
| Pristinamycin yield (mg/L) | 1185.82 | 1194.67 | 1209.41 | 1272.18 | 1298.74 | 1186.38 |
In the control experiment supplemented with an equal amount of water, the pristinamycin yield was 1217.32mg/L after 100h of fermentation culture.
The fermentation medium comprises the following components in percentage by weight: 3.0 parts of soluble starch, 2.0 parts of glucose, 0.5 part of cane sugar, 2.0 parts of soybean cake powder, 0.5 part of yeast powder, 0.2 part of ammonium sulfate, 0.1 part of manganese sulfate, 0.01 part of ferrous sulfate, 0.05 part of monopotassium phosphate and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
The experimental results prove that under the same experimental conditions, different kinds of amino acids such as phenylalanine and the like are supplemented in the fermentation culture process, so that the yield of pristinamycin is hardly increased, and the yield of pristinamycin cannot be increased by short-chain fatty acid sodium such as sodium formate and the like and ethanol. However, n-propanol supplementation greatly increased pristinamycin production.
Example 1: after 30 hours of fermentation culture, the normal propyl alcohol is supplemented at the rate of 0.06 percent per day of volume ratio
(1) Selecting a starting strain: the strain used in the experiment was Streptomyces pristinaespiralis (Streptomyces pristinaespiralis, ATCC 25486).
(2) Slant culture of the strain: a slant strain was obtained by thawing Streptomyces pristinaespiralis (ATCC 25486) in a glycerol tube, inoculating the thawed tube to a slant medium, and culturing at 27. + -. 1.0 ℃ for 165 hours.
The slant culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.1 part of dipotassium hydrogen phosphate, 0.1 part of magnesium sulfate and 2.0 parts of agar, wherein the unit is g/100mL, and the pH value is 7.0.
(3) Strain shake flask culture: digging 2-5cm under aseptic condition 2 And (3) scattering the slant strains prepared in the step (2) by using a homogenizer, inoculating the scattered slant strains into a shake flask culture medium, and culturing the shake flask at the temperature of 27 +/-1.0 ℃ for 28 hours to obtain mature shake flask seed liquid.
The shake flask culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium hydrogen phosphate, 0.2 part of sodium chloride and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
(4) Seed tank culture: 15L of seeds are canned into 10L of seed culture medium, sterilized at 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃. Inoculating the shake flask seed solution obtained in the step (3) into a seed tank culture medium in an inoculation amount of 0.05 percent of volume ratio, controlling the culture temperature to be 27 +/-1.0 ℃, controlling the ventilation rate to be 0.5-1.0VVM, controlling the tank pressure to be 0.045-0.065MPa, controlling the initial stirring rotation speed to be 150RPM, controlling the stirring rotation speed to be 150RPM and 550RPM in the culture process, controlling the dissolved oxygen to be more than 30 percent, and culturing for 35 hours to obtain the seed tank seed solution.
The components and the dosage of the seeding tank culture medium are as follows: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium phosphate, 0.2 part of sodium chloride, 0.3 part of calcium carbonate and 0.05 part of defoaming agent, wherein the unit is g/100mL, and the pH value is 7.0.
(5) Fermentation culture: 50L of fermentation medium is canned for 30L, sterilized at the temperature of 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃, the seed liquid of the seed tank obtained in the step (4) is inoculated into the fermentation medium by the inoculation amount of 5 percent of the volume ratio, the fermentation temperature is controlled to be 27 +/-1.0 ℃, the ventilation amount is controlled to be 0.5-1.0VVM, the tank pressure is 0.045-0.065MPa, the initial stirring rotation speed is 150RPM, the stirring rotation speed is controlled to be 150RPM 550RPM in the culture process, the dissolved oxygen is controlled to be 30-100 percent, and the glucose content is controlled to be 0.2-1.0 percent. After 30h of fermentation culture, continuously supplementing n-propanol into the culture medium at a rate of 0.06%/day of the initial volume of the fermentation culture medium. Sampling and detecting the pristinamycin content in the culture medium after fermentation culture for 80h, 100h, 120h, 140h and 160h, wherein the pristinamycin yield is shown in Table 3:
table 3: production of pristinamycin with different culture periods
| Period (h) | 80 | 100 | 120 | 140 | 160 |
| Pristinamycin yield (mg/L) | 1633.17 | 1846.32 | 1832.66 | 1870.26 | 1910.09 |
The fermentation medium comprises the following components in percentage by weight: 3.0 parts of soluble starch, 2.0 parts of glucose, 0.5 part of cane sugar, 2.0 parts of soybean cake powder, 0.5 part of yeast powder, 0.2 part of ammonium sulfate, 0.1 part of manganese sulfate, 0.01 part of ferrous sulfate, 0.05 part of monopotassium phosphate and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
Example 2: after 40h of fermentation culture, n-propanol was added at a rate of 0.08% per day of the initial volume of the fermentation medium
(1) Selecting a starting strain: the strain used in the experiment was Streptomyces pristinaespiralis (Streptomyces pristinaespiralis, ATCC 25486).
(2) Slant culture of the strain: a slant strain was obtained by thawing Streptomyces pristinaespiralis (ATCC 25486) in a glycerol tube, inoculating the thawed tube to a slant medium, and culturing at 27. + -. 1.0 ℃ for 165 hours.
The slant culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.1 part of dipotassium hydrogen phosphate, 0.1 part of magnesium sulfate and 2.0 parts of agar, wherein the unit is g/100mL, and the pH value is 7.0.
(3) Strain shake flask culture: digging 2-5cm under aseptic condition 2 And (3) scattering the slant strains prepared in the step (2) by using a homogenizer, inoculating the scattered slant strains into a shake flask culture medium, and culturing the shake flask at the temperature of 27 +/-1.0 ℃ for 28 hours to obtain mature shake flask seed liquid.
The shake flask culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium hydrogen phosphate, 0.2 part of sodium chloride and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
(4) Seed tank culture: 15L of seeds are canned into 10L of seed culture medium, sterilized at 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃. Inoculating the shake flask seed solution obtained in the step (3) into a seed tank culture medium in an inoculation amount of 0.05 percent of volume ratio, controlling the culture temperature to be 27 +/-1.0 ℃, controlling the ventilation rate to be 0.5-1.0VVM, controlling the tank pressure to be 0.045-0.065MPa, controlling the initial stirring rotation speed to be 150RPM, controlling the stirring rotation speed to be 150RPM and 550RPM in the culture process, controlling the dissolved oxygen to be more than 30 percent, and culturing for 35 hours to obtain the seed tank seed solution.
The components and the dosage of the seeding tank culture medium are as follows: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium phosphate, 0.2 part of sodium chloride, 0.3 part of calcium carbonate and 0.05 part of defoaming agent, wherein the unit is g/100mL, and the pH value is 7.0.
(5) Fermentation culture: 50L of fermentation medium is canned for 30L, sterilized at the temperature of 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃, the seed liquid of the seed tank obtained in the step (4) is inoculated into the fermentation medium by the inoculation amount of 5 percent of the volume ratio, the fermentation temperature is controlled to be 27 +/-1.0 ℃, the ventilation amount is controlled to be 0.5-1.0VVM, the tank pressure is 0.045-0.065MPa, the initial stirring rotation speed is 150RPM, the stirring rotation speed is controlled to be 150RPM 550RPM in the culture process, the dissolved oxygen is controlled to be 30-100 percent, and the glucose content is controlled to be 0.2-1.0 percent. After fermentation culture for 40h, continuously supplementing n-propanol into the culture medium at a rate of 0.08%/day of the initial volume of the fermentation medium. After fermentation culture for 100h, the pristinamycin content in the culture medium is detected, and the pristinamycin content is 1661.35 mg/L.
The fermentation medium comprises the following components in percentage by weight: 3.0 parts of soluble starch, 2.0 parts of glucose, 0.5 part of cane sugar, 2.0 parts of soybean cake powder, 0.5 part of yeast powder, 0.2 part of ammonium sulfate, 0.1 part of manganese sulfate, 0.01 part of ferrous sulfate, 0.05 part of monopotassium phosphate and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
Example 3: experiment of n-propanol addition amount in fermentation culture process
(1) Selecting a starting strain: the strain used in the experiment was Streptomyces pristinaespiralis (Streptomyces pristinaespiralis, ATCC 25486).
(2) Slant culture of the strain: a slant strain was obtained by thawing Streptomyces pristinaespiralis (ATCC 25486) in a glycerol tube, inoculating the thawed tube to a slant medium, and culturing at 27. + -. 1.0 ℃ for 165 hours.
The slant culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.1 part of dipotassium hydrogen phosphate, 0.1 part of magnesium sulfate and 2.0 parts of agar, wherein the unit is g/100mL, and the pH value is 7.0.
(3) Strain shake flask culture: digging 2-5cm under aseptic condition 2 And (3) scattering the slant strains prepared in the step (2) by using a homogenizer, inoculating the scattered slant strains into a shake flask culture medium, and culturing the shake flask at the temperature of 27 +/-1.0 ℃ for 28 hours to obtain mature shake flask seed liquid.
The shake flask culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium hydrogen phosphate, 0.2 part of sodium chloride and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
(4) Seed tank culture: 15L of seeds are canned into 10L of seed culture medium, sterilized at 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃. Inoculating the shake flask seed solution obtained in the step (3) into a seed tank culture medium in an inoculation amount of 0.05 percent of volume ratio, controlling the culture temperature to be 27 +/-1.0 ℃, controlling the ventilation rate to be 0.5-1.0VVM, controlling the tank pressure to be 0.045-0.065MPa, controlling the initial stirring rotation speed to be 150RPM, controlling the stirring rotation speed to be 150RPM and 550RPM in the culture process, controlling the dissolved oxygen to be more than 30 percent, and culturing for 35 hours to obtain the seed tank seed solution.
The components and the dosage of the seeding tank culture medium are as follows: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium phosphate, 0.2 part of sodium chloride, 0.3 part of calcium carbonate and 0.05 part of defoaming agent, wherein the unit is g/100mL, and the pH value is 7.0.
(5) Fermentation culture: 50L of fermentation medium is canned for 30L, sterilized at the temperature of 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃, the seed liquid of the seed tank obtained in the step (4) is inoculated into the fermentation medium by the inoculation amount of 5 percent of the volume ratio, the fermentation temperature is controlled to be 27 +/-1.0 ℃, the ventilation amount is controlled to be 0.5-1.0VVM, the tank pressure is 0.045-0.065MPa, the initial stirring rotation speed is 150RPM, the stirring rotation speed is controlled to be 150RPM 550RPM in the culture process, the dissolved oxygen is controlled to be 30-100 percent, and the glucose content is controlled to be 0.2-1.0 percent. After fermentation culture for 30h, continuously supplementing n-propanol into the culture medium with different addition amounts, wherein the percentage of the n-propanol addition amount refers to the ratio of the volume of the n-propanol to the initial volume of the fermentation culture medium. After fermentation culture for 100h, a fermentation broth with pristinamycin was obtained, the content of which is shown in table 4:
table 4: effect of n-propanol addition on pristinamycin yield
| N-propanol addition (%/day) | 0.04 | 0.06 | 0.08 | 0.1 | 0.12 | 0.14 |
| Pristinamycin yield (mg/L) | 1515.38 | 1855.81 | 1761.48 | 1405.26 | 1289.84 | 1065.53 |
The fermentation medium comprises the following components in percentage by weight: 3.0 parts of soluble starch, 2.0 parts of glucose, 0.5 part of cane sugar, 2.0 parts of soybean cake powder, 0.5 part of yeast powder, 0.2 part of ammonium sulfate, 0.1 part of manganese sulfate, 0.01 part of ferrous sulfate, 0.05 part of monopotassium phosphate and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
Example 4: experiment of n-propanol addition time in fermentation culture process
(1) Selecting a starting strain: the strain used in the experiment was Streptomyces pristinaespiralis (Streptomyces pristinaespiralis, ATCC 25486).
(2) Slant culture of the strain: a slant strain was obtained by thawing Streptomyces pristinaespiralis (ATCC 25486) in a glycerol tube, inoculating the thawed tube to a slant medium, and culturing at 27. + -. 1.0 ℃ for 165 hours.
The slant culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.1 part of dipotassium hydrogen phosphate, 0.1 part of magnesium sulfate and 2.0 parts of agar, wherein the unit is g/100mL, and the pH value is 7.0.
(3) Strain shake flask culture: digging 2-5cm under aseptic condition 2 And (3) scattering the slant strains prepared in the step (2) by using a homogenizer, inoculating the scattered slant strains into a shake flask culture medium, and culturing the shake flask at the temperature of 27 +/-1.0 ℃ for 28 hours to obtain mature shake flask seed liquid.
The shake flask culture medium comprises the following components in percentage by weight: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium hydrogen phosphate, 0.2 part of sodium chloride and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
(4) Seed tank culture: 15L of seeds are canned into 10L of seed culture medium, sterilized at 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃. Inoculating the shake flask seed solution obtained in the step (3) into a seed tank culture medium in an inoculation amount of 0.05 percent of volume ratio, controlling the culture temperature to be 27 +/-1.0 ℃, controlling the ventilation rate to be 0.5-1.0VVM, controlling the tank pressure to be 0.045-0.065MPa, controlling the initial stirring rotation speed to be 150RPM, controlling the stirring rotation speed to be 150RPM and 550RPM in the culture process, controlling the dissolved oxygen to be more than 30 percent, and culturing for 35 hours to obtain the seed tank seed solution.
The components and the dosage of the seeding tank culture medium are as follows: 2.0 parts of soluble starch, 1.5 parts of glucose, 1.0 part of soybean cake powder, 0.5 part of peptone, 0.2 part of dipotassium phosphate, 0.2 part of sodium chloride, 0.3 part of calcium carbonate and 0.05 part of defoaming agent, wherein the unit is g/100mL, and the pH value is 7.0.
(5) Fermentation culture: 50L of fermentation medium is canned for 30L, sterilized at the temperature of 121.0-123.0 ℃ for 30min and then cooled to 27 +/-1.0 ℃, the seed liquid of the seed tank obtained in the step (4) is inoculated into the fermentation medium by the inoculation amount of 5 percent of the volume ratio, the fermentation temperature is controlled to be 27 +/-1.0 ℃, the ventilation amount is controlled to be 0.5-1.0VVM, the tank pressure is 0.045-0.065MPa, the initial stirring rotation speed is 150RPM, the stirring rotation speed is controlled to be 150RPM 550RPM in the culture process, the dissolved oxygen is controlled to be 30-100 percent, and the glucose content is controlled to be 0.2-1.0 percent. In the fermentation culture process, continuously supplementing n-propanol at 0h, 10h, 20h, 30h, 40h, 50h, 60h, 70h and 80h of the culture period respectively, wherein the supplementing rate is 0.06%/day of the initial volume of the fermentation culture medium. The pristinamycin content in the medium was determined after 100h fermentation culture and is shown in table 5:
table 5: effect of n-propanol addition time on pristinamycin yield
The fermentation medium comprises the following components in percentage by weight: 3.0 parts of soluble starch, 2.0 parts of glucose, 0.5 part of sucrose, 2.0 parts of soybean cake powder, 0.5 part of yeast powder, 0.2 part of ammonium sulfate, 0.1 part of manganese sulfate, 0.01 part of ferrous sulfate, 0.05 part of monopotassium phosphate and 0.3 part of calcium carbonate, wherein the unit is g/100mL, and the pH value is 7.0.
Claims (9)
1. A fermentation process for increasing pristinamycin production, comprising: in the fermentation culture process, n-propanol is supplemented into the fermentation culture medium.
2. Fermentation process according to claim 1, wherein the time of supplementation is between 10 and 60h, preferably between 10 and 50h, more preferably between 20 and 50h, most preferably between 30 and 40h after the start of the fermentation culture.
3. Fermentation process according to claim 1 or 2, wherein the n-propanol is fed in continuously at a rate of 0.04-0.1%/day, preferably 0.06-0.08%/day, of the initial volume of the fermentation medium.
4. The fermentation process of any one of claims 1-3, wherein the fermentation medium comprises: 2-10g/100mL carbon source, 2-5g/100mL nitrogen source, 0.2-1.0g/100mL inorganic salt.
5. Fermentation process according to any one of claims 1 to 4, wherein the carbon source of the fermentation medium is selected from one of glucose, sucrose, lactose, maltose, maltodextrin, soluble starch, sorbitol, glycerol or a combination of the above, preferably a combination of soluble starch, glucose and sucrose.
6. The fermentation method according to any one of claims 1 to 5, wherein the nitrogen source of the fermentation medium is selected from one of soybean cake powder, yeast powder, corn gluten meal, cottonseed cake powder, peanut cake powder, soybean meal, corn steep liquor dry powder, yeast extract, peptone or a combination thereof, preferably a combination of soybean cake powder and yeast powder.
7. The fermentation process of any one of claims 1-6, wherein the fermentation medium comprises: 3.0 plus or minus 0.6 of soluble starch, 2.0 plus or minus 0.4 of glucose, 0.5 plus or minus 0.1 of cane sugar, 2.0 plus or minus 0.4 of soybean cake powder, 0.5 plus or minus 0.1 of yeast powder, 0.2 plus or minus 0.04 of ammonium sulfate, 0.1 plus or minus 0.02 of manganese sulfate, 0.01 plus or minus 0.002 of ferrous sulfate, 0.05 plus or minus 0.01 of potassium dihydrogen phosphate, 0.3 plus or minus 0.06 of calcium carbonate, the unit is g/100mL, and the pH value is 7.0 plus or minus 0.2.
8. Fermentation process according to any one of claims 1 to 7, wherein the fermentation cultivation time is between 80 and 160h, preferably between 100 and 160 h.
9. A fermentation process according to any one of claims 1 to 8, wherein the strain used in the fermentation culture is Streptomyces pristinaespiralis capable of producing pristinamycin, preferably Streptomyces pristinaespiralis, ATCC 25486.
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|---|---|---|---|---|
| CN1607245A (en) * | 2003-10-16 | 2005-04-20 | 浙江大学 | High efficiency fermentation method for pristinamycin |
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| WO2021047431A1 (en) * | 2019-09-12 | 2021-03-18 | 浙江海正药业股份有限公司 | Fermentation culture medium and fermentation method capable of increasing yield of a40926 b0 |
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| CN1607245A (en) * | 2003-10-16 | 2005-04-20 | 浙江大学 | High efficiency fermentation method for pristinamycin |
| CN1876827A (en) * | 2006-04-14 | 2006-12-13 | 浙江大学宁波理工学院 | Method for improving pristinamycine fermentation output |
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| WO2021047431A1 (en) * | 2019-09-12 | 2021-03-18 | 浙江海正药业股份有限公司 | Fermentation culture medium and fermentation method capable of increasing yield of a40926 b0 |
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