CN110551786B - Fermentation medium for increasing yield of A40926B 0 and method thereof - Google Patents
Fermentation medium for increasing yield of A40926B 0 and method thereof Download PDFInfo
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Abstract
The invention provides a fermentation medium and a method for improving the yield of A40926B 0, wherein the fermentation medium comprises an organic carbon source, an organic nitrogen source and inorganic salt, the amount of rapeseed oil in the organic carbon source is controlled to be 1-10g/L, and the amount of soybean cake powder in the organic nitrogen source is controlled to be 50-90 g/L; the fermentation culture method comprises the step of feeding maltodextrin to a fermentation liquid in the fermentation process, so that the yield of the dalbavancin intermediate A40926B 0 is improved. By adopting the fermentation medium and the method thereof, the fermentation unit can reach more than 3800 mg/L.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation culture medium for improving the yield of A40926B 0 and a method thereof.
Background
Dalbavancin (dalbavancin) is a glycopeptide semisynthetic antibiotic, is a few of medicines developed by Durata company and approved by FDA in 2014 5/23 to be marketed in the united states, and is used for treating infectious diseases caused by drug-resistant bacteria such as multidrug-resistant staphylococcus aureus and enterococcus, and has the advantages of wide antibacterial spectrum, long half-life, good tolerance and the like. The bactericidal mechanism is that the combination of dalbavancin and aminoacyl-D-alanyl-D-alanine at the end of cell wall peptidoglycan precursor in the division phase inhibits the synthesis of cell wall and kills bacteria.
The dalbavancin precursor A40926 is produced by fermentation of actinomyces Nonomuraea (sp) ATCC 3975, and is modified by chemical structure to obtain dalbavancin. The fermentation product comprises five components, namely B0, B1, B2, PA and PB, wherein B0 is a main component of A40926 and is a key intermediate for synthesizing dalbavancin.
The structure of A40926B 0 is as follows:
the invention patent CN 106668833A of Shaoxing et al, China, discloses that dalbavancin and acceptable salts thereof have significant anti-HIV activity and low cytotoxicity in the application of preparing drugs for treating AIDS, and explores new indications for dalbavancin.
Chinese invention patent CN103060405A discloses that the yield of A40926 is increased to 720mg/L by feeding glucose, peptone and L-valine after the fermentation culture of A40926 is carried out for 72-112 h. Chinese patent CN107365815A discloses that 40-90 g/L glucose, 90-180 g/L corn steep liquor solution, 3-10 g/L-valine and 1200mg/L of A40926 fermentation unit are supplemented after fermentation culture for 40-120 h. Although related processes of A40926 fermentation are researched more at present in China, the fermentation process is generally complicated, and the fermentation level has no major breakthrough.
Disclosure of Invention
In order to improve the fermentation yield of A40926B 0, the invention provides a fermentation medium for improving the yield of A40926B 0.
A fermentation medium for improving the yield of A40926B 0 comprises an organic carbon source, an organic nitrogen source and inorganic salts, wherein the organic carbon source contains rapeseed oil, the content of the rapeseed oil in the fermentation medium is 1-10g/L, the organic nitrogen source contains soybean cake powder, the content of the soybean cake powder in the fermentation medium is 50-90g/L, preferably, the content of the rapeseed oil in the fermentation medium is 3g/L, and the content of the soybean cake powder in the fermentation medium is 65 g/L.
Preferably, the organic carbon source in the fermentation medium further comprises one or a combination of any two or more selected from the group consisting of glucose, maltodextrin, mannitol, corn starch, corn flour, potato starch, soluble starch, sucrose, flour, preferably a combination of maltodextrin and corn starch.
Preferably, the organic nitrogen source in the fermentation medium further comprises one or a combination of any two or more selected from the group consisting of cottonseed meal, yeast powder, yeast extract, meat peptone, soybean peptone, gluten meal, leucine, valine, glycine, phenylalanine, glutamine, preferably a combination of valine and meat peptone.
Preferably, the inorganic salt in the fermentation medium is selected from one or a combination of any two or more of ammonium sulfate, ammonium nitrate, potassium nitrate, ammonium chloride, sodium nitrate, magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride, sodium chloride and calcium carbonate, preferably a combination of copper sulfate and calcium carbonate.
Preferably, the fermentation medium comprises 50-90g/L of soybean cake powder, 1-10g/L of rapeseed oil, 10-35 g/L of maltodextrin, 5-15 g/L of corn starch, 2.5-3.5 g/L of valine, 15-25 g/L of meat peptone, 5-15 ppm of copper sulfate and 4-6 g/L of calcium carbonate; wherein the corn starch is preferably 10g/L, the valine is preferably 3g/L, the meat peptone is preferably 20g/L, the copper sulfate is preferably 10ppm, and the calcium carbonate is preferably 5 g/L.
A fermentation method for increasing the yield of A40926B 0 comprises inoculating seed liquid of Nonomuraea sp to any one of the above fermentation culture media, and performing liquid fermentation culture.
Preferably, the Nonomuraea (Nonomuraea. sp) is Nonomuraea ATCC 3975 (Nonomuraea. sp ATCC 3975).
Preferably, the culture period of the fermentation culture is 120-168 hours, and when the fermentation culture is carried out for 24-144 hours, the maltodextrin solution is supplemented into the fermentation liquor, wherein the concentration of the supplemented maltodextrin solution is 300-600 g/L.
Preferably, the liquid fermentation is cultured in a shake flask, the culture temperature is 28-30 ℃, the rotation speed of a shaking table is 250rpm, and the culture period is 5-7 days; or the fermentation is carried out in a 50L fermentation tank, the actual loading is 30L, the culture temperature is 28-30 ℃, the stirring speed is 150-600 rpm, the tank pressure is 0.03-0.05 Mpa, the ventilation volume is 0.8-1.2 VVM, and the culture period is 120-168 h.
Preferably, the strategy of supplementing the maltodextrin solution into the fermentation liquor is to control the content of total sugar in the fermentation liquor to be 5-10 g/L, and the determination method of the total sugar is a film volumetric method.
Preferably, the supplementing mode of the maltodextrin is one-time supplementing or feeding, preferably feeding, and the flow rate of the feeding is 0.1-1.0 mL.L-1·h-1。
In the research process, through a plurality of tests, the inventor finds that when the amount of soybean cake powder in a fermentation medium is controlled to be 50-90g/L and the amount of rapeseed oil is controlled to be 1-10g/L, the content of A40926B 0 in a fermentation liquid is obviously improved; and in addition, the effect cannot be achieved in the period, and meanwhile, in the culture process, the fermentation unit of A40926B 0 can be further improved by adding the maltodextrin solution into the fermentation liquor, so that the fermentation unit of A40926B 0 is improved by 200%.
Drawings
FIG. 1 shows a high performance liquid chromatogram of A40926B 0 standard.
FIG. 2 is a high performance liquid chromatogram of the fermentation broth of example 10.
Detailed Description
The technical solutions in the embodiments of the present invention are described below, and the described embodiments are only a part of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the present invention, the producing strain of A40926B 0 is Nonomuraea ATCC 3977 (Nonomuraea. sp ATCC 3975) and is purchased from American type culture Collection.
In the present invention, the material is not particularly limited, and a material source well known in the art may be used.
In the invention, the A40926 fermentation liquid treatment and High Performance Liquid Chromatography (HPLC) detection method comprises the following steps:
the high performance liquid chromatography detection method comprises the following steps: column Hypersil C18 column (4.6 mm. times.250 mm,5 μm) with mobile phase acetonitrile (0.45%) ammonium formate 2: 8; the flow rate is 1.0mL/min, the column temperature is 30 ℃, and the detection wavelength is 207 nm; the amount of the sample was 10. mu.l.
And (3) detection of the standard substance: diluting 50mg of A40926B 0 standard substance with purified water to 100ml, detecting HPLC with standard substance solution, and detecting the standard substance with a chromatogram shown in FIG. 1.
Treating fermentation liquid: sampling three parts of fermentation liquor in parallel, adjusting the pH value to 11.5-12.0 by using 6mol/L sodium hydroxide, sucking 1mL of fermentation liquor, adding 4mL of deionized water for dilution, centrifuging at 4000rpm for 15min, and taking supernatant for detecting HPLC.
Comparative example 1
The preparation method of the slant thallus comprises the following steps: inoculating actinomyces Nomuranus ATCC 3977 to a test tube slant culture medium, wherein the test tube slant culture medium comprises the following components: yeast extract 0.4%, glucose 0.4%, malt extract 1.0%, and water in balance, adjusting pH to 7.0, test tube specification of 20mm 220mm, loading 20ml, and sterilizing at 121 deg.C for 30 min. Standing at 28 deg.C for 7 days to obtain slant thallus of Nonomurae ATCC39727 (Nonomuraea. sp. ATCC 39727).
The preparation method of the seed liquid in the shake flask comprises the following steps: the actinomyces nomureus ATCC39727 (Nonomuraea. sp. ATCC 39727) slant prepared above was inoculated into a shake flask seed medium (1L shake flask was used) with a steel pick: 10g/L of glucose, 10g/L of maltodextrin, 10g/L of corn starch, 5g/L of cottonseed cake powder, 15g/L of soybean cake powder, 1g/L of magnesium sulfate, 1g/L of dipotassium phosphate, 2.5g/L of calcium carbonate and the balance of water, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 30 min. The rotation speed of a shaking table is 250rpm, and the shaking table is cultured for 72 hours at the temperature of 28 ℃ to obtain shaking bottle seed liquid.
Inoculating the shake flask seed solution into a shake flask fermentation medium (adopting a 250mL shake flask) according to the inoculation amount of 10% (volume fraction, the same below): 35g/L of soybean cake powder, 15g/L of maltodextrin, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. And (3) culturing for 7 days at 28 ℃ at the rotation speed of 250rpm of a shaking table to obtain a fermentation liquor, and detecting the fermentation liquor by HPLC, wherein the concentration of A40926B 0 in the fermentation liquor is 1105 mg/L.
Comparative example 2
The seed solution in the shake flask of comparative example 1 was inoculated into a seed tank (the culture medium in the seed tank was the same as the shake flask seed culture medium of comparative example 1) at 3% inoculum size, pH7.0, volume 15L, actual load 10L, and sterilized at 121 ℃ for 30 min. Culturing at 28 deg.C, stirring at 180rpm, under 0.05Mpa, and with ventilation of 1.0VVM for 44h to obtain seed solution in seed tank.
Inoculating the seed solution of the seed tank into a 50L fermentation tank according to the inoculation amount of 10%, wherein the culture medium of the fermentation tank is as follows: 35g/L of soybean cake powder, 15g/L of maltodextrin, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, the actual loading amount is 30L, and the soybean cake powder is sterilized at 121 ℃ for 30 min. Stirring at 200rpm, tank pressure of 0.05Mpa, and ventilation of 1.0VVM, culturing at 28 deg.C for 168 hr to obtain fermentation broth, and detecting by HPLC to obtain fermentation broth with A40926B 0 concentration of 1280 mg/L.
Comparative example 3
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 50g/L of soybean cake powder, 15g/L of maltodextrin, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. The shaking table rotates at 250rpm, the fermentation broth is obtained after 7 days of culture at 30 ℃, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 1492 mg/L.
Comparative example 4
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 65g/L of soybean cake powder, 15g/L of maltodextrin, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. The shaking table is rotated at 250rpm and cultured at 28 ℃ for 7 days to obtain a fermentation broth, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 1224 mg/L.
Comparative example 5
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 65g/L of soybean cake powder, 15g/L of maltodextrin, 15g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized for 30min at the temperature of 121 ℃. The shaking table rotates at 250rpm, the fermentation broth is obtained after the cultivation for 7 days at 28 ℃, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 1646 mg/L.
Comparative example 6
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 100g/L of soybean cake powder, 15g/L of maltodextrin, 1g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. The shaking table rotates at 250rpm, the fermentation broth is obtained after the cultivation for 7 days at 28 ℃, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 1202 mg/L.
Comparative example 7
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 90g/L of soybean cake powder, 15g/L of maltodextrin, 15g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized for 30min at the temperature of 121 ℃. The shaking table rotates at 250rpm, the fermentation broth is obtained after the cultivation for 7 days at 28 ℃, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 1411 mg/L.
Example 1
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 50g/L of soybean cake powder, 35g/L of maltodextrin, 1g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. And (4) culturing the mixture at the rotating speed of 250rpm of a shaking table and the temperature of 28 ℃ for 7 days to obtain a fermentation liquor, and detecting the fermentation liquor by HPLC, wherein the concentration of A40926B 0 is 2006 mg/L.
Example 2
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 90g/L of soybean cake powder, 35g/L of maltodextrin, 1g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. The shaking table rotates at 250rpm, the fermentation broth is obtained after the cultivation for 7 days at 28 ℃, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 2213 mg/L.
Example 3
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 50g/L of soybean cake powder, 35g/L of maltodextrin, 10g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. The shaking table is rotated at 250rpm and cultured at 28 ℃ for 7 days to obtain a fermentation broth, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 2171 mg/L.
Example 4
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 90g/L of soybean cake powder, 35g/L of maltodextrin, 10g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized for 30min at the temperature of 121 ℃. The shaking table rotates at 250rpm, the fermentation broth is obtained after the cultivation for 7 days at 28 ℃, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 2026 mg/L.
Example 5
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 65g/L of soybean cake powder, 35g/L of maltodextrin, 3g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. The shaking table rotates at 250rpm, the fermentation broth is obtained after 7 days of culture at 28 ℃, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 2608 mg/L.
Example 6
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 65g/L of soybean cake powder, 35g/L of maltodextrin, 10g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized for 30min at the temperature of 121 ℃. The shaking table rotates at 250rpm, the fermentation broth is obtained after culturing for 7 days at 28 ℃, and the fermentation broth is detected by HPLC, wherein the concentration of A40926B 0 is 2279 mg/L.
Example 7
The shake flask seed solution of comparative example 1 was inoculated into shake flask fermentation medium (250 mL shake flask was used) at an inoculum size of 10%: 65g/L of soybean cake powder, 15g/L of maltodextrin, 3g/L of rapeseed oil, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, and the soybean cake powder is sterilized at 121 ℃ for 30 min. And (3) culturing at the rotating speed of 250rpm of a shaking table at 28 ℃ for 48h, 72h, 96h and 120h respectively by adding a sterilized maltodextrin solution (500g/L solution) according to the volume fraction of 1%, culturing for 7 days to obtain a fermentation broth, and detecting the fermentation broth by HPLC, wherein the concentration of A40926B 0 is 2992 mg/L.
Example 8
The seed solution in the shake flask of comparative example 1 was inoculated into a seed tank (the culture medium is the same as the shake flask seed culture medium) at a pH of 7.0 and a volume of 15L, and the actual volume of 10L was sterilized at 121 ℃ for 30 min. Culturing at 28 deg.C, stirring at 180rpm, under 0.05Mpa, and with ventilation of 1.0VVM for 46h to obtain seed solution in seed tank.
Inoculating the seed solution of the seed tank into a 50L fermentation tank according to the inoculation amount of 10%, wherein the culture medium of the fermentation tank is as follows: 65g/L of soybean cake powder, 3g/L of rapeseed oil, 15g/L of maltodextrin, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, the actual loading amount is 30L, and the soybean cake powder is sterilized for 30min at the temperature of 121 ℃. Stirring at 200rpm, tank pressure of 0.05Mpa, ventilation of 1.0VVM, culturing at 28 deg.C for 168 hr to obtain fermentation broth, and detecting by HPLC, wherein the concentration of A40926B 0 is 2580 mg/L.
Example 9
The seed solution in the shake flask of comparative example 1 was inoculated into a seed tank (the culture medium is the same as the shake flask seed culture medium) at a pH of 7.0 and a volume of 15L, and the actual volume of 10L was sterilized at 121 ℃ for 30 min. Culturing at 28 deg.C, stirring at 180rpm, under 0.05Mpa, and with ventilation of 1.0VVM for 46h to obtain seed solution in seed tank.
Inoculating the seed solution of the seed tank into a 50L fermentation tank according to the inoculation amount of 10%, wherein the culture medium of the fermentation tank is as follows: 65g/L of soybean cake powder, 3g/L of rapeseed oil, 15g/L of maltodextrin, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, the actual loading amount is 30L, and the soybean cake powder is sterilized for 30min at the temperature of 121 ℃. Stirring at 200rpm, tank pressure of 0.05Mpa, ventilation of 1.0VVM, culturing at 30 deg.C for 30h, 54h, 78h and 102h, respectively, adding maltodextrin solution (500g/L solution) 600ml once, culturing for 160h to obtain fermentation broth, and detecting by HPLC to obtain fermentation broth with A40926B 0 concentration of 3208 mg/L.
Example 10
The seed solution in the shake flask of comparative example 1 was inoculated into a seed tank (the culture medium is the same as the shake flask seed culture medium) at a pH of 7.0 and a volume of 15L, and the actual volume of 10L was sterilized at 121 ℃ for 30 min. Culturing at 28 deg.C, stirring at 180rpm, under 0.05Mpa, and with ventilation of 1.0VVM for 46h to obtain seed solution in seed tank.
Inoculating the seed solution of the seed tank into a 50L fermentation tank according to the inoculation amount of 10%, wherein the culture medium of the fermentation tank is as follows: 65g/L of soybean cake powder, 3g/L of rapeseed oil, 15g/L of maltodextrin, 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate, 5g/L of calcium carbonate and the balance of water, wherein the pH value is 7.0, the actual loading amount is 30L, and the soybean cake powder is sterilized for 30min at the temperature of 121 ℃. Stirring at 200rpm, a tank pressure of 0.05Mpa, an air flow of 1.0VVM, culturing at 30 deg.C for 36 hr, and feeding maltodextrin solution (500g/L solution) at a flow rate of 0.5 ml/L-1·h-1And taking a fermentation sample every 6 hours to detect the total sugar content, controlling the total sugar content to be maintained at 5-10 g/L, culturing for 144 hours to obtain a fermentation liquid, and detecting the fermentation liquid by HPLC, wherein the concentration of A40926B 0 is 3850mg/L, and the HPLC detection map of A40926B 0 in the fermentation liquid is shown in FIG. 2.
Claims (14)
1. A fermentation method for improving the yield of A40926B 0 is characterized in that: inoculating seed liquid of Nonomuraea sp into a fermentation culture medium to perform liquid fermentation culture, wherein the fermentation culture medium comprises an organic carbon source, an organic nitrogen source and inorganic salts, the organic carbon source comprises rapeseed oil, the content of the rapeseed oil in the fermentation culture medium is 1-10g/L, the organic nitrogen source comprises soybean cake powder, the content of the soybean cake powder in the fermentation culture medium is 50-90g/L, and the Nonomuraea sp is Nonomuraea ATCC 3975.
2. The fermentation method for improving the yield of A40926B 0 as claimed in claim 1, wherein: the content of the rapeseed oil in the fermentation culture medium is 3g/L, and the content of the soybean cake powder in the fermentation culture medium is 65 g/L.
3. The fermentation method for improving the yield of A40926B 0 as claimed in claim 1, wherein: the organic carbon source further comprises one or a combination of any two or more selected from glucose, maltodextrin, mannitol, corn starch, corn flour, potato starch, soluble starch, sucrose, flour.
4. The fermentation method for improving the yield of A40926B 0 as claimed in claim 3, wherein: the organic carbon source is a combination of maltodextrin and corn starch.
5. The fermentation method for improving the yield of A40926B 0 as claimed in claim 1, wherein: the organic nitrogen source further contains one or the combination of any two or more of cottonseed cake powder, yeast extract, meat peptone, soybean peptone, gluten powder, wheat gluten, leucine, valine, glycine, phenylalanine and glutamine.
6. The fermentation method for improving the yield of A40926B 0 as claimed in claim 5, wherein: the organic nitrogen source is a combination of valine and meat peptone.
7. The fermentation method for improving the yield of A40926B 0 as claimed in claim 1, wherein: the inorganic salt is selected from one or the combination of any two or more of ammonium sulfate, ammonium nitrate, potassium nitrate, ammonium chloride, sodium nitrate, magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride, sodium chloride and calcium carbonate.
8. The fermentation method for improving the yield of A40926B 0 as claimed in claim 7, wherein: the inorganic salt is a combination of copper sulfate and calcium carbonate.
9. The fermentation method for improving the yield of A40926B 0 according to any one of claims 1 to 8, wherein: the fermentation medium comprises 50-90g/L of soybean cake powder, 1-10g/L of rapeseed oil, 10-35 g/L of maltodextrin, 5-15 g/L of corn starch, 2.5-3.5 g/L of valine, 15-25 g/L of meat peptone, 5-15 ppm of copper sulfate and 4-6 g/L of calcium carbonate.
10. The fermentation method for improving the yield of A40926B 0 as claimed in claim 9, wherein: the fermentation medium comprises 10g/L of corn starch, 3g/L of valine, 20g/L of meat peptone, 10ppm of copper sulfate and 5g/L of calcium carbonate.
11. The fermentation method for improving the yield of A40926B 0 as claimed in claim 1, wherein: the culture period of the fermentation culture is 120-168 hours, and when the fermentation culture is carried out for 24-144 hours, a maltodextrin solution is supplemented into the fermentation liquor, wherein the concentration of the supplemented maltodextrin solution is 300-600 g/L.
12. The fermentation method for improving the yield of A40926B 0 according to claim 11, wherein: the strategy of supplementing the maltodextrin solution into the fermentation liquor is to control the content of total sugar in the fermentation liquor to be 5-10 g/L.
13. The fermentation method for improving the yield of A40926B 0 according to claim 11, wherein: the supplementing mode of the maltodextrin is one-time supplementing or feeding.
14. The fermentation method for improving the yield of A40926B 0 as claimed in claim 13, wherein: the maltodextrin is supplemented in a fed-batch mode, and the flow rate of the fed-batch is 0.1-1.0 mL.L-1·h-1。
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| CN114807275A (en) * | 2022-05-26 | 2022-07-29 | 海正药业(杭州)有限公司 | Fermentation method for increasing pristinamycin yield |
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