CN114381480A - Fermentation method of sirolimus - Google Patents
Fermentation method of sirolimus Download PDFInfo
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- CN114381480A CN114381480A CN202011128702.XA CN202011128702A CN114381480A CN 114381480 A CN114381480 A CN 114381480A CN 202011128702 A CN202011128702 A CN 202011128702A CN 114381480 A CN114381480 A CN 114381480A
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- 230000004151 fermentation Effects 0.000 title claims abstract description 195
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- 241000187844 Actinoplanes Species 0.000 description 1
- HKVAMNSJSFKALM-GKUWKFKPSA-N Everolimus Chemical compound C1C[C@@H](OCCO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 HKVAMNSJSFKALM-GKUWKFKPSA-N 0.000 description 1
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- CBPNZQVSJQDFBE-FUXHJELOSA-N Temsirolimus Chemical compound C1C[C@@H](OC(=O)C(C)(CO)CO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 CBPNZQVSJQDFBE-FUXHJELOSA-N 0.000 description 1
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Classifications
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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
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a sirolimus fermentation method, wherein seed liquid is inoculated into a fermentation tank filled with a fermentation culture medium for culture to obtain a fermentation liquid with high yield of sirolimus, and the fermentation culture medium is subjected to high-intensity sterilization treatment; according to the invention, the culture medium components are changed under the high-pressure sterilization condition of the fermentation process, glucose and phospholipid are supplemented in the fermentation process, sirolimus is distributed in the fermentation liquor in an oily matter state, the phospholipid increases the dispersion of sirolimus and precursors thereof, improves the permeability of cell membranes, promotes the induction of secondary metabolic pathway of sirolimus, and has a certain effect on the feedback inhibition of product accumulation, thereby having a better effect on the conversion of sirolimus and intermediates. The sirolimus fermentation preparation process is simple to operate, and the fermentation content is obviously improved.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a sirolimus fermentation method.
Background
Sirolimus (Sirolimus), also known as Rapamycin (Rapamycin), is a 31-membered macrolide immunosuppressant, is produced by streptomyces hygroscopicus, actinoplanes and other strains, has wide biological activity, and comprises antifungal, immunosuppressive, antitumor, neuroprotection, anti-aging and the like. Meanwhile, sirolimus is used as a precursor, and some chemically modified and synthesized derivatives with novel structures are found to have new treatment effects on the aspects of immunosuppression, cancer resistance, Parkinson disease resistance, AIDS and the like, wherein the synthesized derivatives Temsirolimus, Everolimus and AP23573 are already used as new antitumor targets to be sold on the market or clinically researched, and rapamycin has wide prospects in the field of medical application. In view of the complex structure of sirolimus, which is not suitable for chemical synthesis, the method still depends on fermentation production in the future, so that the development of an excellent production strain is urgently needed, and the fermentation level of sirolimus is improved by combining with the optimization of a fermentation process. With the comprehensive analysis of the biosynthesis pathway and the continuous improvement of the genetic operation system of the sirolimus producing strain, the possibility is provided for the development of fermentation regulation based on the metabolic engineering concept. The synthesis of sirolimus is subject to complex regulation, and some key precursors or metabolic intermediates also have a promoting effect on the synthesis of sirolimus.
According to the metabolic pathway influence patent CN109486877A, the glucose aqueous solution is supplemented in batches every day from fermentation for 96 hours until the fermentation is finished. The fermentation unit can reach over 1000 mug/ml by HPLC method.
In patent CN102199638, amino acid with mass concentration of 0.2-1% is added into the initial fermentation medium, and the fermentation yield can be significantly improved compared with that of a control group, but specific fermentation content is not mentioned. Wherein the improvement range of alanine is obviously lower than that of amino acids such as leucine, valine, glutamic acid, glutamine and the like.
Ginger Wei et al reported in the literature of "optimization research on fed-batch materials during rapamycin fermentation" that 0.35% isoleucine was fed-batch at 72h of fermentation and 0.1% isoleucine was fed-batch at 144h, and the rapamycin fermentation level reached 1360 g/L. Patent CN103555785A reports that the addition of glycerol and phosphate buffer solution in the early stage of fermentation culture process and the addition of L-pipecolic acid and shikimic acid in the synthesis stage achieves the reduction of demethylated derivatives and the increase of rapamycin content in the production stage.
CN109468253A discloses a streptomyces hygroscopicus for high yield of rapamycin, which adopts gene knockout to ensure that the mutant strain of 4083721-4085064 part or all of the sequence in the genome of streptomyces hygroscopicus NRRL 5491 is deleted, so that the fermentation yield of rapamycin reaches 1567.27 mg/L.
Currently, the fermentation yield of sirolimus is still low, the purposeful genetically modified fermentation content only reaches 1567.27mg/L, and further improvement is needed.
Disclosure of Invention
The invention aims to provide a fermentation method of sirolimus with high yield, which is simple to regulate and control, remarkably improves the fermentation unit, simultaneously improves the conversion rate of a key intermediate 29-O-demethylsirolimus, provides convenience for separation and purification, and provides guarantee for improving the international competitiveness of products.
The technical scheme of the invention is as follows:
a fermentation method of high-yield sirolimus comprises the steps of inoculating a seed solution into a fermentation tank filled with a fermentation medium for culture to obtain a fermentation solution of the high-yield sirolimus; the fermentation medium is subjected to high-intensity sterilization treatment.
Preferably, the high-intensity sterilization treatment refers to sterilization of the fermentation medium at 125-130 ℃.
Preferably, the sterilization time of the fermentation medium is 30-60 min.
Preferably, the feeding process comprises a glucose supplementation process.
Preferably, the ratio of the mass of the supplemented glucose to the initial volume of the fermentation system is 10-20 g/L; wherein the mass of the glucose is calculated by g, and the initial volume of the fermentation system is calculated by L.
Preferably, the content of reducing sugar in the fermentation system is detected at regular time during the fermentation process, and glucose is supplemented when the reducing sugar is lower than 1 g/L.
Preferably, the amount of glucose supplemented every time is 1-3 g/L, wherein the mass of glucose supplemented is calculated by g, and the volume of fermentation liquor is calculated by L.
Preferably, the fed glucose is sterilized for 20-30 min at 115-121 ℃.
Preferably, the fermentation process further comprises a phospholipid supplementation process.
Preferably, the ratio of the mass of the added phospholipid to the volume of the fermentation liquid in the fermentation process is 1-3 g/L, wherein the mass of the phospholipid is calculated by g, and the volume of the fermentation liquid is calculated by L.
Preferably, the phospholipid is supplemented in the early stage and/or the middle stage of the fermentation, and further preferably, the phospholipid is supplemented at 66 th to 78 th hours and 114 th to 126 th hours after the fermentation is started.
Preferably, the pH value of the system in the fermentation process is 6.0-6.9, and further preferably 6.0-6.4.
Preferably, the fermentation medium is: 10-25g/L of corn starch, 5-15g/L of soybean cake powder, 5-10g/L of peptone, 5-10g/L of corn steep liquor dry powder, 5-15g/L of glycerol, 20-40g/L of glucose and 3-5g/L, K of piperidine-2-formic acid2HPO4 2-5g/L、KH2PO42-5g/L, MgSO 4.7H 2O 0.02.02-0.06 g/L, 2-5g/L calcium carbonate and 2-5g/L natural sodium benzoate, and the pH value is 6.5-7.0.
Further preferably, the fermentation basal medium is: 13g/L of corn starch, 10g/L of soybean cake powder, 6g/L of peptone, 5g/L of corn steep liquor dry powder, 10g/L of glycerol, 35g/L of glucose and 3g/L, K of piperidine-2-formic acid2HPO4 3g/L、KH2PO43g/L, MgSO4 & 7H2O 0.05.05 g/L, 3g/L calcium carbonate and 3g/L natural sodium silicate, and the pH is 6.9.
Preferably, in the fermentation process, the fermentation condition is that the culture temperature is 28-30 ℃, the pressure of a tank is maintained to be 0.05-0.07 Mpa, and the ventilation volume is 1.4-2.0 m3The rotation speed is 100rpm to 400rpm, the dissolved oxygen is controlled to be 20 to 30 percent, the pH value is 6.0 to 6.9, and the culture time is 216-.
Preferably, Actinoplanes sp.N902-109 is used as the fermentation strain.
Preferably, the fermentation process is to inoculate the seed liquid into the liquid fermentation medium according to the inoculation amount of 8-12% v/v.
The following will illustrate in detail the fermentation process of sirolimus, comprising the following steps:
(1) activating strains: inoculating the strain on a slant culture medium, culturing for 5-8 days in a biochemical incubator at 28-30 ℃, and waiting until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use.
(2) Preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with a seed culture medium, wherein the rotating speed of a shaking table is 200-220 r/m, the temperature is 28-30 ℃, and the culture is carried out for 44-48 h.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with a secondary seed culture medium according to the inoculation amount of 8-12%, wherein the rotating speed of a shaking table is 200-220 r/m, the temperature is 28-30 ℃, and the culture is carried out for 20-30 h.
(4) Seed tank culture: inoculating the secondary seed culture solution into a seed tank filled with the seed culture solution according to the inoculation amount of 1-3%, rotating at 80-100rpm, culturing for 16-24 h at the culture temperature of 28-30 ℃ and the ventilation volume of 0.7-1.4 m3The pot pressure is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: inoculating seed culture solution of a seeding tank into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 8-12%, wherein the culture temperature is 28-30 ℃, the tank pressure is maintained at 0.05-0.07 Mpa, and the ventilation volume is 1.4-2.0 m3The fermentation method comprises the following steps of/h, rotating speed of 100rpm-400rpm, controlling dissolved oxygen at 20-30%, maintaining pH at 6.0-6.9 in the fermentation process, monitoring reducing sugar in the fermentation system in real time in the fermentation process, controlling the content of the reducing sugar in the fermentation system to be not less than 1g/L by supplementing glucose, supplementing phospholipid in the early stage and/or the middle stage of fermentation, and stopping fermentation when the content of sirolimus is not increased any more.
Preferably, the fermentation medium is subjected to high-intensity sterilization treatment at 125-130 ℃ for 30-60 min.
Preferably, the ratio of the glucose supplementing mass to the initial volume of the fermentation system is 10-20 g/L; wherein the mass of the glucose is calculated by g, and the initial volume of the fermentation system is calculated by L.
Preferably, the glucose is supplemented and sterilized for 20-30 min at 115-121 ℃.
Preferably, the ratio of the mass of the added phospholipid to the volume of the fermentation liquid in the fermentation process is 1-3 g/L, wherein the mass of the phospholipid is calculated by g, and the volume of the fermentation liquid is calculated by L.
Preferably, the phospholipid is supplemented in the early stage and/or the middle stage of fermentation, namely the first supplementing is carried out in 66 th-78 th hours after the beginning of fermentation, and the second supplementing is carried out in 114 th-126 th hours after the beginning of fermentation; preferably, the feed is added 72h and 120h after the start of fermentation.
Actinoplanes sp.N902-109 is used as a fermentation strain, and the fermentation initial culture medium is sterilized at different intensities (126 ℃, 60min, 121 ℃, 30min) according to the fermentation method, and fermentation culture is carried out. The effect of the medium on the biomass of the cells under different sterilization conditions was determined by measuring the biomass of the cells during the fermentation process, and the results are shown in Table 1.
TABLE 1 Effect of the Medium on the biomass of the cells under different conditions of Sterilization during fermentation
As can be seen from the data in Table 1, the biomass sterilized at 126 ℃ for 60min had slower growth of the strains at the early and middle stages of fermentation than the biomass sterilized at 121 ℃ for 30min, but did not affect the progress of sirolimus fermentation in the whole fermentation process.
The present invention is not particularly limited to the slant medium, the first-order seed medium, and the second-order seed medium, as long as it is advantageous for the growth of the strain.
For example, the slant culture medium is 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
For example, the seed culture medium is 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH is 7.0.
The fermentation process of the invention changes the components of the culture medium under the condition of high-pressure sterilization, and supposedly mainly comprises the steps of converting glucose into 5-hydroxymethyl furan aldehyde by heating and further decomposing the glucose into acidic substances to have the greatest influence on the fermentation. The sirolimus is distributed in the fermentation liquor in an oily state, the phospholipid increases the dispersion of the sirolimus and the precursor thereof, improves the permeability of cell membranes, promotes the induction of a secondary metabolic pathway of the sirolimus, and has a certain effect on the feedback inhibition of product accumulation, thereby having a better effect on the conversion of the sirolimus and intermediates.
The sirolimus fermentation preparation process is simple to operate, the fermentation content is obviously improved, the conversion rate of the key intermediate (29-O-demethylsirolimus) is improved, and a guarantee is provided for improving the industrial competitive advantage of sirolimus.
Detailed Description
The present invention is further illustrated and described in the following examples, which should be construed as being merely illustrative and not limitative of the remainder of the disclosure. The examples were carried out according to conventional test conditions, unless otherwise specified.
The following examples use Actinoplanes sp.N902-109 as the fermentative species.
Example 1
The slant culture medium is 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
Primary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Secondary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Fermentation tank culture medium: 13g/L of corn starch, 10g/L of soybean cake powder, 6g/L of peptone, 5g/L of corn steep liquor dry powder, 10g/L of glycerol, 35g/L of glucose and 3g/L, K of piperidine-2-formic acid2HPO4 3g/L、KH2PO43g/L, MgSO 4.7H 2O 0.05.05 g/L, 3g/L calcium carbonate and 3g/L natural sodium silicate, and the pH value is 6.9; sterilizing at 121 deg.C for 30 min.
A supplemented medium: glucose 100g/L, sterilizing the glucose solution at 121 deg.C for 30 min.
(1) Activating strains: inoculating the strain on a 30ml slant culture medium, culturing in a biochemical incubator at 28-30 ℃ for 6 days until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use;
(2) preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with 30ml of seed culture medium, and culturing for 48 hours at the rotating speed of a shaking table of 200r/m and the temperature of 28-30 ℃.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with 30ml of secondary seed culture medium according to the inoculation amount of 10%, wherein the rotating speed of a shaking table is 200r/m, the temperature is 28-30 ℃, and culturing is carried out for 24 h.
(4) Seed tank culture: inoculating the secondary seed culture solution into a seed tank filled with the seed culture solution according to the inoculation amount of 1%, rotating at 80-100rpm, culturing for 24h at the culture temperature of 28-30 ℃, and the ventilation volume of 0.7-1.4 m3The pressure in the tank is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: preparing culture medium according to a certain proportion, inoculating seed culture solution into 50L fermentation tank containing 30L culture medium according to 10% inoculum size, culturing at 28-30 deg.C, maintaining tank pressure of 0.05-0.07 Mpa, and ventilation amount of 1.4-2.0 m3The rotation speed is 100rpm-400rpm, the dissolved oxygen is controlled at 20-30%, the pH value is maintained at 6.0-6.4 in the fermentation process, the content of reducing sugar in a fermentation system is monitored every 12 hours in the fermentation process, when the content of the reducing sugar is lower than 1g/L, glucose solution is supplemented, and 0.5L of the reducing sugar is supplemented every time until the fermentation is finished. The fermentation culture time is 264 hours, 4.5L of glucose is supplemented, the sampling detection shows that the content of sirolimus in the fermentation liquid is 1180.6mg/L, and the content of 29-O-demethylsirolimus is 8.92%.
Example 2
The slant culture medium is 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
Primary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Secondary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Fermentation tank culture medium: 13g/L of corn starch, 10g/L of soybean cake powder, 6g/L of peptone, 5g/L of corn steep liquor dry powder, 10g/L of glycerol, 35g/L of glucose and 3g/L, K of piperidine-2-formic acid2HPO4 3g/L、KH2PO43g/L, MgSO 4.7H 2O 0.05.05 g/L, 3g/L calcium carbonate and 3g/L natural sodium silicate, and the pH value is 6.9; sterilizing at 126 deg.C for 60 min.
A supplemented medium: glucose 100g/L, sterilizing the glucose solution at 121 deg.C for 30 min.
(1) Activating strains: inoculating the strain on a 30ml slant culture medium, culturing in a biochemical incubator at 28-30 ℃ for 6 days until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use;
(2) preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with 30ml of seed culture medium, and culturing for 48 hours at the rotating speed of a shaking table of 200r/m and the temperature of 28-30 ℃.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with 30ml of secondary seed culture medium according to the inoculation amount of 10%, wherein the rotating speed of a shaking table is 200r/m, the temperature is 28-30 ℃, and culturing is carried out for 24 h.
(4) Seed tank culture: inoculating the secondary seed culture solution into a seed tank filled with the seed culture solution according to the inoculation amount of 1%, rotating at 80-100rpm, culturing for 24h at the culture temperature of 28-30 ℃, and the ventilation volume of 0.7-1.4 m3The pressure in the tank is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: preparing culture medium according to a certain proportion, inoculating seed culture solution into 50L fermentation tank containing 30L culture medium according to 10% inoculum size, culturing at 28-30 deg.C, maintaining tank pressure of 0.05-0.07 Mpa, and ventilation amount of 1.4-2.0 m3The rotation speed is 100rpm-400rpm, the dissolved oxygen is controlled at 20-30%, the pH value is maintained at 6.0-6.4 in the fermentation process, the content of reducing sugar in a fermentation system is monitored every 12 hours in the fermentation process, when the content of the reducing sugar is lower than 1g/L, glucose solution is supplemented, and 0.5L of the reducing sugar is supplemented every time until the fermentation is finished. The fermentation culture time is 252 hours, 5.0L of glucose is supplemented, the sampling detection shows that the content of sirolimus in the fermentation liquid is 1893.2mg/L, and the content of 29-O-demethylsirolimus is 5.61%.
Example 3
The slant culture medium is 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
Primary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Secondary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Fermentation tank culture medium: 13g/L of corn starch, 10g/L of soybean cake powder, 6g/L of peptone, 5g/L of corn steep liquor dry powder, 10g/L of glycerol, 35g/L of glucose and 3g/L, K of piperidine-2-formic acid2HPO4 3g/L、KH2PO43g/L, MgSO 4.7H 2O 0.05.05 g/L, 3g/L calcium carbonate and 3g/L natural sodium silicate, and the pH value is 6.9; sterilizing at 126 deg.C for 60 min.
A supplemented medium:
glucose 100g/L, sterilizing the glucose solution at 121 deg.C for 30 min.
Weighing 90g of phospholipid, suspending in 3L of water, and sterilizing the phospholipid suspension solution at 121 ℃ for 30min for later use.
(1) Activating strains: inoculating the strain on a 30ml slant culture medium, culturing in a biochemical incubator at 28-30 ℃ for 6 days until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use;
(2) preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with 30ml of seed culture medium, and culturing for 48 hours at the rotating speed of a shaking table of 200r/m and the temperature of 28-30 ℃.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with 30ml of secondary seed culture medium according to the inoculation amount of 10%, wherein the rotating speed of a shaking table is 200r/m, the temperature is 28-30 ℃, and culturing is carried out for 24 h.
(4) Seed tank culture: according to the inoculation amount of 1 percentInoculating the secondary seed culture solution into a seed tank filled with the seed culture solution, culturing at the rotation speed of 80-100rpm for 24h at the culture temperature of 28-30 ℃ and the ventilation volume of 0.7-1.4 m3The pressure in the tank is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: preparing culture medium according to a certain proportion, inoculating seed culture solution into 50L fermentation tank containing 30L culture medium according to 10% inoculum size, culturing at 28-30 deg.C, maintaining tank pressure of 0.05-0.07 Mpa, and ventilation amount of 1.4-2.0 m3The rotation speed is 100rpm to 400rpm, the dissolved oxygen is controlled at 20 percent to 30 percent, the pH value is maintained at 6.0 to 6.4 in the fermentation process, phospholipids are respectively supplemented in the 72 th hour and the 120 th hour after the fermentation is started, and 1.5L of phospholipids are supplemented each time; monitoring the reducing sugar content of the fermentation system every 12h in the fermentation process, and supplementing a glucose solution when the reducing sugar content is lower than 1g/L, wherein 0.5L of the glucose solution is supplemented each time until the fermentation is finished. The fermentation culture time is 264 hours, 5.5L of glucose is supplemented, the sampling detection shows that the content of sirolimus in the fermentation liquid is 2223.5mg/L, and the content of 29-O-demethylsirolimus is 1.98%.
Example 4
The slant culture medium is 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
Primary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Secondary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Fermentation tank culture medium: 10g/L of corn starch, 15g/L of soybean cake powder, 5g/L of peptone, 10g/L of corn steep liquor dry powder, 15g/L of glycerol, 20g/L of glucose and 4g/L, K of piperidine-2-formic acid2HPO4 5g/L、KH2PO4 2g/L、MgSO4·7H20.06g/L of O, 5g/L of calcium carbonate and 5g/L of natural sodium silicate, and the pH value is 7.0; sterilizing at 128 deg.C for 40 min.
A supplemented medium:
glucose 100g/L, sterilizing the glucose solution at 121 deg.C for 30 min.
Weighing 90g of phospholipid, suspending in 3L of water, and sterilizing the phospholipid suspension solution at 121 ℃ for 30min for later use.
(1) Activating strains: inoculating the strain on a 30ml slant culture medium, culturing for 7 days in a biochemical incubator at 28-30 ℃, until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use;
(2) preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with 30ml of seed culture medium, and culturing for 44 hours at the rotating speed of a shaking table of 220r/m and the temperature of 28-30 ℃.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with 30ml of secondary seed culture medium according to the inoculation amount of 8%, wherein the rotating speed of a shaking table is 220r/m, the temperature is 28-30 ℃, and the culture is carried out for 30 h.
(4) Seed tank culture: inoculating the secondary seed culture solution into a seed tank filled with the seed culture solution according to the inoculation amount of 1%, rotating at 80-100rpm, culturing for 20h at the culture temperature of 28-30 ℃, and the ventilation volume of 0.7-1.4 m3The pressure in the tank is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: preparing culture medium according to a certain proportion, inoculating seed culture solution into 50L fermentation tank containing 30L culture medium according to 12% inoculum size, culturing at 28-30 deg.C, maintaining tank pressure of 0.05-0.07 Mpa, and ventilation amount of 1.4-2.0 m3The rotation speed is 100rpm to 400rpm, the dissolved oxygen is controlled at 20 percent to 30 percent, the pH value is maintained at 6.0 to 6.4 in the fermentation process, phospholipids are respectively supplemented in the 78 th hour and the 126 th hour after the fermentation is started, and 1.5L of phospholipids are supplemented each time; monitoring the reducing sugar content of the fermentation system every 12h in the fermentation process, and supplementing a glucose solution when the reducing sugar content is lower than 1g/L, wherein 1L is supplemented each time until the fermentation is finished. The fermentation culture time is 276h, 5L of glucose is supplemented, the sampling detection shows that the content of sirolimus in the fermentation liquid is 2168.5mg/L, and the content of 29-O-demethylsirolimus is 2.39%.
Example 5
Slant culture medium: 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
Primary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Secondary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Fermentation tank culture medium: 25g/L of corn starch, 5g/L of soybean cake powder, 10g/L of peptone, 5g/L of corn steep liquor dry powder, 5g/L of glycerol, 40g/L of glucose and 5g/L, K of piperidine-2-formic acid2HPO4 2g/L、KH2PO45g/L、MgSO4·7H20.02g/L of O, 5g/L of calcium carbonate and 2g/L of natural sodium benzoate, and the pH value is 6.5; sterilizing at 130 deg.C for 30 min.
A supplemented medium:
glucose 100g/L, sterilizing the glucose solution at 115 deg.C for 30 min.
Weighing 90g of phospholipid, suspending in 3L of water, and sterilizing the phospholipid suspension solution at 121 ℃ for 30min for later use.
(1) Activating strains: inoculating the strain on a 30ml slant culture medium, culturing in a biochemical incubator at 28-30 ℃ for 6 days until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use;
(2) preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with 30ml of seed culture medium, and culturing for 48 hours at the rotating speed of a shaking table of 200r/m and the temperature of 28-30 ℃.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with 30ml of secondary seed culture medium according to the inoculation amount of 12%, wherein the rotating speed of a shaking table is 200r/m, the temperature is 28-30 ℃, and culturing is carried out for 20 h.
(4) Seed tank culture: inoculating the secondary seed culture solution into a seed tank filled with the seed culture solution according to the inoculation amount of 3%, rotating at 80-100rpm, culturing for 16h at the culture temperature of 28-30 ℃, and the ventilation volume of 0.7-1.4 m3The pressure in the tank is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: preparing a culture medium according to a proportion, inoculating a seed culture solution into a 50L fermentation tank containing 30L of the culture medium according to an inoculation amount of 8%, wherein the culture temperature is 28-30 ℃, the tank pressure is maintained at 0.05-0.07 Mpa, and the ventilation volume is 1.4-2.0 m3The rotation speed is 100rpm to 400rpm, the dissolved oxygen is controlled at 20 percent to 30 percent, the pH value is maintained at 6.0 to 6.4 in the fermentation process, phospholipids are respectively supplemented in the 66 th hour and the 114 th hour after the fermentation is started, and 1.5L of phospholipids are supplemented each time; monitoring the reducing sugar content of the fermentation system every 12h in the fermentation process, and supplementing a glucose solution when the reducing sugar content is lower than 1g/L, wherein 0.5L of the glucose solution is supplemented each time until the fermentation is finished. The fermentation culture time is 264 hours, 5.5L of glucose is supplemented, the sampling detection shows that the content of sirolimus in the fermentation liquid is 2125.2mg/L, and the content of 29-O-demethylsirolimus is 2.46%.
Example 6
The slant culture medium is 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
Primary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Secondary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Fermentation tank culture medium: 13g/L of corn starch, 10g/L of soybean cake powder, 6g/L of peptone, 5g/L of corn steep liquor dry powder, 10g/L of glycerol, 35g/L of glucose and 3g/L, K of piperidine-2-formic acid2HPO4 3g/L、KH2PO43g/L、MgSO4·7H20.05g/L of O, 3g/L of calcium carbonate and 3g/L of natural sodium benzoate, and the pH value is 6.9; sterilizing at 125 deg.C for 60 min.
A supplemented medium:
glucose 100g/L, sterilizing the glucose solution at 121 deg.C for 30 min.
Weighing 90g of phospholipid, suspending in 3L of water, and sterilizing the phospholipid suspension solution at 121 ℃ for 30min for later use.
(1) Activating strains: inoculating the strain on a 30ml slant culture medium, culturing in a biochemical incubator at 28-30 ℃ for 6 days until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use;
(2) preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with 30ml of seed culture medium, and culturing for 48 hours at the rotating speed of a shaking table of 200r/m and the temperature of 28-30 ℃.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with 30ml of secondary seed culture medium according to the inoculation amount of 10%, wherein the rotating speed of a shaking table is 200r/m, the temperature is 28-30 ℃, and culturing is carried out for 24 h.
(4) Seed tank culture: inoculating the secondary seed culture solution into a seed tank filled with the seed culture solution according to the inoculation amount of 1%, rotating at 80-100rpm, culturing for 24h at the culture temperature of 28-30 ℃, and the ventilation volume of 0.7-1.4 m3The pressure in the tank is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: preparing culture medium according to a certain proportion, inoculating seed culture solution into 50L fermentation tank containing 30L culture medium according to 10% inoculum size, culturing at 28-30 deg.C, maintaining tank pressure of 0.05-0.07 Mpa, and ventilation amount of 1.4-2.0 m3The rotation speed is 100rpm to 400rpm, the dissolved oxygen is controlled at 20 percent to 30 percent, the pH value is maintained at 6.0 to 6.4 in the fermentation process, phospholipid is supplemented in 96h after the fermentation is started, and 3L is supplemented; monitoring the reducing sugar content of the fermentation system every 12h in the fermentation process, and supplementing a glucose solution when the reducing sugar content is lower than 1g/L, wherein 0.6L of the glucose solution is supplemented each time until the fermentation is finished. The fermentation culture time is 260h, 4.8L of glucose is supplemented, the sampling detection shows that the content of sirolimus in the fermentation liquid is 2078.6mg/L, and the content of 29-O-demethylsirolimus is 3.07%.
Example 7
The slant culture medium is 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
Primary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Secondary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Fermentation tank culture medium: 13g/L of corn starch, 10g/L of soybean cake powder, 6g/L of peptone, 5g/L of corn steep liquor dry powder, 10g/L of glycerol, 35g/L of glucose and 3g/L, K of piperidine-2-formic acid2HPO4 3g/L、KH2PO43g/L, MgSO 4.7H 2O 0.05.05 g/L, 3g/L calcium carbonate and 3g/L natural sodium silicate, and the pH value is 6.9; sterilizing at 126 deg.C for 60 min.
A supplemented medium:
glucose 100g/L, sterilizing the glucose solution at 121 deg.C for 30 min.
Weighing 30g of phospholipid, suspending in 3L of water, and sterilizing the phospholipid suspension solution at 121 ℃ for 30min for later use.
(1) Activating strains: inoculating the strain on a 30ml slant culture medium, culturing in a biochemical incubator at 28-30 ℃ for 6 days until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use;
(2) preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with 30ml of seed culture medium, and culturing for 48 hours at the rotating speed of a shaking table of 200r/m and the temperature of 28-30 ℃.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with 30ml of secondary seed culture medium according to the inoculation amount of 10%, wherein the rotating speed of a shaking table is 200r/m, the temperature is 28-30 ℃, and culturing is carried out for 24 h.
(4) Seed tank culture: inoculating the secondary seed culture solution into a seed tank filled with the seed culture solution according to the inoculation amount of 1%, rotating at 80-100rpm, culturing for 24h at the culture temperature of 28-30 ℃, and the ventilation volume of 0.7-1.4 m3The pressure in the tank is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: preparing culture medium according to a certain proportion, inoculating seed culture solution into 50L fermentation tank containing 30L culture medium according to 10% inoculum size, culturing at 28-30 deg.C, maintaining tank pressure of 0.05-0.07 Mpa, and ventilatingThe amount is 1.4-2.0 m3The rotation speed is 100rpm to 400rpm, the dissolved oxygen is controlled at 20 percent to 30 percent, the pH value is maintained at 6.0 to 6.4 in the fermentation process, phospholipids are respectively supplemented in the 72 th hour and the 120 th hour after the fermentation is started, and 1.5L of phospholipids are supplemented each time; monitoring the reducing sugar content of the fermentation system every 12h in the fermentation process, and supplementing a glucose solution when the reducing sugar content is lower than 1g/L, wherein 0.7L of the glucose solution is supplemented each time until the fermentation is finished. The fermentation culture time is 264 hours, 5.6L of glucose is supplemented, the sampling detection shows that the content of sirolimus in the fermentation liquid is 1963.8mg/L, and the content of 29-O-demethylsirolimus is 3.82%.
Example 8
The slant culture medium is 20g/L of soluble starch, 5g/L of yeast extract, 10g/L of glucose, 1g/L of calcium carbonate and 16g/L of agar powder; pH 7.0.
Primary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Secondary seed culture medium: 40g/L of soluble starch, 10g/L of glucose, 5g/L of corn steep liquor dry powder, 5g/L of peptone, 10g/L of yeast extract, 10g/L of peanut meal, 10g/L of glycerol and 2g/L of calcium carbonate, and the pH value is 7.0.
Fermentation tank culture medium: 13g/L of corn starch, 10g/L of soybean cake powder, 6g/L of peptone, 5g/L of corn steep liquor dry powder, 10g/L of glycerol, 50g/L of glucose and 3g/L, K of piperidine-2-formic acid2HPO4 3g/L、KH2PO43g/L, MgSO 4.7H 2O 0.05.05 g/L, 3g/L calcium carbonate and 3g/L natural sodium silicate, and the pH value is 6.5; sterilizing at 126 deg.C for 60 min.
A supplemented medium:
glucose 100g/L, sterilizing the glucose solution at 121 deg.C for 30 min.
Weighing 150g of phospholipid, suspending in 3L of water, and sterilizing the phospholipid suspension solution at 121 ℃ for 30min for later use.
(1) Activating strains: inoculating the strain on a 30ml slant culture medium, culturing in a biochemical incubator at 28-30 ℃ for 6 days until the slant is completely covered by spores, the color is reddish brown, and the shape is full for later use;
(2) preparing a first-level seed solution: and (3) taking a ring of the cultured sirolimus producing strain from the inclined plane by using an inoculation shovel, inoculating the ring of the sirolimus producing strain into a conical flask filled with 30ml of seed culture medium, and culturing for 48 hours at the rotating speed of a shaking table of 200r/m and the temperature of 28-30 ℃.
(3) Preparing a secondary seed liquid: inoculating the primary seed culture solution into a conical flask filled with 30ml of secondary seed culture medium according to the inoculation amount of 10%, wherein the rotating speed of a shaking table is 200r/m, the temperature is 28-30 ℃, and culturing is carried out for 24 h.
(4) Seed tank culture: inoculating the secondary seed culture solution into a seed tank filled with the seed culture solution according to the inoculation amount of 1%, rotating at 80-100rpm, culturing for 24h at the culture temperature of 28-30 ℃, and the ventilation volume of 0.7-1.4 m3The pressure in the tank is 0.05-0.1 MPa.
(5) Culturing in a fermentation tank: preparing culture medium according to a certain proportion, inoculating seed culture solution into 50L fermentation tank containing 30L culture medium according to 10% inoculum size, culturing at 28-30 deg.C, maintaining tank pressure of 0.05-0.07 Mpa, and ventilation amount of 1.4-2.0 m3The rotation speed is 100rpm to 400rpm, the dissolved oxygen is controlled at 20 percent to 30 percent, the pH value is maintained at 6.0 to 6.4 in the fermentation process, phospholipids are respectively supplemented in the 72 th hour and the 120 th hour after the fermentation is started, and 1.5L of phospholipids are supplemented each time; monitoring the reducing sugar content of the fermentation system every 12h in the fermentation process, and supplementing a glucose solution when the reducing sugar content is lower than 1g/L, wherein 0.5L of the glucose solution is supplemented each time until the fermentation is finished. The fermentation culture time is 240 hours, 5.0L of glucose is supplemented, the sampling detection shows that the content of sirolimus in the fermentation liquid is 1907.6mg/L, and the content of 29-O-demethylsirolimus is 2.23%.
Claims (10)
1. A fermentation method of sirolimus comprises inoculating a seed solution into a fermentation tank filled with a fermentation medium for culture to obtain a fermentation solution of high-yield sirolimus; it is characterized in that the fermentation medium is subjected to high-intensity sterilization treatment.
2. The fermentation method according to claim 1, wherein the high-intensity sterilization treatment is sterilization of the fermentation medium at 125 to 130 ℃.
3. The fermentation method according to claim 2, wherein the sterilization time is 30 to 60 min.
4. A fermentation process according to any one of claims 1 to 3, wherein glucose is fed during the fermentation.
5. The fermentation method according to claim 4, wherein the ratio of the mass of the supplemented glucose to the initial volume of the fermentation system is 10-20 g/L; wherein the mass of the glucose is calculated by g, and the initial volume of the fermentation system is calculated by L.
6. The fermentation process of claim 4, wherein glucose is supplemented when the reducing sugars are less than 1g/L during the fermentation.
7. The fermentation method according to claim 4, wherein the glucose is sterilized at 115 to 121 ℃ for 20 to 30 min.
8. A fermentation process according to any one of claims 1 to 3, wherein phospholipids are additionally fed during the fermentation.
9. The fermentation method according to claim 8, wherein the ratio of the mass of the supplemented phospholipids to the volume of the fermentation broth is 1-3 g/L, wherein the mass of the phospholipids is in g and the volume of the fermentation broth is in L.
10. Fermentation process according to claim 8, wherein the phospholipids are supplemented at the early and/or middle stage of the fermentation, preferably at 66 to 78 hours and 114 to 126 hours after the start of the fermentation.
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