A culture medium for fermenting Didennin B and its preparation method

Abstract

This invention belongs to the field of microbial fermentation and discloses a culture medium for preparing Didemnin B using *Tistrella mobilis* and its preparation method, which can be used for industrial-scale production of Didemnin B. Specifically, this invention discloses a culture medium for fermenting Didemnin B. By optimizing the formulation and composition of the culture medium and adding L-isoleucine, the potency level in a 50L fermenter can be increased to 332 mg / L. Through further optimization of the preparation process, staged control of dissolved oxygen, and development of a fed-batch process, the fermentation level in a 50L fermenter can be increased to 544 mg / L. In industrial-scale production with a 4-ton fermenter, the fermentation level can reach 489 mg / L. This method is expected to overcome the current situation of low Didemnin B potency and provide methodological support for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, specifically relating to a culture medium for fermenting Didemnin B and its preparation method. Background Technology

[0002] Didemnins are a class of natural products derived from marine microorganisms, first discovered in Mediterranean sea squirts (Aplidium albicans), possessing complex cyclic peptide structures. Currently, more than 20 structures of didemnins have been identified, including didemnin B, dehydrodidemnin B, nordidemnin B, and didemnin X / Y. They are produced by bacteria such as *Tistrella mobilis* and *Tistrella bauzanensis*, belonging to the phylum Alpha-Proteobacteria, and possess antitumor, antiviral, and immunosuppressive activities. Didemnin B was the first marine natural product to enter clinical trials. It exerts its antitumor effect by inhibiting the replication of RNA and DNA in tumor cells and the synthesis of proteins in tumor cells, thus showing good therapeutic effects against malignant tumors such as gastric cancer, esophageal cancer, and colon cancer. However, didemnin B has significant toxic side effects, thus its clinical use is limited. In recent years, to increase the utilization rate of didemnin B, its derivative, dehydrodidemnin B, has been modified clinically, exhibiting significantly greater anticancer activity and lower toxicity than didemnin B.

[0003] JP2012240974A reported a motile strain of Citrella mobilis YIT12409 (FERMAP~22080) that was capable of producing Didemnin B with a calculated potency of approximately 20 mg / L.

[0004] US9644005B2 provides a novel *Tistrella mobilis* strain with accession number NRRL B-50531, which can yield more than 10 mg / L of didemnin B and nordidemnin B from fermentation of *Tistrella mobilis* using a simple culture medium.

[0005] The literature *Bacterial Production of the Tunicate-Derived Antitumor Cyclic Depsipeptide Didemnin B* reports a didemnin B potency of approximately 3.2 mg / L produced by α-proteobacterium Tistrella mobilis; the literature *Bacterial Biosynthesis and Maturation of the Didemnin Anti-cancer Agents* also reports obtaining 0.2 mg / L of didemnin B through fermentation of the microorganism *T. mobilis* KA081020-065, but the yields are both low.

[0006] In summary, the existing technology for obtaining Didemnin B through fermentation suffers from low fermentation yield and high cost, making it unsuitable for industrial production. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a culture medium for fermenting Didennin B and its preparation method, which can be used for the industrial production of Didennin B.

[0008] On the one hand, the present invention provides a culture medium for fermenting Didemnin B, characterized in that the culture medium contains L-isoleucine, wherein the content of L-isoleucine is not less than 0.05%, preferably 0.05-0.3%, and more preferably 0.15%.

[0009] Specifically, the culture medium contains a carbon source selected from one or any combination of glycerol, dextrin, starch, glucose, lactose, sucrose, maltose, mannitol, and sorbitol, preferably glycerol, dextrin, starch, lactose, or any combination thereof.

[0010] The culture medium contains a nitrogen source, which is selected from yeast nitrogen source, peptone, casein, soy protein isolate, soy protein concentrate, pea protein, cottonseed meal, or any combination thereof; preferably yeast nitrogen source, peptone, cottonseed meal, or any combination thereof.

[0011] The culture medium also contains inorganic salts selected from sea salts, hydrochlorides, sulfates, nitrates, citrates, or sodium, magnesium, cobalt, zinc, ferrous, manganese, copper, molybdate ions, or any combination thereof; preferably hydrochlorides, citrates, sulfates, or sodium, magnesium, cobalt, zinc ions, or any combination thereof; more preferably sodium chloride, sodium citrate, magnesium sulfate, zinc sulfate, cobalt chloride, or any combination thereof.

[0012] Preferably, the culture medium comprises or is composed of: 0-2% corn starch, 0-4% maltodextrin, 0-4% glycerol, 0-3% yeast powder, 0-4% cottonseed meal, 0.05-0.3% L-isoleucine, 0-3% sodium chloride, 0-0.3% magnesium sulfate heptahydrate, 0-0.2% sodium citrate, 0-0.0002% cobalt chloride, 0-0.0004% zinc sulfate, and 0.05% defoamer.

[0013] More preferably, the culture medium consists of 1% corn starch, 1.5% maltodextrin, 2% glycerol, 1% yeast powder, 2% cottonseed meal, 0.15% L-isoleucine, 1.8% sodium chloride, 0.2% magnesium sulfate heptahydrate, 0.2% sodium citrate, 0.0002% cobalt chloride, 0.0004% zinc sulfate, and 0.05% defoamer.

[0014] On the other hand, the present invention provides a method for preparing Didemnin B by fermentation, characterized in that the fermentation uses the culture medium described above, and during the fermentation culture, dissolved oxygen is controlled in three stages, and / or ammonia water and maltodextrin are added.

[0015] Specifically, during the 0-24h fermentation stage, the dissolved oxygen level is controlled to be no less than 30%; during the 24-48h fermentation stage, the dissolved oxygen level is controlled to be 15-30%; and during the 48h fermentation stage until before discharge, the dissolved oxygen level is controlled to be 5-20%.

[0016] Specifically, after 24 hours of fermentation, carbon and nitrogen sources are added, preferably maltodextrin and ammonia. More preferably, the daily ammonia volume is 0.05% to 0.4% of the fermentation broth volume, until fermentation ends. The total sugar concentration is controlled as follows: from the start of feeding to about 96 hours, the total sugar concentration is controlled at 1% to 2%; from about 96 hours to before unloading, the total sugar concentration is controlled at 0.5% to 1.5%.

[0017] Specifically, the preparation method includes inoculating the seed liquid into an optimized fermenter at a ratio of 10%, culturing the fermentation broth at 28–32°C, controlling the pH during the fermentation stage at 6.5–8.0, and maintaining a tank pressure of 0.05 MPa.

[0018] Specifically, the strain used for fermentation is Tistrella mobilis or its mutagenized strain.

[0019] Preferably, the present invention provides a method for preparing Didemnin B by fermentation using *Tistrella mobilis* or its mutagenic strain, characterized in that the fermentation medium comprises 1% corn starch, 1.5% maltodextrin, 2% glycerol, 1% yeast powder, 2% cottonseed meal, 0.15% L-isoleucine, 1.8% sodium chloride, 0.2% magnesium sulfate heptahydrate, 0.2% sodium citrate, 0.0002% cobalt chloride, 0.0004% zinc sulfate, and 0.05% defoamer; the preparation method includes, after 24 hours of fermentation, adding ammonia water, with the daily ammonia water volume being 0.05% to 0.4% of the fermentation medium volume, until the end of fermentation; further comprising, inoculating the seed liquid into the fermenter, controlling the dissolved oxygen level to be not lower than 30% during the 0-24 hours of fermentation, controlling the dissolved oxygen level to be 15-30% during the 24-48 hours, and controlling the dissolved oxygen level to be 5-20% from 48 hours until discharge from the fermenter. Throughout the fermentation process, the fermentation temperature was controlled at 28–32℃, the pH during the fermentation stage was controlled at 6.5–8.0, and the tank pressure was 0.05 MPa.

[0020] The present invention also provides a method for preparing Dehydrodidemnin B, characterized in that the preparation method includes the above-described method for preparing Didennin B, and then Dehydrodidemnin B is prepared from Didennin B.

[0021] Beneficial effects:

[0022] This invention discloses a culture medium for preparing Didemnin B using *Tistrella mobilis* fermentation and its preparation method. Specifically, this invention discloses a culture medium for fermenting Didemnin B. By optimizing the culture medium's formulation and composition and adding L-isoleucine, a concentration of 332 mg / L can be achieved in a 50L fermenter. Further optimization of the fermentation method, controlling dissolved oxygen levels and feeding processes during fermentation, can increase the fermentation concentration to 544 mg / L. For industrial-scale production on a 4-ton tank scale, a concentration of 489 mg / L can be achieved. This invention overcomes the previous low Didemnin B potency, stably and significantly improving its potency, providing methodological support for industrial-scale production. Detailed Implementation

[0023] The present invention will be described in detail below with reference to specific embodiments, but the scope of the present invention is not limited thereto.

[0024] In the following specific embodiments, unless otherwise specified, the reagents and instruments used are commonly used in the art and can be obtained commercially; the methods used are conventional methods in the art, and those skilled in the art can know how to implement the methods and obtain the corresponding results based on the content of the embodiments.

[0025] The following examples use the tunicaebenone B strain with accession number CGMCC NO.22003 as an illustration. This strain is deposited at the China General Microbiological Culture Collection Center and disclosed in CN113174344A. The seed preparation process before fermentation culture is the same in all the following examples, as follows:

[0026] 1. Preparation of the inclined plane:

[0027] Take the glycerol tube containing the working culture of the isolated and purified CGMCC NO.22003 strain, thaw it, and then serially dilute it to 10⁻⁶ with sterile physiological saline (diluent). ～2 Take 0.1 mL of the diluted solution and inoculate it onto the modified slant culture medium, spread it evenly, and incubate it in the dark at 28℃ and 70% relative humidity for 3 days to obtain slant bacterial growth.

[0028] 2. Primary Seed Preparation:

[0029] Take a mature slant culture, add 10 mL of sterile physiological saline, scrape off the bacterial growth, break it up, and obtain a bacterial suspension. Take 10 mL of the bacterial suspension and inoculate it into a 500 mL primary shake flask containing seed culture medium. After sealing, place it on a shaker at 30℃ and 220 rpm for 2 days.

[0030] The primary shake flask seed culture medium consists of: 1.2% lactose, 0.4% glucose, 0.6% yeast extract, 1% peptone, and 2% sodium chloride. The pH of the culture medium is adjusted to 7.0 before sterilization, and the sterilization conditions are 121–123°C for 30 min.

[0031] 3. Secondary seed preparation:

[0032] After the primary seed culture matures, take 50 mL of seed culture solution and inoculate it into a seed tank containing 10 L of secondary seed culture medium. The culture temperature is 28–30 °C, dissolved oxygen is controlled at ≥30%, and the culture period is 2 days.

[0033] The secondary seed culture medium consists of: 2% glucose, 1% yeast extract, 0.5% peptone, 2% sodium chloride, and 0.05% antifoaming agent. The pH of the culture medium is adjusted to 7.0 before sterilization, and the sterilization conditions are 121-123℃ for 30 minutes.

[0034] 4. Fermentation culture

[0035] The secondary seed culture was inoculated into the optimized 50L (or industrial scale 4T) fermentation medium in a fermenter at a ratio of 10%, and fermentation was carried out under the conditions of the different examples below. The potency level of Didemnin B in the fermentation broth was determined.

[0036] Before the determination, the sample processing procedure was as follows: After the fermentation was completed, 1 mL of fermentation broth was taken, 3 mL of anhydrous ethanol was added, the mixture was mixed, and the mixture was sonicated for 2 h. After mixing again, the mixture was centrifuged at 14000 rpm for 10 min, filtered through a 0.45 μm filter membrane, and then analyzed by liquid chromatography.

[0037] Sample liquid chromatography analysis methods:

[0038] Chromatographic column: C18, 50 × 4.6 mm, 1.8 μm

[0039] Wavelength: 210nm

[0040] Column temperature: 40℃

[0041] Flow rate: 1 mL / min

[0042] Injection volume: 5 μL

[0043] Run time: 5 minutes

[0044] Mobile phase: 59% acetonitrile.

[0045] Furthermore, in this invention, the content of all culture medium components is calculated based on the volume ratio of the total fermentation broth unless otherwise specified. For example, 1.2% lactose means that there is 1.2g of lactose in every 100mL of fermentation broth.

[0046] The specific implementation method is as follows:

[0047] Example 1.

[0048] The fermentation formula is as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0049] Adjust the pH to 7.0 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.

[0050] The secondary seed culture was inoculated into the fermentation medium in a 50L fermenter at a ratio of 10%. The fermentation temperature was 28-32℃, the initial stirring was 200rpm, the initial air flow rate was 0.5vvm, the tank pressure was 0.05MPa, the dissolved oxygen was controlled at 5%-15%, and the pH was controlled between 6.5 and 8.0 during the process. The fermentation culture was completed in 5-7 days.

[0051] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the Didennin B content in the fermentation broth obtained under this implementation scheme was confirmed to be 242 mg / L.

[0052] Example 2.

[0053] The fermentation formula is as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, L-lysine 0.1%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0054] Adjust the pH to 7.0 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.

[0055] The secondary seed culture was inoculated into the optimized 50L fermenter fermentation medium at a ratio of 10%. The fermentation temperature was 28-32℃, the initial stirring was 200 rpm, the initial air flow rate was 0.5 vvm, the tank pressure was 0.05 MPa, the dissolved oxygen level was controlled at 5%-15%, and the pH was controlled at 6.5-8.0 during the process. The fermentation culture lasted for 5-7 days.

[0056] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the Didemnin B content in the fermentation broth obtained under this implementation scheme was confirmed to be 251 mg / L.

[0057] Example 3.

[0058] The fermentation formula is as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, L-isoleucine 0.05%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0059] Adjust the pH to 7.0 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.

[0060] The secondary seed culture was inoculated into the optimized 50L fermenter fermentation medium at a ratio of 10%. The fermentation temperature was 28-32℃, the initial stirring was 200 rpm, the initial air flow rate was 0.5 vvm, the tank pressure was 0.05 MPa, the dissolved oxygen was controlled at 5-15%, and the pH was controlled at 6.5-8.0 during the process. The fermentation culture lasted for 5-7 days.

[0061] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the Didennin B content in the fermentation broth obtained under this implementation scheme was confirmed to be 322 mg / L.

[0062] Example 4.

[0063] The fermentation formula is as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, L-isoleucine 0.3%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0064] Adjust the pH to 7.0 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.

[0065] The secondary seed culture was inoculated into the optimized 50L fermenter fermentation medium at a ratio of 10%. The fermentation temperature was 28-32℃, the initial stirring was 200 rpm, the initial air flow rate was 0.5 vvm, the tank pressure was 0.05 MPa, the dissolved oxygen level was controlled at 5-15%, and the pH was controlled at 6.5-8.0 during the process. The fermentation culture was completed in 5-7 days.

[0066] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the Didennin B content in the fermentation broth obtained under this implementation scheme was confirmed to be 327 mg / L.

[0067] Example 5.

[0068] The fermentation formula is as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, L-isoleucine 0.15%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0069] The pH of the culture medium was adjusted to 6.8 before sterilization, and the sterilization conditions were 121-123℃ for 30 minutes.

[0070] The secondary seed culture was inoculated into the optimized 50L fermenter fermentation medium at a ratio of 10%. The fermentation temperature was 28-32℃, the initial stirring was 200 rpm, the initial air flow rate was 0.5 vvm, the tank pressure was 0.05 MPa, and the dissolved oxygen control strategy was as follows. During the process, the pH was controlled at 6.5-8.0, and the fermentation culture was completed in 5-7 days.

[0071] Dissolved oxygen control strategy:

[0072] After transplanting, dissolved oxygen was corrected to 100%. During fermentation, dissolved oxygen levels were controlled within the following ranges by adjusting air flow, stirring speed, and tank pressure:

[0073] ① Constant range: Dissolved oxygen level is controlled at ≥30% throughout the process;

[0074] ② Constant range: Oxygen levels are controlled between 5% and 15% throughout the entire process;

[0075] ③ Staged control: During fermentation from 0 to 24 hours, the dissolved oxygen level should be controlled at no less than 30%; during fermentation from 24 to 48 hours, the dissolved oxygen level should be controlled at 15% to 30%; from 48 hours of fermentation until before being discharged from the tank, the dissolved oxygen level should be controlled at 5% to 20%.

[0076] The experimental conditions and results are as follows:

[0077] Serial Number Dissolved oxygen control scheme Potency of canning (mg / L) Can volume L 1 ① 301 35.5 2 ② 332 35.6 3 ③ 428 35.7

[0078] The comparison shows that the fermentation broth obtained by the three-stage oxygen control scheme in Experiment ③ had the highest Didennin B content, which was 428 mg / L.

[0079] Example 6.

[0080] The fermentation formula is as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, L-isoleucine 0.15%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0081] The pH of the culture medium was adjusted to 6.8 before sterilization, and the sterilization conditions were 121-123℃ for 30 minutes.

[0082] The secondary seed culture was inoculated into the optimized 50L fermenter culture medium at a ratio of 10%. The fermentation temperature was 28–32℃, the initial stirring speed was 200 rpm, the initial air flow rate was 0.5 vvm, the tank pressure was 0.05 MPa, the dissolved oxygen was controlled at 5–15%, and the pH was controlled at 6.5–8.0 during the process.

[0083] After 24 hours of fermentation, fed-batch feeding was started, with maltodextrin added to control the total sugar concentration as follows: from the start of feeding to about 96 hours, the total sugar concentration was controlled at 1-2%; from about 96 hours to before unloading, the total sugar concentration was controlled at 0.5-1.5%. Ammonia water was added, with the daily feeding amount being 0.05-0.4% of the formula volume, and the dissolved ammonium value was controlled at 200-400 ppm. Fermentation was completed in 5-7 days.

[0084] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the Didemnin B content in the fermentation broth obtained under this implementation scheme was confirmed to be 477 mg / L.

[0085] Example 7.

[0086] The fermentation formula is as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, L-isoleucine 0.15%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0087] The pH of the culture medium was adjusted to 6.8 before sterilization, and the sterilization conditions were 121-123℃ for 30 minutes.

[0088] The secondary seed culture was inoculated into the optimized 50L fermenter culture medium at a ratio of 10%. The fermentation temperature was 28–32℃, the initial stirring speed was 200 rpm, the initial air flow rate was 0.5 vvm, the tank pressure was 0.05 MPa, the dissolved oxygen was controlled at 5–15%, and the pH was controlled at 6.5–8.0 during the process.

[0089] After 24 hours of fermentation, fed-batch feeding was started. The total sugar concentration of maltodextrin was controlled as follows: 1-2% from the start of feeding to about 96 hours; 0.5-1.5% from about 96 hours to before discharge. Ammonia was added: the daily feeding amount was 0.05-0.4% of the formula volume, and the dissolved ammonium value was controlled at 400-600 ppm. Fermentation was completed in 5-7 days.

[0090] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the Didennin B content in the fermentation broth obtained under this implementation scheme was confirmed to be 468 mg / L.

[0091] Example 8.

[0092] The fermentation formula is as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, L-isoleucine 0.15%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0093] The pH of the culture medium was adjusted to 6.8 before sterilization, and the sterilization conditions were 121-123℃ for 30 minutes.

[0094] The secondary seed culture was inoculated into the optimized 50L fermenter fermentation medium at a ratio of 10%. The fermentation temperature was controlled at 28-32℃, the initial stirring was 200 rpm, the initial air flow rate was 0.5 vvm, the tank pressure was 0.05 MPa, and the dissolved oxygen and feeding were controlled as follows.

[0095] (1) Staged dissolved oxygen control:

[0096] After transplanting, the dissolved oxygen level is adjusted to 100%. During fermentation, the dissolved oxygen level is controlled within the following ranges by adjusting the air flow rate, stirring speed, and tank pressure: 0-24h fermentation, not less than 30%; 24-48h fermentation, 15-30%; 48h fermentation until before being removed from the tank, 5-20%.

[0097] (2) Material replenishment control:

[0098] After 24 hours of fermentation, fed-batch feeding was started. The total sugar concentration of maltodextrin was controlled as follows: 1-2% from the start of feeding to about 96 hours; 0.5-1.5% from about 96 hours to before discharge. Ammonia was added: the daily feeding amount was 0.05-0.4% of the formula volume, and the dissolved ammonium value was controlled at 200-600 ppm. Fermentation was completed in 5-7 days.

[0099] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the Didemnin B content in the fermentation broth obtained under this implementation scheme was confirmed to be 544 mg / L.

[0100] Example 9.

[0101] Scale-up of fermentation process in 4T tanks

[0102] The secondary seed culture was inoculated into the fermentation medium of a 4T fermenter at a ratio of 10%. The optimized fermentation medium composition was as follows: corn starch 1%, maltodextrin 1.5%, glycerol 2%, yeast powder 1%, cottonseed meal powder 2%, L-isoleucine 0.15%, sodium chloride 1.8%, magnesium sulfate heptahydrate 0.2%, sodium citrate 0.2%, cobalt chloride 0.0002%, zinc sulfate 0.0004%, and defoamer 0.05%.

[0103] The pH of the culture medium was adjusted to 6.8 before sterilization, and the sterilization conditions were 121-123℃ for 30 minutes.

[0104] Fermentation temperature is controlled at 28–32℃, initial stirring is 200 rpm, initial air flow is 0.5 vvm, tank pressure is 0.05 MPa, and dissolved oxygen and feeding are controlled as follows.

[0105] (1) Staged dissolved oxygen control:

[0106] After transplanting, the dissolved oxygen level is adjusted to 100%. During fermentation, the dissolved oxygen level is controlled within the following ranges by adjusting the air flow rate, stirring speed, and tank pressure: 0-24h fermentation, not less than 30%; 24-48h fermentation, 15-30%; 48h fermentation until before being removed from the tank, 5-20%.

[0107] (2) Material replenishment control:

[0108] After 24 hours of fermentation, fed-batch feeding was started. The total sugar concentration of maltodextrin was controlled as follows: 1-2% from the start of feeding to about 96 hours; 0.5-1.5% from about 96 hours to before discharge. Ammonia was added: the daily feeding amount was 0.05-0.4% of the formula volume, and the dissolved ammonium value was controlled at 200-600 ppm. Fermentation was completed in 5-7 days.

[0109] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the Didemnin B content in the fermentation broth obtained under this implementation scheme was confirmed to be 489 mg / L.

[0110] At an industrial scale of 4T, three batches of trials were repeated. The potency of the other two batches was 477mg / L and 485mg / L, respectively. The process showed good stability, indicating that the process is suitable for stable industrial production.

[0111] Although the present invention has described the above embodiments, this does not limit the scope of patent protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the innovative concept of the present invention, or any equivalent structural or procedural transformations made using the content of the present invention specification, that directly or indirectly apply the above technical solutions to other related technical fields, are all included within the scope of patent protection of the present invention.

Claims

1. A fermentation medium for the fermentation preparation of Didennin B, characterized in that... The culture medium contains L-isoleucine, and the content of said L-isoleucine is not less than 0.05%; the culture medium is used to culture *Teslina motilityis* (…). Tistrella mobileis The sample is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCCNO.22003.

2. The culture medium as described in claim 1, characterized in that... The content of L-isoleucine is 0.05% to 0.3%.

3. The culture medium as described in claim 2, characterized in that... The content of L-isoleucine is 0.15%.

4. The culture medium as described in claim 1, characterized in that... The culture medium also contains a carbon source, a nitrogen source, and inorganic salts; the carbon source is selected from one or any combination of glycerol, dextrin, starch, glucose, lactose, sucrose, maltose, mannitol, and sorbitol; The nitrogen source is selected from one or any combination of yeast nitrogen source, peptone, casein, soy protein isolate, soy protein concentrate, pea protein, and cottonseed meal. The inorganic salt is selected from one or any combination of sea salt, hydrochloride, sulfate, nitrate, and citrate.

5. The culture medium as described in claim 4, characterized in that... The carbon source is selected from one or any combination of glycerol, dextrin, starch, and lactose; The nitrogen source is selected from one or any combination of yeast-based nitrogen sources, peptone, and cottonseed meal. The inorganic salt is selected from one or any combination of hydrochloride, citrate, and sulfate.

6. The culture medium as described in claim 5, characterized in that... The inorganic salt is selected from one or any combination of sodium chloride, sodium citrate, magnesium sulfate, zinc sulfate, and cobalt chloride.

7. The culture medium as described in claim 1, characterized in that, The culture medium comprises: 1-2% corn starch, 1.5-4% maltodextrin, 2-4% glycerol, 1-3% yeast powder, 2-4% cottonseed meal, 0.05-0.3% L-isoleucine, 1.8-3% sodium chloride, 0.2-0.3% magnesium sulfate heptahydrate, 0.2-0.2% sodium citrate, 0.0002% cobalt chloride, 0.0004% zinc sulfate, and 0.05% defoamer.

8. The culture medium as described in claim 1, characterized in that... The culture medium consists of 1% corn starch, 1.5% maltodextrin, 2% glycerol, 1% yeast powder, 2% cottonseed meal, 0.15% L-isoleucine, 1.8% sodium chloride, 0.2% magnesium sulfate heptahydrate, 0.2% sodium citrate, 0.0002% cobalt chloride, 0.0004% zinc sulfate, and 0.05% defoamer.

9. A method for preparing Didennin B by fermentation, characterized in that, The fermentation used the culture medium described in any one of claims 1 to 8, and the strain was *Teslinium motilityum* (…). Tistrella mobilis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.22003.

10. The preparation method according to claim 9, characterized in that, Dissolved oxygen is controlled in three stages during fermentation.

11. The preparation method according to claim 9, characterized in that, During the fermentation stage, from 0 to 24 hours, the dissolved oxygen level should be controlled at no less than 30%; from 24 to 48 hours, the dissolved oxygen level should be controlled at 15 to 30%; and from 48 hours to before discharge, the dissolved oxygen level should be controlled at 5 to 20%.

12. The preparation method according to claim 9, characterized in that... After 24 hours of fermentation, carbon and nitrogen sources were added.

13. The preparation method according to claim 9, characterized in that... After 24 hours of fermentation, maltodextrin and ammonia were added.

14. The preparation method according to claim 13, characterized in that... The daily ammonia water replenishment volume is 0.05%~0.4% of the fermentation broth culture medium volume, until the fermentation is completed.

15. The preparation method according to claim 13, characterized in that... The total sugar concentration should be controlled as follows when adding maltodextrin: from the start of feeding to 96 hours, the total sugar concentration should be controlled at 1-2%; from 96 hours to before unloading, the total sugar concentration should be controlled at 0.5-1.5%.

16. The preparation method according to any one of claims 9 to 15, characterized in that, The preparation method includes controlling the fermentation temperature at 28~32℃ and controlling the pH during the fermentation culture stage at 6.5~8.

0.

17. A method for preparing Dehydrodidemnin B, characterized in that, The preparation method includes the method for preparing Didemnin B as described in any one of claims 9 to 16.