Fermentation medium for preparing rifamycin O and fermentation method
By using β-cyclodextrin fermentation medium and optimized fermentation control methods, the problems of multiple fermentation steps and low yield of rifamycin were solved, achieving efficient and low-cost preparation of rifamycin O and increasing the fermentation potency to 5541 mg/L.
Patent Information
- Application Number
- CN202510977503.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-10-31
AI Technical Summary
The existing rifamycin fermentation process involves many steps and has a low yield, resulting in high production costs and making it difficult to achieve efficient and low-cost industrial production.
Rifamycin O was prepared in a one-step process using a fermentation medium containing β-cyclodextrin and by controlling dissolved oxygen and supplementing with ammonia and glucose.
The fermentation potency of rifamycin O was increased from 471 mg/L to 5541 mg/L, simplifying the process steps, reducing production costs, and providing a foundation for industrial production.
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Figure CN120866441A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation, specifically relating to a culture medium and preparation method for fermenting rifamycin O. Background Technology
[0002] Rifamycin is an ansamycin class of antibiotics with antimycobacterial activity, produced by *Amycolatoposis mediterranei*, and widely used to treat tuberculosis, leprosy, and AIDS-related mycobacterial infections. Sensi et al. first reported the discovery of rifamycin in 1959. In 1962, rifamycin SV, a derivative of rifamycin B, was first used clinically. In 1963, Prelogde et al. determined the structure of rifamycin. In 1967, Hartmann et al. first discovered the mechanism of action of rifamycin—specific inhibition of bacterial RNA polymerase. Simultaneously, after years of collaboration, the Swiss company Ciba-Geigy and the Italian company Leptit successfully synthesized a new semi-synthetic oral rifamycin—rifampin—which has been widely used in the treatment of tuberculosis.
[0003] There are two main industrial-scale rifamycin fermentation processes: rifamycin B fermentation and rifamycin SV fermentation. Both processes are currently in production in China. Rifamycin B fermentation is monophase, meaning that the product is synthesized simultaneously with mycelial growth. The fermentation unit is relatively high; the fermentation unit of Ciba-Geigy in Switzerland is around 24,000 u / ml. However, the high viscosity of the fermentation broth hinders extraction, and the synthesis of rifampin involves multiple reaction steps and low yield. After purification and separation, rifamycin B is oxidized to rifamycin O. The purified rifamycin O then undergoes various reactions to yield rifamycin S and other rifamycin-related compounds. This involves various synthetic chemical reactions, and the multiple steps increase the production cost of the final product.
[0004] Reports on the direct synthesis of rifamycin O are currently very limited. This invention is based on a strain of bacteria obtained through screening and mutagenesis that can directly produce rifamycin O, and includes a culture medium and preparation method for rifamycin O. The fermentation-produced rifamycin O can achieve a potency of 5541 mg / L. The mild microbial fermentation conditions reduce process steps, providing a foundation for high-efficiency and low-cost large-scale production of rifamycin O. Summary of the Invention
[0005] The purpose of this invention is to address the problems of multiple reaction steps and low yield in the fermentation of rifamycin in the prior art, and to provide a fermentation medium for preparing rifamycin O, as well as a method for preparing rifamycin O in one step using the fermentation medium.
[0006] To achieve the above objectives, the present invention provides a method for preparing rifamycin O by fermentation using Amycolatoposismediterranei or its mutagenic strains.
[0007] In a first aspect, the present invention provides a fermentation medium for preparing rifamycin O, comprising β-cyclodextrin, wherein the content of said β-cyclodextrin is 2% to 8% (w / v), preferably 6% (w / v).
[0008] Specifically, the culture medium further includes a carbon source, a nitrogen source, and inorganic salts; the carbon source is selected from one or any combination of glucose, maltose, corn starch, maltodextrin, sucrose, and lactose, preferably glucose, maltose, maltodextrin, or any combination of these.
[0009] The nitrogen source is selected from soybean flour, cottonseed flour, yeast nitrogen source, peptone, polypeptide powder, casein, soy protein isolate, methyl oleate, butyl oleate, or any combination thereof; preferably yeast peptone, polypeptide powder, methyl oleate, or any combination thereof.
[0010] The inorganic salt is selected from sea salt, hydrochloride, sulfate, nitrate, citrate, or sodium, potassium, magnesium, cobalt, zinc, ferrous, manganese, copper, molybdate ions, or any combination thereof; preferably sea salt, phosphate, sulfate, nitrate, or sodium, potassium, magnesium, cobalt, zinc ions, or any combination thereof; more preferably sea salt, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium nitrate, or any combination thereof.
[0011] Preferably, the culture medium contains or is composed of: 0-12% glucose, 2-8% β-cyclodextrin, 0-4% maltose, 0-3% polypeptide powder, 0-4% yeast peptone, 0-2% methyl oleate, 0-0.5% sea salt, 0-0.5% magnesium sulfate heptahydrate, 0-0.5% potassium dihydrogen phosphate, 0-2% sodium nitrate, and 0.05-0.1% defoamer.
[0012] More preferably, the culture medium is composed of: 10% glucose, 6% β-cyclodextrin, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 0.5% sodium nitrate, and 0.05% defoamer.
[0013] Secondly, the present invention provides a method for preparing rifamycin O by fermentation, wherein the fermentation uses the culture medium described in the first aspect, and the strain used is Amycolatoposis mediterranei or its mutagenesis.
[0014] As a preferred embodiment, dissolved oxygen is controlled in two stages during fermentation culture, and / or ammonia and glucose are added.
[0015] Specifically, fermentation is carried out for 0-60 hours. After the dissolved oxygen level drops to 15%, the dissolved oxygen level is controlled at 15-25%. After 60 hours of fermentation, the tank is removed, and pure oxygen is intermittently introduced to control the dissolved oxygen level at 25-50%.
[0016] Specifically, after 20 hours of fermentation, the pH of the fermentation broth was controlled at 6.0–7.5 by adding ammonia.
[0017] Specifically, after 60 hours of fermentation, a carbon source was added, preferably a glucose solution. Further optimization involved adding glucose to maintain the residual sugar concentration at 0.5–1.5% (w / v).
[0018] Specifically, the preparation method includes controlling the fermentation temperature at 27–30°C and controlling the pH during the fermentation culture stage at 6.0–7.5.
[0019] Preferably, this invention provides a method for preparing rifamycin O by fermentation using *Amycolatoposismediterranei* or its mutagenic strains. The fermentation medium comprises: 10% glucose, 6% β-cyclodextrin, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 0.5% sodium nitrate, and 0.05% antifoaming agent. The preparation method includes: inoculating the seed culture into a fermenter, fermenting for 0-60 hours, and controlling the dissolved oxygen level to 15-25% after the dissolved oxygen level drops to 15%; after 60 hours of fermentation, removing the fermenter and intermittently introducing pure oxygen to control the dissolved oxygen level at 25-50%; after 20 hours of fermentation, automatically controlling the pH at 6.0-7.5 with ammonia water; after 60 hours of fermentation, starting to supplement glucose to control the residual sugar concentration at 0.5-1.5%. The fermentation temperature was controlled at 27–30℃, the pH during the fermentation stage was controlled at 6.0–7.5, and the tank pressure was 0.05 MPa.
[0020] The beneficial effects of this invention are as follows: This invention discloses a culture medium and fermentation process for preparing rifamycin O using *Amycolatoposismediterranei*. Specifically, by optimizing the formulation and composition of the culture medium for fermenting rifamycin O and adding β-cyclodextrin, the potency level in a 50L fermenter can be increased to 1830 mg / L. Further optimization of the process, by controlling dissolved oxygen in stages and intermittently introducing pure oxygen during fermentation, can the fermentation level in a 50L fermenter be increased to 5541 mg / L. This method achieves one-step direct fermentation to obtain rifamycin O, with a simple and controllable fermentation process, providing methodological support for industrial production. Attached Figure Description
[0021] Figure 1 This is the HPLC (High Performance Liquid Chromatography) chromatogram of the fermentation broth sample from Example 6. Detailed Implementation
[0022] 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.
[0023] 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 specifically implement the methods and achieve the corresponding results based on the content of the embodiments.
[0024] The following examples use rifamycin O strain with accession number CGMCC No. 34584 as an illustration. This strain is deposited at the China General Microbiological Culture Collection Center (classified as: *Amycolatopsis mediterranei*, deposited on May 19, 2025). The seed preparation process prior to fermentation culture is the same in all the following examples, as follows:
[0025] 1. Preparation of the inclined plane:
[0026] Take the working culture glycerol tube of the isolated and purified CGMCC No.34584 strain, thaw it, take 0.1 ml and inoculate it onto a plate, spread it evenly, and incubate it at 25℃ for 7 days to obtain slant culture.
[0027] 2. Primary seed preparation:
[0028] Take a 2cm section of the cultured, matured slant. 2The bacterial culture was inoculated into a 500ml Erlenmeyer flask containing 100ml of seed culture medium, wrapped, and placed on a shaker at 28℃ and 250rpm for 48h, with the pH of the seed culture controlled at 6.0-7.0.
[0029] The primary shake flask seed culture medium consists of: 1.5% glucose, 1.5% corn starch, 0.5% yeast extract, 0.5% wheat peptone, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and 0.2% calcium carbonate. The pH of the culture medium is adjusted to 6.0 before sterilization, and the sterilization conditions are 121-123℃ for 30 minutes.
[0030] 3. Secondary seed preparation:
[0031] 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.
[0032] The secondary seed culture medium consists of: 1.5% glucose, 1.5% corn starch, 0.5% yeast extract, 0.5% wheat peptone, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.2% calcium carbonate, and 0.05% defoamer. The pH of the culture medium is adjusted to 6.0 before sterilization, and the sterilization conditions are 121-123℃ for 30 minutes.
[0033] 4. Fermentation culture:
[0034] The secondary seed culture was inoculated into the optimized 50L fermenter fermentation medium at a ratio of 10% (v / v), and fermentation was carried out under the conditions of the different examples below. The potency level of the product rifamycin O in the fermentation broth was determined.
[0035] Before the determination, the sample processing procedure was as follows: After the fermentation was completed, 1 mL of fermentation broth was taken, anhydrous methanol was added, the mixture was mixed, and the mixture was sonicated for 30 min. Then, it was centrifuged at 14000 rpm for 10 min, filtered through a 0.45 μm filter membrane, and then analyzed by liquid chromatography.
[0036] Sample liquid chromatography analysis method: Column: C18, 250×4.6mm, 5μm; Wavelength: 254nm; Column temperature: 30℃; Flow rate: 1mL / min; Injection volume: 20μL; Run time: 15min; Mobile phase: 50mM ammonium formate:methanol:acetonitrile = 25:50:25.
[0037] 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, 1.5% glucose means that there is 1.5g of glucose per 100mL of fermentation broth.
[0038] Example 1
[0039] The fermentation medium includes: 10% glucose, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, and 0.05% defoamer;
[0040] Adjust the pH to 7.2 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.
[0041] The secondary seed culture was inoculated into the fermentation medium in a 50L fermenter at a ratio of 10% (v / v). The fermentation temperature was 27-30℃, the initial stirring was 200rpm, the initial air flow rate was 0.5vvm, the tank pressure was 0.05MPa, and the dissolved oxygen was controlled above 25%.
[0042] After 20 hours of fermentation, the pH was automatically controlled at 6.0–7.5 using ammonia. Fermentation was completed in 5–7 days.
[0043] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the rifamycin O content in the fermentation broth obtained from this implementation scheme (basic fermentation formula + β-cyclodextrin-free + ammonia supplement) was confirmed to be 471 mg / L.
[0044] Example 2
[0045] The fermentation medium includes: 10% glucose, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 0.5% sodium nitrate, and 0.05% defoamer;
[0046] Adjust the pH to 7.2 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.
[0047] The secondary seed culture was inoculated into the fermentation medium in a 50L fermenter at a ratio of 10% (v / v). The fermentation temperature was 27-30℃, the initial stirring was 200rpm, the initial air flow rate was 0.5vvm, the tank pressure was 0.05MPa, and the dissolved oxygen was controlled above 25%.
[0048] After 20 hours of fermentation, the pH was automatically maintained at 6.0–7.5 using ammonia. After 60 hours of fermentation, glucose was added to maintain the residual sugar concentration at 0.5–1.5%. Fermentation was completed in 7–9 days.
[0049] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the rifamycin O content in the fermentation broth obtained from this implementation scheme (basic fermentation formula + β-cyclodextrin-free + ammonia supplement + glucose supplement) was confirmed to be 671 mg / L.
[0050] Example 3
[0051] The fermentation medium includes: 10% glucose, 6% β-cyclodextrin, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 0.5% sodium nitrate, and 0.05% defoamer;
[0052] Adjust the pH to 7.2 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.
[0053] The secondary seed culture was inoculated into the fermentation medium in a 50L fermenter at a ratio of 10% (v / v). The fermentation temperature was 27-30℃, the initial stirring was 200rpm, the initial air flow rate was 0.5vvm, the tank pressure was 0.05MPa, and the dissolved oxygen was controlled above 25%.
[0054] After 20 hours of fermentation, the pH was automatically maintained at 6.0–7.5 using ammonia. After 60 hours of fermentation, glucose was added to maintain the residual sugar concentration at 0.5–1.5%. Fermentation was completed in 7–10 days.
[0055] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, it was confirmed that the rifamycin O content in the fermentation broth obtained by this implementation scheme (basic fermentation formula + β-cyclodextrin + ammonia supplementation + glucose supplementation) was 1830 mg / L, which significantly improved the potency level of the 50L fermenter compared to Example 2 (without β-cyclodextrin).
[0056] Example 4
[0057] The fermentation medium includes: 10% glucose, 2% β-cyclodextrin, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 0.5% sodium nitrate, and 0.05% defoamer;
[0058] Adjust the pH to 7.2 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.
[0059] The secondary seed culture was inoculated into a 50L fermenter at a ratio of 10% (v / v) into the fermentation medium. The fermentation temperature was 27–30℃, the initial stirring speed was 200 rpm, the initial air flow rate was 0.5 vvm, and the tank pressure was 0.05 MPa. Fermentation was carried out for 0–60 h. After the dissolved oxygen level dropped to 15%, the dissolved oxygen level was controlled at 15–25%. After 60 h of fermentation, the tank was removed, and pure oxygen was intermittently introduced to control the dissolved oxygen level at 25–50%.
[0060] After 20 hours of fermentation, the pH was automatically maintained at 6.0–7.5 using ammonia. After 60 hours of fermentation, glucose was added to maintain the residual sugar concentration at 0.5–1.5%. Fermentation was completed in 7–10 days.
[0061] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the rifamycin O content in the fermentation broth obtained from the implementation scheme (basic fermentation formula + 2% β-cyclodextrin + ammonia supplement + glucose supplement + pure oxygen to control dissolved oxygen) was confirmed to be 4688 mg / L.
[0062] Example 5
[0063] The fermentation medium includes: 10% glucose, 8% β-cyclodextrin, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 0.5% sodium nitrate, and 0.05% defoamer;
[0064] Adjust the pH to 7.2 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.
[0065] The secondary seed culture was inoculated into a 50L fermenter at a ratio of 10% (v / v) into the fermentation medium. The fermentation temperature was 27–30℃, the initial stirring speed was 200 rpm, the initial air flow rate was 0.5 vvm, and the tank pressure was 0.05 MPa. Fermentation was carried out for 0–60 h. After the dissolved oxygen level dropped to 15%, the dissolved oxygen level was controlled at 15–25%. After 60 h of fermentation, the tank was removed, and pure oxygen was intermittently introduced to control the dissolved oxygen level at 25–50%.
[0066] After 20 hours of fermentation, the pH was automatically maintained at 6.0–7.5 using ammonia. After 60 hours of fermentation, glucose was added to maintain the residual sugar concentration at 0.5–1.5%. Fermentation was completed in 7–10 days.
[0067] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the rifamycin O content in the fermentation broth obtained from the implementation scheme (basic fermentation formula + 8% β-cyclodextrin + ammonia supplement + glucose supplement + pure oxygen to control dissolved oxygen) was confirmed to be 5362 mg / L.
[0068] Example 6
[0069] The fermentation medium includes: 10% glucose, 6% β-cyclodextrin, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 0.5% sodium nitrate, and 0.05% defoamer;
[0070] Adjust the pH to 7.2 before disinfection, and disinfection conditions are 121-123℃ for 30 minutes.
[0071] The secondary seed culture was inoculated into a 50L fermenter at a ratio of 10% (v / v) into the fermentation medium. The fermentation temperature was 27–30℃, the initial stirring speed was 200 rpm, the initial air flow rate was 0.5 vvm, and the tank pressure was 0.05 MPa. Fermentation was carried out for 0–60 h. After the dissolved oxygen level dropped to 15%, the dissolved oxygen level was controlled at 15–25%. After 60 h of fermentation, the tank was removed, and pure oxygen was intermittently introduced to control the dissolved oxygen level at 25–50%.
[0072] After 20 hours of fermentation, the pH was automatically maintained at 6.0–7.5 using ammonia. After 60 hours of fermentation, glucose was added to maintain the residual sugar concentration at 0.5–1.5%. Fermentation was completed in 7–10 days.
[0073] Using the aforementioned high-performance liquid chromatography (HPLC) detection method, the rifamycin O content in the fermentation broth obtained under this implementation scheme (basic fermentation formula + 6% β-cyclodextrin + ammonia supplementation + glucose supplementation + pure oxygen for dissolved oxygen control) was confirmed to be 5541 mg / L. The HPLC chromatogram of the fermentation broth is shown below. Figure 1 As shown.
[0074] 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 preparing rifamycin O, characterized in that, The fermentation medium contains β-cyclodextrin.
2. The fermentation medium as described in claim 1, characterized in that, The content of the β-cyclodextrin is 2% to 8% w / v.
3. The fermentation medium as described in claim 2, characterized in that, The preferred content of the β-cyclodextrin is 6% w / v.
4. The fermentation medium as described in claim 1, characterized in that, The fermentation medium also includes a carbon source, a nitrogen source, and inorganic salts; The carbon source is selected from one or any combination of glucose, maltose, corn starch, maltodextrin, sucrose, and lactose, preferably glucose, maltose, maltodextrin, or any combination thereof; The nitrogen source is selected from soybean flour, cottonseed flour, yeast nitrogen source, peptone, polypeptide powder, casein, soy protein isolate, methyl oleate, butyl oleate, or any combination thereof; preferably yeast peptone, polypeptide powder, methyl oleate, or any combination thereof. The inorganic salt is selected from sea salt, hydrochloride, sulfate, nitrate, citrate, or sodium, potassium, magnesium, cobalt, zinc, iron, ferrous, manganese, copper, molybdate ions or any combination thereof; preferably sea salt, phosphate, sulfate, nitrate, and sodium, potassium, magnesium, cobalt, zinc ions or any combination thereof; more preferably sea salt, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium nitrate or any combination thereof.
5. The fermentation medium as described in claim 1, characterized in that, The fermentation medium comprises or is composed of: 0-12% glucose, 2-8% β-cyclodextrin, 0-4% maltose, 0-3% polypeptide powder, 0-4% yeast peptone, 0-2% methyl oleate, 0-0.5% sea salt, 0-0.5% magnesium sulfate heptahydrate, 0-0.5% potassium dihydrogen phosphate, 0-2% sodium nitrate, and 0.05-0.1% defoamer; the preferred medium composition is: 10% glucose, 6% β-cyclodextrin, 2% maltose, 2% polypeptide powder, 2% yeast peptone, 1% methyl oleate, 0.1% sea salt, 0.2% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 0.5% sodium nitrate, and 0.05% defoamer.
6. A method for preparing rifamycin O by fermentation, characterized in that, The fermentation was carried out using the fermentation medium described in any one of claims 1 to 5, and the strain used was Amycolatoposis mediterranei or a mutant strain thereof.
7. The method as described in claim 6, characterized in that, Dissolved oxygen is controlled in two stages during fermentation.
8. The method as described in claim 6 or 7, characterized in that, During the fermentation culture from 0 to 60 hours, after the dissolved oxygen drops to 15%, the dissolved oxygen level is controlled at 15-25%. From 60 hours of fermentation culture until before being discharged from the tank, pure oxygen is intermittently introduced to control the dissolved oxygen level at 25-50%.
9. The method as described in claim 6, characterized in that, During fermentation, the temperature was controlled at 27–30℃; after 20 hours of fermentation, the pH was controlled at 6.0–7.5 by adding ammonia.
10. The method as described in claim 6, characterized in that, After 60 hours of fermentation, a carbon source was added, preferably glucose, to control the residual sugar concentration at 0.5–1.5% w / v.