Method for reducing content of M impurity in fusidic acid fermentation liquor

Abstract

The invention discloses a method for reducing the content of an M impurity in fusidic acid fermentation liquor. The generation of the M impurity is inhibited from a fermentation source by optimizing a fermentation culture medium and regulating and controlling pH in stages. A fermentation culture medium adopts a composite organic nitrogen source, contains soybean cake powder with specific grease content, and is matched with a yeast extract and corn steep liquor, so that a key precursor can be provided for fusidic acid synthesis, and the enzyme activity can be regulated; the pH is regulated in the early stage of fermentation to adapt to the proliferation requirement of the strain, M impurity synthesis is preliminarily inhibited, the pH is regulated in the middle and later stages to strengthen the activity of a target product synthetase, and M impurity synthetase expression is remarkably inhibited. The process is simple, subsequent complex purification is not needed, the yield of fusidic acid is increased, M impurities are reduced to 2% or below, and the method is suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and more specifically, relates to a method for reducing the content of M impurities in fusidic acid fermentation broth. Background Technology

[0002] Fusidic acid is a steroidal antibiotic with significant antibacterial activity against Gram-positive bacteria, especially Staphylococcus aureus, and is widely used clinically. Currently, fusidic acid is mainly produced through liquid fermentation by the filamentous fungus *Fusidium coccineum*. However, during fermentation, the strain's metabolism often produces a byproduct—the M impurity—that has a structure similar to fusidic acid but with lower or no antibacterial activity. This byproduct lacks an α-hydroxyl group (α-OH) at the C11 position. The European Pharmacopoeia and industry standards explicitly stipulate that the M impurity content in fusidic acid products should be less than 2%. The M byproduct content in the crude fusidic acid extract obtained through high-yield fermentation with *Fusidium coccineum* is typically around 7.0–8.0%. The presence of the M impurity not only reduces the yield and purity of the target product, fusidic acid, but also poses significant challenges to subsequent separation and purification processes, increases production costs, and affects the quality of the final drug.

[0003] In existing technologies, to address the issue of fusidic acid (M) impurities, the focus is typically on the extraction and purification stages after fermentation, using methods such as multi-solvent extraction and column chromatography. While these methods are effective, they are complex, use large amounts of organic solvents, are costly, and can easily lead to the loss of fusidic acid itself. For example, Chinese patent CN109535218 A discloses a method for extracting and purifying fusidic acid. This method utilizes the characteristic that fusidic acid is readily soluble in organic solvents such as methanol, ethanol, and ethyl acetate. The fermentation broth is first separated to obtain mycelium, which is then dried and extracted with organic solvents. Impurities are then removed through extraction, decolorization, concentration, crystallization, and recrystallization. Another example is Chinese patent CN 110540570 A, which discloses a method for separating and purifying fusidic acid using ion exchange resins. The process involves filtering the fermentation broth, adsorbing the filtrate with ion exchange resins such as KT-30, D303, or D315, and then eluting with a 0.7 mol sodium chloride aqueous solution. The eluent is then concentrated, extracted with ethyl acetate, crystallized, and purified to obtain the final product. These methods all remove M impurities after fermentation, but do not inhibit the production of M impurities at the source of fermentation.

[0004] Therefore, there is an urgent need for a method that can effectively suppress the generation of M impurities from the source, namely during the fermentation process, thereby simplifying subsequent processes and improving production efficiency and product quality. Summary of the Invention

[0005] This invention discloses a method for reducing the content of muscarinic impurities (M impurities) in fusidic acid fermentation broth. By optimizing the fermentation medium and adjusting the pH in stages, the generation of M impurities is inhibited from the fermentation source. The fermentation medium uses a composite organic nitrogen source, containing soybean meal with a specific oil content, combined with yeast extract and corn steep liquor, which provides key precursors for fusidic acid synthesis and regulates enzyme activity. In the early stage of fermentation, the pH is adjusted to suit the strain's proliferation needs, while initially inhibiting M impurity synthesis. In the middle and late stages, the pH is adjusted to enhance the activity of the target product synthase, significantly inhibiting the expression of M impurity synthase. This invention features a simple process, requiring no complex subsequent purification, and reduces M impurities to 2% or below while increasing fusidic acid yield, making it suitable for large-scale industrial production.

[0006] This invention provides a method for reducing the content of M impurities in fusidic acid fermentation broth, wherein seed liquid is added to fermentation medium for fermentation; and the pH of the medium is controlled at 7.2~7.8 during the fermentation process.

[0007] Furthermore, the pH of the fermentation medium is controlled at 7.2-7.5 in the early stage of fermentation and at 7.5-7.8 in the middle and late stages of fermentation; the early stage of fermentation refers to the first 0 to 48 hours of fermentation, and the middle and late stages of fermentation refer to the period from 48 hours to the end of fermentation.

[0008] This invention utilizes phased pH regulation as a key method to reduce M impurities, precisely matching the metabolic patterns of the bacterial strain. In the early stages of fermentation, the pH is controlled at 7.2–7.5 to match the strain's proliferation requirements, promoting robust mycelial growth and laying the foundation for the synthesis of the target product, while simultaneously initially inhibiting the M impurity synthesis pathway. In the middle and later stages of fermentation, the pH is increased to 7.5–7.8, precisely enhancing fusidic acid synthase activity and significantly inhibiting the expression of M impurity synthase (a key enzyme catalyzing the deletion of α-OH at C11).

[0009] Furthermore, the fermentation medium contains a complex organic nitrogen source, which includes soybean meal powder, and the oil content of the soybean meal powder is 5-10%.

[0010] Preferably, the oil content of the soybean meal is 5-7%.

[0011] In this invention, the specific oil content of soybean meal is a key synergistic factor in reducing M impurities, forming a synergistic effect with the ratio of complex organic nitrogen sources and staged pH regulation. The oil can be decomposed into fatty acids, which serve as key precursors for the synthesis of fusidic acid steroid structures, enhancing the metabolic flux of the target product, and also regulate enzyme activity, inhibiting the expression of M impurity synthases. The specific oil content needs to be adapted to the metabolic requirements of the strain; too low a content will lead to insufficient precursors, activating the impurity synthesis pathway; too high a content will result in insufficient dissolved oxygen, increased metabolic burden, and weakened inhibitory effect.

[0012] Existing technologies rarely focus on reducing the content of fusidic acid (M) impurities at the fermentation source. Instead, they emphasize removing M impurities after fermentation through methods such as solvent extraction and column chromatography. These methods are complex, consume large amounts of organic solvents, are costly, and are prone to fusidic acid loss. Furthermore, the pH is mostly maintained at 6.5–7.0 throughout the fermentation process, without considering the differences in the metabolic needs of different strains at different fermentation stages. Additionally, existing technologies only use soybean meal as a nitrogen source supplement, neglecting the impact of its oil content, or directly using oil-free nitrogen sources (such as yeast extract), leading to insufficient fatty acid precursors and active M impurity synthesis pathways (content 7.0–8.0%). For example, CN 110863027 A discloses a method for reducing the content of fusidic acid byproducts through biotransformation. This method first produces fusidic acid through conventional fermentation (with the M impurity content in the fermentation broth still as high as 7.72%), then extracts and concentrates the fermentation product to obtain a concentrated solution containing M impurities, and finally puts it into a culture medium of transforming bacteria (Rhizopus nigricans / Rhizopus oryzae) for secondary fermentation to convert the M impurities into fusidic acid. This method still does not reduce the M impurity content at the fermentation source, but rather reduces it by concentrating the fermentation product and subjecting it to secondary fermentation. This process does not involve the regulation of the original fusidic acid fermentation process; it relies solely on the 11α-hydroxylase activity of the transforming bacteria to replenish the C11 α-OH of the M impurity, converting it into fusidic acid. This requires additional transforming bacteria, oxygen carriers, and transformation time to reduce the M impurity content to below 2% (compared to fusidic acid), and the fusidic acid potency only increases to 3293 μg / ml (approximately 3.29 g / L).

[0013] This invention optimizes the fermentation medium and adjusts the pH in stages to directly adapt to the metabolic patterns of fusidic acid producing strains. The amino acids provided by the compound organic nitrogen source and the fatty acids from the decomposition of soybean meal oil form a precursor library for steroid synthesis. The two work synergistically to block the synthesis pathway of M impurities, inhibiting the synthesis of M impurities from the source of the strain's metabolic pathway, reducing their content to 2% or less. The reduction of impurity content does not require subsequent complex purification processes, simplifying the production process. At the same time, the fusidic acid potency can reach 7.3 g / L.

[0014] The early stage of fermentation is the period of strain proliferation, while the middle and late stages of fermentation are the period of product synthesis. Current technologies usually use a single pH value or natural pH value throughout the entire fermentation process, which cannot adapt to the metabolic differences of strains at different growth stages.

[0015] Furthermore, the composite organic nitrogen source also includes yeast extract and corn steep liquor; the mass ratio of soybean meal, yeast extract and corn steep liquor is 2~3:1~2:1~2; the amount of composite organic nitrogen source added is 4~7%.

[0016] In conventional fermentation processes, the organic nitrogen source in the fermentation medium is usually corn steep liquor and yeast extract, which are nutritionally limited (e.g., yeast extract lacks a complete range of amino acids, and corn steep liquor has a limited peptide content), leading to metabolic imbalances in the strains and the accumulation of micronutrient impurities (M). In the compound organic nitrogen source of this invention: soybean meal provides abundant plant protein and peptides, yeast extract provides vitamins and growth factors, and corn steep liquor provides amino acids and minerals. The three are combined in a ratio of 2-3:1-2:1-2, resulting in a more balanced nutritional profile. This can inhibit the synthetic metabolic pathways of M impurities while promoting fusidic acid synthesis, reducing the M impurity content to 2% or below, and increasing its potency by at least 10%.

[0017] Preferably, the mass ratio of soybean meal powder, yeast extract and corn steep liquor is 3:2:2.

[0018] Preferably, the amount of the composite organic nitrogen source added is 7%.

[0019] Furthermore, the pH of the early and middle stages of fermentation is controlled by adding an alkaline solution to the fermentation medium.

[0020] The alkaline solution is added directly by a flow-feed method, and the pH can be adjusted in real time by an automatic replenishment system, which has higher precision (maintained within ±0.1 range). The alkaline solution is also easy to volatilize or recover, and will not leave impurities in the fermentation broth, thus ensuring product purity.

[0021] Furthermore, the alkaline solution includes any one of sodium hydroxide solution, sodium carbonate solution, and ammonia water.

[0022] The sodium hydroxide, sodium carbonate, and ammonia are moderately corrosive and easy to handle under sterile conditions. The ammonia can be removed by evaporation through aeration, and the sodium carbonate and sodium hydroxide can be separated through subsequent purification steps, leaving no residue in the product.

[0023] Furthermore, the fermentation temperature in the early stage of fermentation is 25~28℃, and the aeration rate is 1.0~1.2 vvm; the fermentation temperature in the middle and late stages of fermentation is 25~28℃, and the aeration rate is 1.2~1.5 vvm.

[0024] The early stage of fermentation is the proliferation phase of the bacterial strain, with low oxygen demand; the middle and late stages of fermentation are the product synthesis phase of the bacterial strain, with high oxygen demand. This invention adjusts the aeration rate at different stages of fermentation based on the metabolic differences of the bacterial strain at each stage.

[0025] Furthermore, the fermentation medium also includes 5-8% sucrose, 0.15% ammonium sulfate, 0.1% potassium dihydrogen phosphate, and 0.05% magnesium sulfate.

[0026] Furthermore, the seed culture is obtained by two expansion cultures of fusidic acid-producing strains; the fusidic acid-producing strains include Clostridium liposomum and its mutant strains with the same metabolic characteristics.

[0027] Furthermore, before expanding the culture, the fusidic acid producing strain needs to be activated. The activation steps are as follows: Take the fusidic acid producing strain, inoculate the strain into an agar slant medium (component: 40g / L PDA medium), and incubate it statically in an incubator at 25~30℃ for 7~10 days to obtain the activated strain.

[0028] Further, the steps for the first scale-up culture are as follows: Select activated cells from the slant culture and inoculate them into seed culture medium (components: yeast peptone 1.0%, corn steep liquor 2.0%, glucose 2.5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.2%, pH 6.5~7.0), with an inoculation amount of 2% (v / v), and culture in a shaker at 25~30℃ and 220rpm for 70~75h to obtain the primary seed culture.

[0029] Further, the second expansion culture is carried out as follows: the primary seed culture is transferred to a seed tank (the culture medium composition is the same as that of the primary seed culture) at an inoculation rate of 5-8% (v / v), and cultured at 25-30℃, aeration rate of 0.08-1.0 vvm, and stirring speed of 200-250 rpm for 48-60 h to obtain the secondary seed culture.

[0030] Furthermore, the seed liquid is inoculated into a fermenter containing the fermentation medium at an inoculation rate of 8-10%, and the initial volume of the fermentation liquid in the fermenter is 60-70% of the total volume of the tank.

[0031] The present invention has the following beneficial effects: 1. Effectively inhibits the generation of M impurities from the source: Through the optimization of compound organic nitrogen sources, the regulation of specific oil content in soybean meal, and the synergistic effect of staged pH, the synthesis of M impurities is directly inhibited from the source of fermentation, reducing the M impurity content to 2% or less (meeting the European Pharmacopoeia and industry standards), without relying on complex solvent extraction, column chromatography, or secondary conversion processes after fermentation.

[0032] 2. Excellent nutritional system adaptability and efficient strain metabolism: The compound organic nitrogen source (soybean meal powder: yeast extract: corn steep liquor = 2~3:1~2:1~2) provides a balanced supply of protein, amino acids, vitamins and fatty acid precursors. Combined with staged pH regulation, it not only meets the strain's proliferation needs but also enhances product synthesis, avoiding metabolic imbalances caused by a single nitrogen source or fixed pH. The strain's growth status is stable, and the fermentation efficiency remains high.

[0033] 3. Significantly improved fusidic acid potency: The optimized nutrient system and metabolic regulation enhance the synthesis flow of the target product, increasing the fusidic acid potency to over 7.3 g / L, which is at least 10% higher than traditional fermentation. This achieves the dual goals of "reduced impurities" and "improved potency", significantly increasing production benefits.

[0034] 4. Stable process and uniform product quality: Key parameters such as fermentation medium components, pH control range, aeration rate, and inoculum size are clearly defined. The process is highly standardized and reproducible, which can effectively avoid the problem of uneven product purity caused by process fluctuations in existing technologies and ensure batch-to-batch quality consistency.

[0035] 5. Simplified process and improved production efficiency: The fermentation process is simple. The compound organic nitrogen source (soybean meal powder, yeast extract, corn steep liquor), alkaline solution and carbon source used are all commonly used industrial raw materials, which are inexpensive and easy to obtain. After fermentation, the fermentation liquid only needs simple purification to produce the finished product, which shortens the production cycle, reduces equipment investment and operation complexity, and is suitable for large-scale industrial production. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0037] Example 1: A method for reducing the content of M impurities in fusidic acid fermentation broth 1. Activation of microbial strains and preparation of seed culture (1) Activation of strain: Take the fusidic acid production strain. In this example, the strain is Clostridium liposomalidum. Inoculate the strain into slant culture medium (component: 40g / L PDA medium) and incubate it in a 25~30℃ incubator for 7~10 days to obtain the activated strain. (2) Seed culture preparation: Select activated mycelium from the slant culture and inoculate it into seed culture medium (components: yeast peptone 1.0%, corn steep liquor 2.0%, glucose 2.5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.2%, pH 6.5~7.0), with an inoculation amount of 2% (v / v). Incubate at 25~30℃ and 220rpm in a shaker for 70~75h to obtain primary seed culture; the primary seed culture pre-transfer indexes are pH 5.5~6.5, robust mycelium with deep staining, and obvious mycelial abundance. Increase the bacterial concentration to ≥5%, and ensure no contamination. Transfer the primary seed culture to a seed tank (with the same culture medium composition as the primary seed culture) at an inoculation rate of 5-8% (v / v) (preferably 8%), and incubate at 25-30℃, aeration rate of 0.08-1.0 vvm, and stirring speed of 200-250 rpm (preferably 250 rpm) for 48-60 hours to obtain the secondary seed culture. Before transfer, the indicators should be pH 5.5-6.5, thick and deeply stained hyphae, abundant hyphae, bacterial concentration ≥10%, and no contamination.

[0038] 2. Fermentation (1) Inoculation: The secondary seed liquid is inoculated into a fermenter containing fermentation medium at an inoculation rate of 8-10% (v / v) (preferably 10%). The initial volume of the fermenter is 60-70% of the total volume of the tank. The fermentation medium consists of 5-8% sucrose (preferably 8%), 4-7% compound organic nitrogen source (preferably 7%), 0.15% ammonium sulfate, 0.1% potassium dihydrogen phosphate, and 0.05% magnesium sulfate. The compound organic nitrogen source is prepared from soybean meal, corn steep liquor, and yeast extract in a mass ratio of 2-3:1-2:1-2 (preferably soybean meal: corn steep liquor: yeast extract = 3:2:2). The oil content of the soybean meal is 5-7% (preferably 5%). (2) Early stage of fermentation (0~48h): control the fermentation temperature at 25~28℃, the aeration rate at 1.0~1.2vvm, the stirring speed at 250~300rpm (preferably 250rpm), and maintain the pH value at 7.2~7.5 by automatically adding 10% (w / v) ammonia water; (3) Mid-to-late stage of fermentation (48h to end of fermentation): control the fermentation temperature at 25-28℃, increase the aeration rate to 1.2-1.5vvm, the stirring speed at 300-350rpm (preferably 300rpm), and maintain the pH value at 7.5-7.8; during this period, control the residual sugar content of the fermentation broth at 10-15g / L by adding 50% (w / v) glucose solution until the fusidic acid titer in the fermentation broth no longer increases (the fermentation cycle is usually 200-260h).

[0039] Furthermore, the potency of fusidic acid and the content of its M impurity were determined using the following method: the fermentation broth was diluted with 5 times methanol, shaken and soaked for 30 min, then centrifuged and the supernatant was collected for HPLC detection; HPLC conditions: mobile phase: H3PO4 aqueous solution (1.15 mL / L): acetonitrile: methanol = 16:64:20; chromatographic column: C18, 4.6 × 250 mm, 5 µm; detection wavelength: 235 nm, column temperature: 40 ℃, flow rate: 0.8 mL / min, injection volume: 10 µL; the experiment was repeated 3 times, and the average value of the results was taken.

[0040] The test showed that the potency of fusidic acid was 7.3 g / L and the content of impurity M was 2%.

[0041] Example 2: Effect of pH in the early and middle stages of fermentation on the content of M impurities in fusidic acid fermentation broth The pH was changed during the early and middle-late stages of fermentation in Example 1, and the experimental groups were set up accordingly: Control group: pH was not controlled, and the entire fermentation process was carried out at the natural pH. Experimental group 1: The pH value was maintained at 6.5~7.0 throughout the fermentation process; Experimental group 2: The pH value was maintained at 7.2~7.5 throughout the fermentation process; Experimental group 3: Same as Example 1, the pH value was maintained at 7.2~7.5 in the early stage of fermentation and at 7.5~7.8 in the middle and late stages of fermentation; Experimental group 4: The pH value was maintained at 7.5~7.8 throughout the fermentation process.

[0042] The remaining conditions for the above experimental groups were the same as in Example 1, with the proportions of the components in the fermentation medium selected from the preferred schemes. After fermentation, the potency of fusidic acid and the content of M impurities (relative to fusidic acid) in the fermentation broth were measured. Each experiment was repeated three times, and the average value of the results was taken. The test results are shown in Table 1 below.

[0043] Table 1. Effects of different pH values ​​on the potency of fusidic acid and the content of M impurities in the fermentation broth during fermentation. According to the data in Table 1, compared with the control group, controlling the pH during fermentation can improve the potency of fusidic acid and reduce the content of M impurities. However, only when the pH value is maintained at 7.2-7.5 in the early stage of fermentation and at 7.5-7.8 in the middle and late stages of fermentation can the potency of fusidic acid be increased by more than 10% while the content of M impurities can be reduced to 2% or below, thus achieving the dual goals of "reducing impurities" and "increasing potency".

[0044] Example 3: Effect of the types and ratios of organic nitrogen sources in the fermentation medium on the content of M impurities in fusidic acid fermentation broth This embodiment changes the type and ratio of organic nitrogen sources in Example 1, as shown in Table 2 below.

[0045] Table 2. Types and proportions of organic nitrogen sources Ten fermentation media were prepared using the ten organic nitrogen sources described above, following the method in Example 1, and fermentation was carried out. The organic nitrogen sources were added at 7% (optimal). After fermentation, the potency of fusidic acid and the content of M impurities (relative to fusidic acid) in the fermentation broth were measured. The detection method was the same as in Example 1. Each experiment was repeated three times, and the average value of the results was taken. The detection results are shown in Table 3 below.

[0046] Table 3. Fusidic acid potency and M impurity content in fermentation broth after fermentation using different organic nitrogen sources According to the data in Table 3, compared with Group 1 (the original fermentation medium nitrogen source scheme), the fermentation media prepared with single nitrogen sources in Groups 2-4 and two combined nitrogen sources in Groups 5-7 generally had lower or almost the same fusidic acid potency and higher M impurity content. However, compared with Group 1, only Group 8, with its combination of soybean meal, yeast extract, and corn steep liquor as the organic nitrogen source, showed a more than 10% increase in fusidic acid potency and a decrease in M ​​impurity content to 2% or below. In contrast, Groups 8-9 ultimately showed a decrease in fusidic acid potency and an increase in M ​​impurity content.

[0047] Fermentation media prepared with a single nitrogen source are nutritionally unbalanced. Corn steep liquor lacks vitamins and growth factors, yeast extract lacks sufficient fatty acid precursors, and soybean meal lacks mineral supplementation, leading to metabolic imbalance in the strains, active M-impurity synthesis pathways, and inhibited synthesis of the target product. Fermentation media prepared with two or three combined nitrogen sources show inconsistent results. Only when the combined nitrogen source is soybean meal, yeast extract, and corn steep liquor can the nutritional system of the fermentation medium be balanced, meeting the needs of strain proliferation while enhancing the synthesis of the target product and inhibiting impurity formation.

[0048] Therefore, the organic nitrogen source selected in the fermentation medium is a combination of soybean meal, yeast extract and corn steep liquor.

[0049] Example 4: Effect of oil content in soybean meal on the content of M impurities in fusidic acid fermentation broth The oil content in soybean meal affects the M impurity content in fusidic acid fermentation broth. In this embodiment, the oil content of soybean meal in Example 1 is changed. Soybean meal containing different oils is shown in Table 4 below.

[0050] Table 4. Soybean cake powder containing different oils The soybean meal powders with different oil contents were prepared into different fermentation media according to the method in Example 1 and fermented. After fermentation, the potency of fusidic acid and the content of M impurities (relative to fusidic acid) in the fermentation broth were detected. The detection method was the same as in Example 1. Each group of experiments was repeated 3 times, and the average value of the results was taken. The detection results are shown in Table 5 below.

[0051] Table 5. Fusidic acid potency and M impurity content in fermentation broth after fermentation of soybean meal powder with different oil contents According to the data in Table 5, when the oil content of soybean meal is 5-10% (groups 1-3), the M impurity content can be reduced to 3.1% or below, which is far lower than the 7.0-8.0% of traditional fermentation. However, only group 1 has the highest fusidic acid potency and the M impurity content is reduced to 2% or below. When the oil content of soybean meal is below 2%, although the final fusidic acid potency is higher than that of groups 2-3 and the M impurity content is lower than that of groups 2-3, it is still lower than that of group 1.

[0052] When the oil content of soybean meal is 5-7%, the fatty acids produced by oil decomposition can not only fully meet the precursor requirements for fusidic acid steroid synthesis, enhancing the metabolic flux of the target product, but also moderately regulate enzyme activity, effectively inhibiting the expression of M impurity synthases. Simultaneously, this content does not cause insufficient dissolved oxygen or metabolic burden, forming an optimal synergistic effect with the complex organic nitrogen source and staged pH regulation, achieving the dual optimal goals of "highest potency + lowest impurities." When the oil content of soybean meal is 8-10%, excessive oil increases the viscosity of the fermentation broth, leading to insufficient dissolved oxygen (due to the aerobic nature of bacterial metabolism), while also increasing the metabolic burden on the cells and weakening the activity of the target product synthases. Furthermore, excessive fatty acids may activate some impurity synthesis pathways, resulting in a decline in the M impurity inhibition effect. When the oil content of soybean meal is 6-8%, this oil content leads to a slight imbalance between precursor supply and metabolic balance. While sufficient precursors can still support a high potency, slightly excessive oil begins to affect dissolved oxygen efficiency, causing a slight weakening of the M impurity inhibition effect. When the oil content of soybean meal is less than 2%, although the balanced nutrition of the compound organic nitrogen source and the staged pH regulation can still maintain a high synthesis efficiency of the target product (the potency is only 0.2 g / L lower than that of group 1), the lack of fatty acid precursors will slightly activate the M impurity synthesis branch, resulting in an impurity content higher than that of the 5-7% oil range group.

[0053] Therefore, the preferred oil content of soybean meal is 5-7%.

[0054] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for reducing the content of M impurities in fusidic acid fermentation broth, characterized in that, The seed culture was added to the fermentation medium for fermentation; the pH of the medium was controlled at 7.2-7.8 during the fermentation process.

2. The method as described in claim 1, characterized in that, The pH of the fermentation medium was controlled at 7.2-7.5 in the early stage of fermentation and at 7.5-7.8 in the middle and late stages of fermentation. The early stage of fermentation refers to the first 0 to 48 hours of fermentation, and the middle and late stages of fermentation refers to the last 48 hours of fermentation until the end of fermentation.

3. The method as described in claim 1, characterized in that, The fermentation medium contains a complex organic nitrogen source, which includes soybean meal powder, and the oil content of the soybean meal powder is 5-10%.

4. The method as described in claim 2, characterized in that, The compound organic nitrogen source also includes yeast extract and corn steep liquor; the mass ratio of soybean meal, yeast extract and corn steep liquor is 2~3:1~2:1~2; the amount of compound organic nitrogen source added is 4~7%.

5. The method as described in claim 3, characterized in that, The pH of the early and middle stages of fermentation is controlled by adding an alkaline solution to the fermentation medium.

6. The method as described in claim 5, characterized in that, The alkaline solution includes any one of sodium hydroxide solution, sodium carbonate solution, and ammonia water.

7. The method as described in claim 5, characterized in that, The fermentation temperature in the early stage of fermentation is 25~28℃, and the aeration rate is 1.0~1.2 vvm; the fermentation temperature in the middle and late stages of fermentation is 25~28℃, and the aeration rate is 1.2~1.5 vvm.

8. The method as described in claim 1, characterized in that, The fermentation medium also includes 5-8% sucrose, 0.15% ammonium sulfate, 0.1% potassium dihydrogen phosphate, and 0.05% magnesium sulfate.

9. The method as described in claim 1, characterized in that, The seed culture was obtained by two expansion cultures of a fusidic acid-producing strain; the fusidic acid-producing strain includes Clostridium liposomum and its mutant strains with the same metabolic characteristics.

10. The method as described in claim 9, characterized in that, The seed liquid is inoculated into a fermenter containing the fermentation medium at an inoculation rate of 8-10%, and the initial volume of the fermentation liquid in the fermenter is 60-70% of the total volume of the tank.

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