Preparation method and system of enramycin high-purity fine powder

Abstract

The invention discloses a preparation method and system of enramycin high-purity fine powder, and belongs to the technical field of separation and purification. The method comprises the following steps: heating and inactivating fermentation liquor, cooling, flocculating and filtering, flash drying, extracting with a methanol or ethanol mixed solvent, adjusting acidity, decolorizing with activated carbon, growing grains in a neutral state, and drying in vacuum to obtain the fine powder. The process parameters are optimized as follows: the inactivation temperature is 50-80 DEG C, the concentration of an extraction solvent is 60%-90%, the crystal growing temperature is 4-6 DEG C and the like, and the solvent and the activated carbon can be recycled. The matching system comprises a fermentation liquor treatment unit, an extraction unit, a decoloration unit, a crystal growing unit and a drying unit, the extraction unit is provided with pH and constant temperature control, and the crystal growing unit is provided with a low-temperature constant-temperature and stirring device. The purity of the refined powder prepared by the method is greater than or equal to 98%, the yield is greater than or equal to 95%, and the method has the advantages of low cost, simplicity and convenience in operation, high safety, good environmental friendliness and the like and is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, specifically to a method and system for preparing high-purity enramycin concentrate using physicochemical methods. Background Technology

[0002] Enramycin, a polypeptide antibiotic, is produced by fermentation of *Streptomyces fungicidious* NO. B-5477. It exhibits potent inhibitory activity against Gram-positive bacteria (especially the harmful intestinal bacterium *Clostridium*), and possesses advantages such as high stability, low toxicity, low residue, and low likelihood of inducing drug resistance. It is widely used in the field of animal feed additives. Since its development by Takeda Pharmaceutical Company Ltd. of Japan in 1966, its official registration in Japan in 1974, and its registration in my country in 1993, its global demand has continued to grow.

[0003] However, the fermentation broth of enramycin has a complex composition. Even after simple pretreatment, the crude product contains only 9.0%-10.0% enramycin, and numerous byproducts make separation and purification difficult. Currently, commercially available products are mainly premixes, while high-purity concentrates are scarce and expensive, limiting their application in high-end feed additives and other fields.

[0004] Existing separation and purification technologies have significant limitations: Resin separation method: For example, Chinese invention patent (CN102311486B) uses macroporous weak acid cation exchange resin for purification. Although separation can be achieved, the resin is expensive and difficult to recycle. The recovery rate of enramycin is only 73.1%, which is difficult to meet the cost control requirements of large-scale industrial production.

[0005] Chromatographic extraction method: The Chinese invention patent (CN105037497B) uses chromatographic technology, which requires the use of flammable and explosive organic reagents such as petroleum ether and ethyl acetate. This not only poses safety hazards but also causes environmental pollution, which is inconsistent with the concept of green production and limits its industrial application.

[0006] Therefore, developing a low-cost, easy-to-operate, high-yield, and environmentally friendly separation and purification process is key to overcoming the bottleneck in the industrial production of high-purity enramycin powder. This invention addresses the shortcomings of existing technologies, such as low purity, low yield, and high cost, by proposing an integrated process based on organic solvent extraction, acid-base adjustment, antisolvent crystallization, and cryo-crystallization. This aims to achieve efficient separation and purification of enramycin, providing a new pathway for its industrial production. Summary of the Invention

[0007] The purpose of this invention is to address the problems of low product purity, low yield, high cost, and poor safety in existing enramycin separation and purification technologies, and to provide a low-cost, simple-to-operate, high-yield, and high-purity enramycin powder preparation method to meet the needs of large-scale industrial production. To solve the above problems, this invention provides a method for preparing high-purity enramycin powder.

[0008] To achieve the above objectives, the first aspect of the present invention provides a method for preparing high-purity enramycin powder. The preparation process includes the following steps: (1) Inactivating enramycin fermentation broth by heating and then cooling it, adding a flocculant for flocculation, filtering through a plate and frame filter to obtain wet mycelium, and flash evaporating the wet mycelium to obtain dry flash powder; (2) Adding an extraction solvent to the dry flash powder, adjusting the pH to acidic, and then extracting, and filtering to obtain an extract; (3) Adjusting the pH of the extract to acidic, adding activated carbon for decolorization, and filtering to obtain a decolorized solution; (4) Adjusting the pH of the decolorized solution to neutral, adding pure water, and then growing crystals at a constant temperature of 0-10℃, filtering to obtain wet crystals, and then vacuum drying and grinding to obtain high-purity enramycin powder.

[0009] Further, in step (1), the enramycin fermentation broth is heated to 50-80℃ for 20-50 minutes to inactivate it, and then cooled to 30-50℃ after inactivation.

[0010] Further, the flocculant mentioned in step (1) is polyaluminum chloride or polyacrylamide, and the amount used is 0.1% to 0.5% of the fermentation broth mass.

[0011] Further, the extraction solvent in step (2) is a mixed organic solvent formed by methanol or ethanol and water, wherein the concentration of methanol or ethanol in the mixed organic solvent is 60%~90%, and the mass ratio of the extraction solvent to the dried flash powder is (5-10):1.

[0012] Further, in step (2), the pH is adjusted to 1.0~5.0, the extraction temperature is 30~40℃, and the extraction time is 1~3 hours.

[0013] Further, in step (3), the pH is adjusted to 1.0~5.0, the amount of activated carbon used is 1.0%~3.0% of the mass of the extract, and the decolorization time is 0.5~2 hours.

[0014] Furthermore, in step (4), the volume ratio of pure water to extract is (0.5~2):1, crystallization is carried out at a constant temperature of 4-6℃ for 4~6 hours.

[0015] Further, in step (2), the pH is adjusted to 3.0~4.0, the extraction temperature is 32.5℃, and the extraction time is 1.5 hours.

[0016] Further, the extraction solvent in step (2) is a 70% methanol aqueous solution, and the mass ratio of the methanol aqueous solution to the dried flash powder is 7:1.

[0017] Furthermore, the acidic solution used to adjust the pH value in step (2) is a 10% acetic acid solution or a 3% hydrochloric acid solution.

[0018] Further, in step (3), the alkaline solution for adjusting the pH value is a 10% sodium hydroxide solution or a sodium bicarbonate solution, and the pH is adjusted to 4.0~5.0; the amount of activated carbon used is 1.5% of the mass of the extract, and the decolorization time is 1 hour.

[0019] Furthermore, in step (4), the volume ratio of pure water to extract is 1:1, crystallization is carried out at a constant temperature of 5°C, and the crystallization time is 4.5 hours.

[0020] Further, the temperature for inactivation in step (1) is 62.5°C and the inactivation time is 32 minutes; the flocculant is polyaluminum chloride and the amount used is 0.27% of the mass of the fermentation broth.

[0021] Further, the feature is that in step (2), the mycelium after extraction is subjected to at least one repeated extraction, and the extracts are combined to improve the yield.

[0022] Furthermore, the characteristic feature is that the reagent used to adjust the pH in step (4) is a 10% sodium hydroxide solution.

[0023] Furthermore, the vacuum drying conditions are: temperature 40~60℃, vacuum degree -0.08~-0.1MPa, and drying time 2~4 hours.

[0024] Furthermore, the method is characterized by including a step of recovering and reusing the organic solvent and the decolorized activated carbon in the leaching solution.

[0025] Furthermore, the extraction solvent is recovered by distillation, and the activated carbon is reused after elution.

[0026] Furthermore, the characteristic feature is that the final enramycin concentrate has a purity of ≥98% and a yield of ≥95%.

[0027] Secondly, the present invention provides a system for preparing high-purity enramycin powder, comprising a fermentation broth treatment unit, an extraction unit, a decolorization unit, a crystal growth unit and a drying unit connected in sequence. The extraction unit is equipped with a pH adjustment module and a constant temperature control module, and the crystal growth unit is equipped with a low-temperature constant temperature device and a stirring device.

[0028] Compared with existing technologies, the present invention has the following advantages: (1) High efficiency: By optimizing the extraction, decolorization and crystallization parameters, the purity of enramycin is ≥98% and the yield is ≥95%, which is significantly better than existing technologies (such as the resin method with a recovery rate of 73.1%).

[0029] (2) Low cost: Using conventional solvents such as methanol, the cost is lower than that of macroporous resins; the solvent and activated carbon can be recycled and reused, reducing the cost of consumables.

[0030] (3) Safety and environmental protection: Avoid the use of flammable and explosive organic solvents (such as petroleum ether) to improve production safety; reduce chemical reagent emissions to meet green production requirements.

[0031] (4) Process convenience: The process is simple (only four steps: pretreatment, extraction, decolorization and crystallization), the operation time is short, and it is suitable for continuous industrial production; key links (such as pH adjustment and constant temperature control) can be precisely controlled by automated equipment to improve process stability. Attached Figure Description

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The structural formulas of enramycin A and B are shown. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] In a first aspect, the present invention provides a method for preparing high-purity enramycin powder, comprising the following steps: (1) After the enramycin fermentation broth is heated to inactivate it, it is cooled down, flocculant is added for flocculation, wet mycelium is obtained by plate and frame filtration, and dry flash powder is obtained after the wet mycelium is flash evaporated. (2) Add extraction solvent to the dried flash powder, adjust the pH to acidic, extract, and filter to obtain the extract; (3) Adjust the pH of the extract to acidic, add activated carbon for decolorization, and filter to obtain the decolorized solution; (4) Adjust the pH of the decolorizing solution to neutral, add pure water and grow crystals at a constant temperature of 0-10℃, filter to obtain wet crystals, and then vacuum dry and grind to obtain high-purity enramycin powder.

[0036] In this invention, the preparation process of high-purity enramycin powder is divided into 4 steps, and the resulting high-purity enramycin powder has a purity of ≥98% and a yield of ≥95%.

[0037] In some embodiments, in step (1), the enramycin fermentation broth is heated to 50-80°C for 20-50 minutes to inactivate it, and then cooled to 30-50°C. For example, the temperature can be selected from 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C, the inactivation time can be selected from 20, 25, 30, 35, 40, 45, and 50 minutes, and the cooling temperature after inactivation can be selected from 30, 35, 40, 45, and 50°C.

[0038] In some embodiments, the flocculant in step (1) is polyaluminum chloride or polyacrylamide, and the amount used is 0.1% to 0.5% of the fermentation broth mass. For example, the amount of polyaluminum chloride added is 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% of the fermentation broth mass, etc., and the amount of polyacrylamide added is 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% of the fermentation broth mass, etc.

[0039] In some embodiments, the extraction solvent in step (2) is an aqueous methanol solution, and the concentration of methanol can be 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc., or it can be an aqueous ethanol solution, and the concentration of ethanol can be 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc., and the mass ratio of the extraction solvent to the dried flash powder is 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0040] In some implementations, step (2) can adjust the pH to 1.0, 2.0, 3.0, 4.0, or 5.0, the extraction temperature to 30, 32, 34, 36, 38, or 40°C, and the extraction time to 1, 1.5, 2, 2.5, or 3 hours.

[0041] In some embodiments, step (3) can adjust the pH to 1.0, 2.0, 3.0, 4.0, or 5.0, and the amount of activated carbon can be 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, or 3.0% of the extract mass, etc., and the decolorization time can be 0.5, 1, 1.5, or 2 hours, etc.

[0042] In some embodiments, the volume ratio of pure water to extract in step (4) is 0.5:1, 1:1, 1.5:1, 2:1, etc., and crystals can be grown at a constant temperature of 4, 5, or 6°C, and the crystal growth time can be 4, 4.5, 5, 5.5, or 6 hours, etc.

[0043] In one embodiment, step (2) adjusts the pH to 3.5, the extraction temperature to 32.5°C, and the extraction time to 1.5 hours.

[0044] In one embodiment, the extraction solvent in step (2) is a 70% methanol aqueous solution, and the mass ratio of the methanol aqueous solution to the dried flash powder is 7:1.

[0045] In one embodiment, the acidic solution used to adjust the pH value in step (2) is a 10% acetic acid solution or a 3% hydrochloric acid solution.

[0046] In one embodiment, the alkaline solution used to adjust the pH in step (3) is a 10% sodium hydroxide solution or a 10% sodium bicarbonate solution, and the pH is adjusted to 4.5; the amount of activated carbon used is 1.5% of the mass of the extract, and the decolorization time is 1 hour.

[0047] In one embodiment, the volume ratio of pure water to extract in step (4) is 1:1, crystal growth is carried out at a constant temperature of 5°C, and the crystal growth time is 4.5 hours.

[0048] In one embodiment, the inactivation temperature in step (1) is 62.5°C and the inactivation time is 32 minutes; the flocculant is polyaluminum chloride and the amount used is 0.27% of the mass of the fermentation broth.

[0049] In one embodiment, step (2) involves repeated extraction of the extracted mycelium twice.

[0050] In one embodiment, the reagent used to adjust the pH in step (4) is a 10% sodium hydroxide solution.

[0051] In one embodiment, the vacuum drying conditions are: temperature 50°C, vacuum degree 0.09 MPa, and drying time 3 hours.

[0052] Secondly, the present invention provides a system for preparing high-purity enramycin powder, comprising a fermentation broth treatment unit, an extraction unit, a decolorization unit, a crystal growth unit and a drying unit connected in sequence. The extraction unit is equipped with a pH adjustment module and a constant temperature control module, and the crystal growth unit is equipped with a low-temperature constant temperature device and a stirring device.

[0053] Thirdly, the present invention provides a method for preparing high-purity enramycin powder, which can also recover and reuse the methanol or ethanol aqueous solution and the decolorized activated carbon in the extraction solution. The extraction solvent is recovered by distillation, and the activated carbon is reused after elution.

[0054] The present invention will be further described in detail below through specific embodiments.

[0055] The raw material information used in the various embodiments and comparative examples of this invention is as follows: Enramycin fermentation broth: provided by the production department of Jiangsu Moxi Biotechnology Co., Ltd.

[0056] Polyaluminum chloride: PAC-2, Aisen Flocculant Co., Ltd.

[0057] Polyacrylamide: APAM, Aisen Flocculant Co., Ltd.

[0058] Methanol: Industrial methanol, Chengdu Kelong Chemical Co., Ltd.

[0059] Ethanol: Industrial ethanol, Chengdu Kelong Chemical Co., Ltd.

[0060] Hydrochloric acid solution: Sinopharm Group Chemical Reagent Co., Ltd.

[0061] Acetic acid solution: Sinopharm Group Chemical Reagent Co., Ltd.

[0062] Sodium hydroxide: Inner Mongolia Junzheng Energy Chemical Group Co., Ltd.

[0063] Sodium bicarbonate: Food grade, Chongqing Tenghua Chemical Co., Ltd.

[0064] Activated carbon: food grade, 325 mesh, Henan Cardiff Activated Carbon Co., Ltd.

[0065] Example 1: (1) The enramycin fermentation broth was heated to 62.5℃ and inactivated for 32 minutes. After inactivation, the temperature was lowered to 40℃. Polyaluminum chloride of 0.27% of the fermentation broth mass was added for flocculation. The wet mycelium was obtained by plate and frame filtration. The wet mycelium was flash-evaporated at 55℃ to obtain dry flash-evaporated powder. (2) Add 7 times the mass of 70% methanol aqueous solution to the dry flash powder, adjust the pH to 3.5 with 3% hydrochloric acid solution and then extract. The extraction temperature is 32.5℃ and the extraction time is 1.5 hours. Filter to obtain the extract; repeat the extraction once and combine the extracts.

[0066] (3) Adjust the pH of the extract to 4.5 with 10% sodium hydroxide solution, add 1.5% activated carbon (by mass of the extract) for decolorization for 1 hour, and filter to obtain the decolorized solution; (4) Adjust the pH of the decolorizing solution to 7.0 with 10% sodium hydroxide solution, add 1:1 mass of pure water, and grow crystals at 5℃ for 4.5 hours. Filter to obtain wet crystals, and obtain high-purity enramycin powder after vacuum drying and grinding. The vacuum drying conditions are: temperature 50℃, vacuum degree 0.09MPa, and drying time 3 hours.

[0067] Example 2 The preparation method of this embodiment is basically the same as that of Example 1, except that in step (2), 7 times the mass of 60% methanol aqueous solution is added to the dried flash powder.

[0068] Example 3 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (2), 7 times the mass of 80% methanol aqueous solution is added to the dried flash powder.

[0069] Example 4 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (2), 5 times the mass of 70% methanol aqueous solution is added to the dried flash powder.

[0070] Example 5 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (2), 9 times the mass of 70% methanol aqueous solution is added to the dried flash powder.

[0071] Example 6 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (2), the pH is adjusted to 2.5 by 3% hydrochloric acid solution before extraction.

[0072] Example 7 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (2), the pH is adjusted to 4.5 by 3% hydrochloric acid solution before extraction.

[0073] Example 8 The preparation method of this embodiment is basically the same as that of Example 1, except that the extraction temperature in step (2) is 30°C.

[0074] Example 9 The preparation method of this embodiment is basically the same as that of Example 1, except that the extraction temperature in step (2) is 36°C.

[0075] Example 10 The preparation method of this embodiment is basically the same as that of Example 1, except that the extraction temperature in step (2) is 40°C.

[0076] Example 11 The preparation method of this embodiment is basically the same as that of Example 1, except that the extraction time in step (2) is 1 hour.

[0077] Example 12 The preparation method of this embodiment is basically the same as that of Example 1, except that the extraction time in step (2) is 3 hours.

[0078] Example 13 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (3), 1.0% of activated carbon is added to the extract for decolorization for 1 hour, and the decolorized liquid is obtained by filtration.

[0079] Example 14 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (3), 3.0% of activated carbon is added to the extract for decolorization for 1 hour, and the decolorized liquid is obtained by filtration.

[0080] Example 15 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (4), the pH of the decolorizing solution is adjusted to 7.0 with 10% sodium hydroxide solution, and 0.5:1 mass of pure water is added. The solution is then kept at 5°C for 4.5 hours to grow crystals, and then filtered to obtain wet crystals.

[0081] Example 16 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (4), the pH of the decolorizing solution is adjusted to 7.0 with 10% sodium hydroxide solution, and then pure water is added in a ratio of 2:1 by mass. The solution is then kept at 5°C for 4.5 hours to grow crystals, and then filtered to obtain wet crystals.

[0082] Example 17 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (4), the pH of the decolorizing solution is adjusted to 7.0 with 10% sodium hydroxide solution, and pure water is added in a 1:1 mass ratio. The solution is then kept at 5°C for 4 hours to grow crystals, and wet crystals are obtained by filtration.

[0083] Example 18 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (4), the pH of the decolorizing solution is adjusted to 7.0 with 10% sodium hydroxide solution, and pure water is added in a 1:1 mass ratio. The solution is then kept at 5°C for 6 hours to grow crystals, and wet crystals are obtained by filtration.

[0084] Comparative Example 1 The preparation method of the high-purity enramycin powder in this comparative preparation example differs from that in Example 1 in that: Enramycin high-purity powder was prepared by methanol solution extraction: Step (1) Extraction: Enramycin mycelium was extracted three times using methanol solution as the extraction solvent under low pH and 30℃ conditions.

[0085] Step (2) Concentration and Decolorization: Combine the extracts and concentrate under reduced pressure, then add activated carbon for decolorization.

[0086] Step (3) Crystallization and recrystallization: The pH of the concentrate is adjusted to neutral by adding alkali, and crude crystals precipitate out. The crude product is recrystallized with acetone solution to obtain refined enramycin powder.

[0087] Comparative Example 2 The preparation method of the high-purity enramycin powder in this comparative preparation example differs from that in Example 1 in that: Enramycin high-purity powder was prepared by acidic acetone extraction. Step (1) Acidic extraction: Crude enramycin was extracted with acidic acetone aqueous solution for 2 h.

[0088] Step (2) Distillation: Acetone was removed by vacuum distillation of the extract to precipitate crude enramycin extract; the supernatant was removed by centrifugation, the precipitate was washed with water and dried at 60°C to obtain refined enramycin powder.

[0089] Comparative Example 3 The preparation method of the high-purity enramycin powder in this comparative preparation example differs from that in Example 1 in that: Enramycin high-purity powder was prepared by silica gel column chromatography: Step (1) Preparation of crude extract: Enramycin flash powder is dissolved in methanol solution, pH is adjusted to 3.5, and after centrifugation, the supernatant is neutralized to pH 7.5.

[0090] Step (2) Silica gel column separation: The extract was mixed with silica gel and dried before being loaded onto the column by dry method; impurities were eluted sequentially with petroleum ether-ethyl acetate-butyl acetate-methanol gradient; the target component was eluted with hydrochloric acid-methanol solution, neutralized and then dried by rotary evaporation to obtain enramycin refined powder.

[0091] The preparation methods of high-purity enramycin powder provided in Examples 1-18 and Comparative Examples 1-3 yielded the high-purity enramycin powder.

[0092] Performance testing methods: 1. Purity detection (HPLC method) (1) Objective: To quantitatively analyze the content of principal components and impurities.

[0093] (2) Method: A, Chromatographic conditions: B. Column: C18 reversed-phase column (250 × 4.6 mm, 5 μm) C. Mobile phases: Phase A: 0.1% trifluoroacetic acid aqueous solution; Phase B: 0.1% trifluoroacetic acid acetonitrile solution. D, gradient procedure: Time / min Phase B / % 0 20 30 80 35 20 Flow rate: 1.0 mL / min Detection wavelength: 220 nm Column temperature: 30℃ Injection volume: 20 μL (3) Sample preparation: Accurately weigh approximately 20 mg of enramycin sample, dilute to 10 mL with mobile phase A, dissolve by sonication, and filter through a 0.22 μm filter membrane.

[0094] (4) Calculation: A, Purity (%) = Main peak area / Sum of all peak areas * 100% B, Impurity limits: Single impurity ≤ 1.0%, total impurities ≤ 5.0% (refer to EP standard).

[0095] 2. Potency testing (microbiological assay) (1) Purpose: To determine the bioactivity (unit: μg / mg or IU / mg).

[0096] (2) Method: Tube and saucer method (cup and saucer method) A. Test strain: Bacillus subtilis (CMCC 63501) B, Culture medium: Bottom layer: Nutrient agar (20 mL / plate) Bacterial layer: Nutrient agar containing 0.5% bacterial solution (5 mL / plate) (3) Standard solution: Enramycin standard (potency known), dissolved in pH 6.0 phosphate buffer, and diluted to 0.5, 1.0, 2.0, 4.0, 8.0 IU / mL.

[0097] (4) Test solution: Dilute to approximately 2.0 IU / mL as expected using the same method.

[0098] (5) Procedure: Place stainless steel tubes evenly in a petri dish and add standard / test solution (200 μL / tube) to each. Incubate at 37℃ for 16-18 h and measure the diameter of the inhibition zone.

[0099] (6) Calculation (two-dose method): Plot a standard curve with the logarithmic concentration of the standard as the x-axis and the diameter of the inhibition zone as the y-axis; Potency (IU / mg) = Potency of the test sample / Sample weight * Dilution factor of the sample 3. Yield Calculation (1) Purpose: To evaluate the efficiency of the production process.

[0100] (2) Formula: Yield (%) = Total potency of pure enramycin (IU) / Theoretical total potency (IU) * 100% step: Theoretical total potency: calculated based on fermentation feed amount and historical potency data of the strain.

[0101] Total potency of pure product: final product weight (g) * measured potency (IU / g) Test results: Table 1: Example purity(%) Yield (%) Potency of extract (μg / ml) Example 1 99.5 97.8 15356 Example 2 98.3 95.2 14253 Example 3 97.6 96.5 13664 Example 4 98.6 95.5 15336 Example 5 93.2 95.6 12925 Example 6 98.3 96.5 14232 Example 7 98.6 92.5 13368 Example 8 97.6 96.8 14376 Example 9 98.2 93.3 14362 Example 10 98.1 90.2 14633 Example 11 93.0 91.5 13856 Example 12 98.3 97.7 14322 Example 13 98.3 97.8 14523 Example 14 98.9 93.2 13963 Example 15 91.3 91.6 14332 Example 16 98.6 97.6 14256 Example 17 98.4 96.3 14523 Example 18 98.5 97.7 14393 Comparative Example 1 67.3 69.6 9263 Comparative Example 2 60.5 63.2 6475 Comparative Example 3 59.6 59.8 5432 As shown in Table 1, compared with Comparative Examples 1-3, Examples 1-18 exhibit the following significant advantages: Significant purity advantage: The purity of all examples is ≥91.3%, with Example 1 reaching 99.5%, and the purity of most examples concentrated in the 98%-99% range. In contrast, the highest purity of Comparative Examples 1-3 is only 67.3% (Comparative Example 1), and the rest are all below 61%. This indicates that the present invention effectively removes impurities and improves product purity by optimizing parameters such as extraction pH and crystal growth temperature.

[0102] The yield was significantly improved: the lowest yield in the examples was 90.2% (Example 10), and the highest was 97.8% (Example 1), with an overall yield of ≥90%. In contrast, the yields of Comparative Examples 1-3 were all <70%, with Comparative Example 3 only reaching 59.8%. This is due to the repeated extraction process of the present invention (such as two extractions in Example 1) and precise control of crystal growth conditions, which reduced the loss of enramycin.

[0103] The extracts exhibited higher potency: the lowest potency of the extracts in the examples was 12925 μg / ml (Example 5), and the highest was 15356 μg / ml (Example 1), significantly higher than that of Comparative Example 1 (9263 μg / ml), Comparative Example 2 (6475 μg / ml), and Comparative Example 3 (5432 μg / ml). This indicates that the mixed solvent concentration (e.g., 70% methanol aqueous solution) and extraction temperature (around 32.5°C) used in this invention can more efficiently dissolve enramycin and improve extraction efficiency.

[0104] High process stability: Even when adjusting a single parameter (such as solvent concentration, pH value, crystal growth time, etc.) in the examples, the purity and yield remain at a high level. For example, the purity fluctuation range in Examples 2-18 is only 91.3%-98.9%, and the yield fluctuation is 90.2%-97.7%, proving the strong robustness of the process parameters. In contrast, the comparative examples, which use traditional methods (such as silica gel column chromatography and acidic acetone extraction), are affected by reagent toxicity and process complexity, resulting in large fluctuations in indicators and low overall performance.

[0105] In summary, this invention, by integrating technologies such as organic solvent extraction, precise acid-base adjustment, low-temperature crystal growth, and solvent recovery, overcomes the bottlenecks of low purity and low yield in existing methods, and achieves efficient purification and industrial production of enramycin.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing high-purity enramycin powder, characterized in that, The following steps are involved: (1) After the enramycin fermentation broth is heated to inactivate it, it is cooled down, flocculant is added for flocculation, wet mycelium is obtained by plate and frame filtration, and dry flash powder is obtained after the wet mycelium is flash evaporated. (2) Add extraction solvent to the dried flash powder, adjust the pH to acidic, extract, and filter to obtain the extract; (3) Adjust the pH of the extract to acidic, add activated carbon for decolorization, and filter to obtain the decolorized solution; (4) Adjust the pH of the decolorizing solution to neutral, add pure water and grow crystals at a constant temperature of 0-10℃, filter to obtain wet crystals, and then vacuum dry to obtain high-purity enramycin powder.

2. The preparation method according to claim 1, characterized in that, In step (1), the enramycin fermentation broth is heated to 50-80℃ for 20-50 minutes to inactivate it, and then cooled to 20-40℃. The flocculant is polyaluminum chloride or polyacrylamide, and the dosage is 0.1%~0.5% of the fermentation broth mass. The flash evaporation temperature is 50-75℃.

3. The preparation method according to claim 1, characterized in that, The extraction solvent in step (2) is a mixed organic solvent formed by methanol or ethanol and water, wherein the concentration of methanol or ethanol in the mixed organic solvent is 60% to 90%, and the mass ratio of the extraction solvent to the dried flash powder is (5-10):1; the pH is adjusted to 1.0 to 5.0, the extraction temperature is 30 to 40°C, and the extraction time is 1 to 3 hours.

4. The preparation method according to claim 1, characterized in that, In step (3), the pH is adjusted to 1.0 to 5.0, the amount of activated carbon used is 1.0% to 3.0% of the mass of the extract, and the decolorization time is 0.5 to 2 hours.

5. The preparation method according to claim 1, characterized in that, In step (4), the volume ratio of pure water to extract is (0.5~2):

1. Crystallization is carried out at a constant temperature of 4-6℃ for 4~6 hours. The reagent used to adjust the pH is 10% sodium hydroxide solution.

6. The preparation method according to claim 1 or 3, characterized in that, In step (2), the pH is adjusted to 3.0-4.0, the extraction temperature is 32.5℃, and the extraction time is 1.5 hours. The extraction solvent is a 70% methanol aqueous solution, and the mass ratio of the methanol aqueous solution to the dried flash powder is 7:

1. The acidic solution for adjusting the pH value is a 10% acetic acid solution or a 3% hydrochloric acid solution. The mycelium after extraction is subjected to at least one repeated extraction, and the extracts are combined to improve the yield.

7. The preparation method according to claim 1 or 4, characterized in that, The alkaline solution used to adjust the pH value in step (3) is a 10% sodium hydroxide solution or a sodium bicarbonate solution, and the pH is adjusted to 4.0~5.0; the amount of activated carbon used is 1.5% of the mass of the extract, and the decolorization time is 1 hour.

8. The preparation method according to claim 1 or 5, characterized in that, In step (4), the volume ratio of pure water to extract is 1:1, crystal growth is carried out at a constant temperature of 5℃ for 4.5 hours, and the vacuum drying conditions are: temperature 40~60℃, vacuum degree -0.08~-0.1MPa, drying time 2~4 hours.

9. The preparation method according to claim 1 or 2, characterized in that, The inactivation temperature in step (1) is 62.5℃ and the inactivation time is 32 minutes; the flocculant is polyaluminum chloride and the amount used is 0.27% of the fermentation liquid mass.

10. A system for the preparation method according to any one of claims 1-9, characterized in that, The device includes a fermentation broth treatment unit, an extraction unit, a decolorization unit, a crystal growth unit, and a drying unit connected in sequence. The extraction unit is equipped with a pH adjustment module and a constant temperature control module, and the crystal growth unit is equipped with a low-temperature constant temperature device and a stirring device. The device also includes a step of recovering and reusing the organic solvent in the extraction solution and the activated carbon after decolorization. The extraction solvent is recovered by distillation, and the activated carbon is reused after elution. The final enramycin concentrate has a purity of ≥98% and a yield of ≥95%.

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