Organic solvent-free kitasamycin extraction process
By employing a solvent-free extraction process for tylosin, utilizing steps such as pH adjustment with oxalic acid, flocculation filtration, vacuum evaporation, dynamic crystallization, and activated carbon decolorization, the safety hazards, health risks, and high costs associated with existing technologies have been resolved, achieving high-purity and high-yield extraction of tylosin.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-29
- Publication Date
- 2026-03-27
AI Technical Summary
The existing extraction process for tylosin uses organic solvents, which leads to safety hazards, health risks, high costs, and significant environmental pressure, as well as low purity and yield.
An organic solvent-free extraction process is employed, involving steps such as pH adjustment with oxalic acid, flocculation filtration, vacuum evaporation, dynamic crystallization, activated carbon decolorization, and gradient crystallization-countercurrent washing, to achieve high purity and high yield of tylosin.
It has achieved the extraction of tylosin with high purity (≥99.5%) and high yield (92-95%), reducing production costs and environmental pressure, and improving the stability and safety of the process.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of separation and purification technology, specifically to a process for extracting high-purity tylosin from tylosin fermentation broth, which is particularly suitable for industrial production scenarios that are low-cost and environmentally friendly and do not involve organic solvents. Background Technology
[0002] Kitasamycin, also known as columnar cytosine, is a sixteen-membered macrolide antibiotic. Its antibacterial spectrum primarily covers Gram-positive bacteria and some Gram-negative bacteria, exhibiting significant antibacterial activity against Gram-positive pathogens such as Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus viridans. Furthermore, this compound also demonstrates clear antibacterial activity against Gram-negative pathogens such as Neisseria gonorrhoeae and Bordetella pertussis.
[0003] Existing technology: The industrial extraction process of tylosin typically follows this technical flow: First, the fermentation broth undergoes pretreatment (including cell disruption and impurity precipitation), followed by solid-liquid separation using centrifugation or membrane filtration to obtain a clear filtrate. Then, liquid-liquid extraction is performed using a hydrophobic organic solvent (such as butyl acetate), with pH adjustment (usually controlled at 8.0-9.0) to promote the distribution of tylosin to the organic phase. The organic phase containing the loaded product is then back-extracted with an acidic buffer solution to transfer the target compound to the aqueous phase. Next, crystallization purification is achieved using isoelectric point crystallization (adjusting the system pH to an alkaline range), followed by a gradient cooling process to obtain a high-purity crystalline product. Finally, residual solvent is removed by oven drying to obtain tylosin raw material that meets pharmacopoeia standards.
[0004] Technical problems with existing technologies: The above-mentioned methods for preparing tylosin use a large amount of organic solvents, such as butyl acetate, which brings a series of problems: 1. Organic solvents are mostly flammable and explosive liquids. When mixed with air, they may form explosive mixtures, requiring various explosion-proof equipment and significantly increasing the cost of plant construction and maintenance; 2. Long-term exposure to organic solvents may cause health hazards to operators, requiring strict protective measures and increasing occupational health management costs; 3. High-purity organic solvents are expensive, and the solvents are volatile during extraction and other operations, requiring frequent replenishment of new solvents, which drives up raw material costs; 4. Organic solvent extraction and back-extraction require recovery processes such as distillation, which are energy-intensive and require large equipment investments. If the recovery rate is insufficient, additional wastewater treatment costs are required; 5. The use of organic solvents requires more attention to environmental pressures such as air pollution and water pollution, requiring the installation of corresponding exhaust gas treatment systems and wastewater treatment systems, increasing post-treatment costs.
[0005] Therefore, developing a high-yield, high-purity, and low-cost extraction process for tylosin without organic solvents has become a key need to address the pain points of existing technologies. Summary of the Invention
[0006] Based on the above technical problems and in view of the shortcomings of existing organic solvent extraction processes, this invention provides a solvent-free extraction process for tylosin. By optimizing the process steps and parameters, the product purity is ≥99.5% and the total yield is 92-95%, while reducing production costs and environmental pressure.
[0007] This invention discloses a process for extracting tylosin without organic solvents, characterized by comprising the following sequentially implemented technical steps: (1) Primary processing stage: Add 5-8% oxalic acid solution to the tylosin fermentation broth to adjust the pH, control the pH value of the fermentation broth to 3.5±0.2, maintain the stirring speed at 120-150 rpm for 30±2 minutes for acidification treatment; add flocculant to a final concentration of 0.15-0.25 g / L, allow to stand for flocculation, and then filter using a plate and frame filter press to obtain filter residue and primary filtrate.
[0008] (2) Filtrate concentration stage: The pH of the primary filtrate was adjusted to the range of 5.0-5.5 using a 10% NaOH solution. Then, vacuum film evaporation was carried out under vacuum conditions of 0.09±0.005MPa and 60±2℃ to concentrate the filtrate to 1 / 5±0.02 of its original volume.
[0009] (3) Primary crystallization purification: Add 4% NaOH solution to the concentrate to adjust the pH to 8.4±0.1, and carry out dynamic crystallization in a constant temperature water bath at 50±1℃ for 30±5 minutes. Separate the primary crystal crude product and the mother liquor containing residual products by vacuum filtration. The mother liquor is returned to the pre-concentration treatment unit in step 2 via a pipeline circulation system for product recovery.
[0010] (4) Acidic dissolution and refining: The primary crystalline crude product was dissolved in a buffer system containing 0.6% sodium oxalate and 2.2% oxalic acid by mass fraction, and the pH of the system was precisely adjusted to 3.1±0.1 by adding 0.5 mol / L oxalic acid solution in a gradient. The dissolution process was carried out under ultrasonic assistance (40kHz, 300W), and the solution and insoluble residue were obtained by filtration through a 0.22μm microporous membrane. (5) Adsorption decolorization treatment: Add 0.3% (w / w) of activated carbon adsorbent to the solution, perform dynamic decolorization at 55±2℃ for 30±2 minutes, and obtain the decolorized and clarified solution through multi-stage filtration using a multi-layer filter paper assembly.
[0011] (6) Final crystallization purification: The pH of the decolorizing solution was adjusted to 9.2±0.2 using a 5% NaOH solution, and high-purity tylosin crystals were obtained by gradient cooling crystallization under constant temperature conditions of 55±1℃. The product was subjected to three countercurrent top washing processes using a dilute alkaline solution with a pH of 9.8±0.2, and finally vacuum dried to obtain the purified tylosin product with a purity of ≥99.5%.
[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: No organic solvents, safe and environmentally friendly: No organic solvents such as butyl acetate are used throughout the process, eliminating the need for explosion-proof equipment and exhaust gas / wastewater treatment systems. Plant construction costs are reduced by 30-50%, environmental protection costs are reduced by 20-25%, and health risks to operators are eliminated.
[0013] Significantly improved yield and purity: Through processes such as mother liquor recycling, ultrasonic-assisted dissolution, and gradient crystallization-countercurrent top washing, the total yield reaches 92-95% (an improvement of 18-22 percentage points compared to existing technologies), the purity of the final product is ≥99.5% (an improvement of 9.5-14.5 percentage points compared to existing technologies), the residue on ignition is ≤0.2%, and the potency is ≥1500 IU / mg, fully complying with the standards of the Chinese Pharmacopoeia.
[0014] Significant cost reduction: By eliminating the costs of purchasing organic solvents and distillation recovery, the production cost per batch (1000L fermentation broth) has been reduced from RMB 85,000-90,000 to RMB 50,000-55,000, a reduction of RMB 35,000-40,000; at the same time, activated carbon can be recycled, further reducing raw material costs.
[0015] Strong process stability: Through comparison and verification between Examples 1-10 and Comparative Examples 1-6, even when adjusting a single parameter (such as pH or type of flocculant), the product purity remains ≥97% and the yield is ≥92%, proving that the process parameters are highly stable and suitable for large-scale industrial production. Detailed Implementation
[0016] 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.
[0017] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.
[0018] The genistein fermentation broth used in this example was derived from the fermentation broth produced in our large-scale production tanks. All other experimental equipment and reagents used were sourced from our production and post-processing departments. All raw materials were commercially available products.
[0019] Example
[0020] 1. Initial processing stage Take 1000L of tylosin fermentation broth (initial pH 6.7~7.1), add 5% oxalic acid solution at a uniform speed under uniform mechanical stirring, adjust the pH to 3.5, and continue acidification treatment for 30 min.
[0021] Add 200g of ferrous sulfate to the acidification solution, control the final concentration to 0.2 g / L, and let stand for 30 min to allow the flocs to settle completely.
[0022] Solid-liquid separation was carried out using a plate and frame filter press (filter cloth pore size ≤ 5 μm, operating pressure 0.4~0.6 MPa), the primary filtrate was collected, and the filter residue was treated as hazardous waste.
[0023] 2. Filtrate Concentration Stage The primary filtrate was transferred to a vacuum membrane evaporation system, and a 10% NaOH solution was slowly added under uniform mechanical stirring to adjust the pH to 5.0.
[0024] Start the vacuum system, control the vacuum level to 0.09 MPa, and heat the temperature to 60℃ to evaporate and concentrate to 1 / 5 of the original volume.
[0025] After the concentrate is concentrated, it is transferred to a crystallization tank.
[0026] 3. Primary crystallization purification Add a 4% NaOH solution to the concentrate at a uniform rate, adjust the pH to 8.4, and heat to 50℃ and maintain the temperature.
[0027] Turn on the dynamic crystallization mode, mechanically stir at a constant speed for 30 minutes to precipitate primary crystals of tylosin.
[0028] The vacuum filtration system is turned on to separate the crude crystals and the mother liquor. The mother liquor is returned to the concentration unit in step 2 through the pipeline circulation system, while the crude crystals are transferred to the next process.
[0029] 4. Acidic dissolution and refining The primary crystalline crude product was mixed with oxalate-sodium oxalate buffer (containing 0.6% NaOH and 2.2% oxalic acid) at a mass ratio of 1:10, and 0.5 mol / L oxalic acid solution was added dropwise in a gradient to precisely adjust the pH to 3.1.
[0030] The primary crystalline crude product was completely dissolved under ultrasonic-assisted conditions (frequency 40 kHz, power 300 W, treatment time 20 min).
[0031] High-purity solution and insoluble residue were separated by filtration through a 0.22 μm microporous membrane.
[0032] 5. Adsorption and decolorization treatment Add 0.3% activated carbon to the solution, heat to 55℃, and stir dynamically for 30 minutes to decolorize.
[0033] The vacuum filtration system is activated to separate the activated carbon and the decolorizing liquid. The separated activated carbon is recycled, and the decolorizing liquid enters the next process.
[0034] 6. Final crystallization purification Add a 5% NaOH solution to the decolorizing solution to adjust the pH to 9.2. Start the dynamic crystallization mode at a constant temperature of 55℃ for 30 minutes. After crystallization, separate the crystals and mother liquor by vacuum filtration. The mother liquor enters the concentration treatment unit in step 2 through the pipeline circulation system.
[0035] The resulting crystals were subjected to three countercurrent top washes with a dilute alkaline solution at pH 9.8 to remove impurities.
[0036] After vacuum drying (temperature 45℃, vacuum degree 0.08 MPa), white powdery tylosin crystals were obtained. The purity was 99.6% as determined by HPLC, and the residue on ignition was ≤0.2%.
[0037] 1000L of tylosin fermentation broth was processed using the above process, and 9.4kg of refined tylosin product was finally obtained, with a total yield ≥93%, purity 99.6%, and residue on ignition ≤0.2%, which meets the standards of the Chinese Pharmacopoeia.
[0038] Example 2 The preparation method of this embodiment is basically the same as that of Example 1, except that the pH of the fermentation broth is adjusted to 3.2 in step 1-1.
[0039] Example 3 The preparation method of this embodiment is basically the same as that of Example 1, except that the flocculant in steps 1-2 is changed from ferrous sulfate to zinc sulfate.
[0040] Example 4 The preparation method of this embodiment is basically the same as that of Example 1, except that in steps 1-2, the flocculant is changed to a mixture of 100g of ferrous sulfate and 100g of zinc sulfate.
[0041] Example 5 The preparation method in this embodiment is basically the same as that in Example 1, except that the pH is adjusted to 5.5 in step 2-1.
[0042] Example 6 The preparation method in this embodiment is basically the same as that in Example 1, except that in step 3-1, a 4% NaOH solution is used to adjust the pH of the concentrate to 8.5.
[0043] Example 7 The preparation method in this embodiment is basically the same as that in Example 1. The difference is that in step 4-1, during the acidic dissolution and purification stage, 0.5 mol / L oxalic acid solution is added dropwise to adjust the pH to 3.2.
[0044] Example 8 The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step 5-1, during the adsorption and decolorization treatment stage, the amount of activated carbon added is adjusted to 0.25% by mass.
[0045] Example 9 The preparation method in this embodiment is basically the same as that in Example 1, except that in step 6-1, a 5% NaOH solution is used to adjust the pH of the decolorizing solution to 9.0.
[0046] Example 10 The preparation method of this embodiment is basically the same as that of Example 1, except that in step 6-2, a dilute alkaline solution with a pH of 9.5 is used for two countercurrent top washes.
[0047] Comparative Example 1: The preparation method in this embodiment is basically the same as that in Example 1, except that zinc sulfate / ferrous sulfate flocculant is not added in steps 1-2, and the mixture is directly filtered.
[0048] Comparative Example 2: The preparation method in this embodiment is basically the same as that in Example 1. The difference is that in the primary crystallization and purification stage of step 3-1, the pH of the concentrate is adjusted to 7.5 (below the optimal range) using a 4% NaOH solution.
[0049] Comparative Example 3: The preparation method in this embodiment is basically the same as that in Example 1. The difference is that in step 4-2, the acidic dissolution and purification stage, ultrasonic-assisted treatment is omitted, and the primary crystalline crude product is dissolved only by mechanical stirring.
[0050] Comparative Example 4: The preparation method of this embodiment is basically the same as that of Example 1. The difference is that the amount of activated carbon used in the decolorization stage of step 5-1 is reduced to 0.1%, and the temperature control is not strict (40℃).
[0051] Comparative Example 5: The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step 6-1, the final crystallization and purification stage, the pH of the decolorizing solution is adjusted to 8.0 (below the optimal range) using a 5% NaOH solution.
[0052] Comparative Example 6: The preparation method in this embodiment is basically the same as that in Example 1, except that in the final crystallization purification stage of step 6-2, countercurrent washing is not performed.
[0053] The organic solvent-free extraction processes for tylosin provided in Examples 1-10 and Comparative Examples 1-6 yielded high-purity tylosin powder.
[0054] Performance testing methods: 1. Purity detection (HPLC method) (1) Objective: To quantitatively analyze the content of principal components and impurities.
[0055] (2) Method: Chromatographic conditions: Chromatographic column: C18 column (150×4.6mm, 5μm); Mobile phase: Phase A: 0.1 mol / L ammonium acetate (pH 4.5); Phase B: methanol; Phase C: acetonitrile (A:B:C = 40:55:5, v / v / v); Flow rate: 1.0 mL / min, detection wavelength: 231 nm, column temperature: 60 ℃, injection volume: 10 μL; (3) Sample preparation: Accurately weigh approximately 20 mg of tylosin sample, dilute to 10 mL with mobile phase A, dissolve by sonication, and filter through a 0.22 μm filter membrane.
[0056] (4) Calculation: Purity (%) = Main peak area / Sum of all peak areas * 100%; Impurity limits: Calculated by the peak area of tylosin A5 using the external standard method, tylosin A5 should be 35%~70%, A4 should be 5%~25%, and A1 and A13 should both be 3%~15%; the sum of the main components of tylosin A9, A8, A7, A6, A5, A4, A1, A3, and A13 should not be less than 85%.
[0057] 2. Potency testing (microbiological assay) (1) Purpose: To determine the bioactivity (unit: μg / mg or IU / mg).
[0058] (2) Method: Tube and saucer method (cup and saucer method) A. Test strain: Bacillus subtilis (CMCC 63501); B, Culture medium: Bottom layer: Nutrient agar (20mL / plate); Bacterial layer: Nutrient agar containing 0.5% bacterial solution (5mL / plate). (3) Standard solution: Tadalafil standard (potency known), dissolved in phosphate buffer at pH 6.0, and diluted to 0.5, 1.0, 2.0, 4.0, and 8.0 IU / mL.
[0059] (4) Test solution: Dilute to approximately 2.0 IU / mL as expected using the same method.
[0060] (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.
[0061] (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.
[0062] (2) Formula: Yield (%) = Total potency of pure tylosin (IU) / Theoretical total potency (IU) * 100%; step: Theoretical total potency: calculated based on fermentation feed amount and historical potency data of the strain.
[0063] Total potency of pure product: final product weight (g) * measured potency (IU / g) The test results are shown in Table 1: Table 1:
[0064] As shown in Table 1, compared with Comparative Examples 1-5, Examples 1-10 exhibit the following significant advantages: Significant purity advantage: The purity of all examples is ≥97%, with Example 1 reaching 99.6%, and the purity of most examples concentrated in the 98%-99% range. In contrast, the highest purity of the comparative examples is only 81.2% (Comparative Example 1), and the rest are all below 80%, indicating that the present invention effectively removes impurities and improves product purity by optimizing various steps.
[0065] The yield was significantly improved: the yield of the examples ranged from a minimum of 92% to a maximum of 95%, with an overall yield of ≥92%. In contrast, the yields of the comparative examples were all <72%, with Comparative Example 1 only reaching 61%.
[0066] Higher potency of pure product: The potency of pure products in the examples ranged from a minimum of 1573 u / mg to a maximum of 1732 u / mg, significantly higher than that of Comparative Example 1. This indicates that the acidic buffer dissolution system and gradient crystallization-countercurrent top washing coupling process used in this invention can dissolve tylosin more efficiently, thus improving extraction efficiency.
[0067] Strong process stability: Even when adjusting a single parameter (such as solvent concentration, pH value, flocculant, etc.) in the examples, the purity and yield remain at a high level, proving that the process parameters are robust.
[0068] In summary, this invention overcomes the bottlenecks of low purity and low yield in existing methods by integrating mother liquor recycling mechanisms, gradient crystallization-countercurrent top washing coupling processes, and acidic buffer dissolution systems, thus achieving efficient purification and industrial production of tylosin.
[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 process for extracting tylosin without organic solvents, characterized in that, Includes the following steps: The fermentation broth of tylosin was treated in the following stages: primary treatment, filtrate concentration, primary crystallization purification, acidic dissolution purification, adsorption decolorization, and final crystallization purification. The high-purity tylosin crystals were subjected to three countercurrent top washing treatments with dilute alkaline solution and finally vacuum dried to obtain the refined tylosin product.
2. The organic solvent-free extraction process for tylosin according to claim 1, characterized in that, The first step (1) primary treatment stage: the tylosin fermentation broth is subjected to primary treatment to obtain primary filtrate; Step (2) Filtrate Concentration Stage: The primary filtrate is concentrated to obtain a concentrated solution; The primary crystallization purification in step (3) involves adding an alkaline solution to the concentrate, performing dynamic crystallization in a constant temperature water bath, and separating the primary crystal crude product and the mother liquor containing residual products by vacuum filtration; the residual mother liquor is returned to step (2) for repeated processing. Step (4) Acidic dissolution and purification: The primary crystalline crude product is dissolved in a buffer system, and the pH value is adjusted by adding oxalic acid solution in a gradient. Dissolution was performed under ultrasonic assistance, and a high-purity solution and insoluble residue were obtained by filtration through a microporous membrane. The step (5) adsorption decolorization treatment: Activated carbon adsorbent is added to the high-purity solution for dynamic decolorization, and multi-layer filter paper is used for multi-stage filtration to obtain a decolorized and clarified solution; Step (6) final crystallization purification: the pH value of the decolorized and clarified solution is adjusted using NaOH solution, and gradient cooling crystallization is carried out to obtain high-purity tylosin crystals.
3. The organic solvent-free extraction process for tylosin according to claim 1 or 2, characterized in that, Step (1) Primary treatment stage: Add 5-8% oxalic acid solution to the tylosin fermentation broth for pH adjustment, control the pH value of the fermentation broth to the range of 3.5±0.2, maintain the stirring speed of 120-150 rpm / min for acidification treatment for 30±2 minutes; add flocculant to the final concentration of 0.15-0.25 g / L, let it stand for flocculation, and then filter it using a plate and frame filter press to obtain filter residue and primary filtrate respectively; the flocculant is ferrous sulfate and / or zinc sulfate.
4. The organic solvent-free extraction process for tylosin according to claim 1 or 2, characterized in that, In step (2) the filtrate concentration stage, the pH value of the primary filtrate is adjusted to the range of 5.0-5.5 by using a 10% NaOH solution. The filtrate is then concentrated to 1 / 5 ± 0.02 of its original volume under vacuum conditions of 0.09 ± 0.005 MPa and 60 ± 2 °C to obtain the concentrated filtrate.
5. The organic solvent-free extraction process for tylosin according to claim 1 or 2, characterized in that, Step (3) Primary crystallization purification: Add 4% NaOH solution to the concentrate to adjust the pH to 8.4±0.1, and carry out dynamic crystallization in a constant temperature water bath at 50±1℃ for 30±5 minutes. Separate the primary crystal crude product and the mother liquor containing residual products by vacuum filtration. The mother liquor is returned to the pre-concentration treatment unit of step 2 through the pipeline circulation system for product recovery.
6. The organic solvent-free extraction process for tylosin according to claim 1 or 2, characterized in that, Step (4) Acidic dissolution and purification process: The primary crystalline crude product is dissolved in a buffer system containing 0.6% sodium oxalate and 2.2% oxalic acid by mass, and the buffer system is 50% of the initial fermentation liquid volume; The pH of the system was precisely adjusted to 3.1 ± 0.1 by adding 0.5 mol / L oxalic acid solution in a gradient, resulting in the treated acidic solution.
7. The organic solvent-free extraction process for tylosin according to claim 6, characterized in that, Step (4) Acidic dissolution and purification process: The acidic solution is dissolved under ultrasonic assistance (40kHz, 300W) and filtered through a 0.22μm microporous membrane to obtain a high-purity solution and insoluble residue.
8. The organic solvent-free extraction process for tylosin according to claim 1 or 2, characterized in that, Step (5) Adsorption and decolorization treatment: Add 0.3% by mass of activated carbon adsorbent to the high-purity solution, and perform dynamic decolorization at 55±2℃ for 30±2 minutes. Use a three-layer filter paper group for multi-stage filtration to obtain decolorized and clarified solution.
9. The organic solvent-free extraction process for tylosin according to claim 1 or 2, characterized in that, Step (6) Final crystallization purification: The pH of the decolorized and clarified solution was adjusted to 9.2±0.2 using a 5% NaOH solution. Gradient cooling crystallization was carried out at a constant temperature of 55±1℃ to obtain high-purity tylosin crystals.
10. The organic solvent-free extraction process for tylosin according to claim 9, characterized in that, Step (6) Final crystallization purification: The high-purity tylosin crystals are subjected to three countercurrent top washing treatments with sodium hydroxide solution at pH 9.8±0.2, and finally vacuum dried to obtain the refined tylosin product with a purity ≥99.5%.