A method for extracting and purifying spinosyn

Abstract

The present application belongs to the technical field of biological pharmacy, and particularly relates to a method for extracting and purifying spinosad. The method for extracting and purifying spinosad from saccharopolyspora spinosa fermentation broth by combining enzymolysis and acid extraction reduces the amount of organic solvent; the membrane separation technology is used to purify the spinosad extract, the microfiltration and ultrafiltration are used to remove the bacterial bodies, insoluble proteins, colloid, pigments and other macromolecular impurities, and the nanofiltration is used to remove small molecular water-soluble impurities, so as to realize the purification of the extract; the two-phase extraction is used to realize the second purification of the spinosad; and the evaporation crystallization is used to realize the third purification of the spinosad. Compared with the prior art, the method for extracting and purifying spinosad has the advantages of simple process, high efficiency, environmental protection, low consumption, and the like.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a method for extracting and purifying spinosad. Background Technology

[0002] Spinosad, also known as spinosad, is a secondary metabolite produced by the soil actinomycete *Spirosporium spp.* through aerobic fermentation. It is a macrolide-based, pollution-free, and highly effective biological insecticide. Its main active ingredients are spinosad A (85%-90%) and spinosad D (10%-15%). Its mechanism of action involves the action of nicotinic acid acetylcholine receptors, which continuously activate nicotinic acetylcholine receptors in target insects, rapidly paralyzing and ultimately killing them. Its insecticidal speed is comparable to that of chemical pesticides. However, it has relatively poor stability, readily decomposes in light, hydrolyzes rapidly, and has a short half-life, making it a low-toxicity, highly effective, low-residue, and broad-spectrum insecticide. It is safe for beneficial insects and mammals, making it particularly suitable for pollution-free vegetable and fruit production.

[0003] Spinosad is a light gray or white solid crystalline powder with a slightly stale, earthy odor. It is poorly soluble in water but readily soluble in low-carbon alcohols, esters, acetone, and dichloromethane, among other organic solvents. Current extraction methods mainly include solvent extraction and adsorption.

[0004] The solvent extraction method utilizes the selectivity of solvents to extract spinosad from bacterial cells, effectively removing large-molecule proteins and colloids. However, as an intracellular product, spinosad suffers from low extraction efficiency and high solvent consumption during solvent extraction, resulting in high energy consumption and pollution. The purification process employs a combination of extraction and back-extraction, which is cumbersome, requires sophisticated equipment, and suffers significant losses, leading to a low spinosad yield. Furthermore, the pretreatment of the fermentation broth involves the addition of large amounts of salt filter aids, especially zinc sulfate, generating substantial amounts of zinc-containing wastewater, significantly increasing the difficulty and cost of wastewater treatment.

[0005] The adsorption method uses adsorption materials (resin, activated carbon, diatomaceous earth, silica gel) to adsorb and desorb spinosad. However, the process involves large amounts of solvent, low efficiency, and low recovery rate, which is not environmentally friendly. In addition, it requires high-end equipment and is difficult to achieve large-scale production. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides a method for the extraction and purification of spinosad. Spinosad extracts spinosad from *Polyspora spp.* using a combination of enzymatic hydrolysis and acid extraction, reducing the amount of organic solvent used. Membrane separation technology is employed to purify the spinosad extract, using microfiltration and ultrafiltration to remove large molecular impurities such as bacterial cells, insoluble proteins, colloids, and pigments, and nanofiltration to remove small molecular water-soluble impurities, thus achieving purification of the extract. A second purification of spinosad is achieved through two-phase extraction, and a third purification is achieved through evaporation and crystallization. Compared to existing technologies that suffer from large solvent consumption, cumbersome processes, long cycles, high equipment requirements, severe environmental pollution, and high production costs, the spinosad extraction and purification method provided by this invention is simple, efficient, environmentally friendly, and low-consumption.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0008] A method for extracting and purifying spinosad includes the following steps:

[0009] 1) Enzymatic hydrolysis: Add biological enzymes to the fermentation broth and hydrolyze at 40℃-50℃ for 0.5-2 hours;

[0010] 2) Acid extraction: Adjust the pH of the fermentation broth after enzymatic hydrolysis to 2-3, and extract by stirring at room temperature for 0.5-2 hours;

[0011] 3) Membrane separation: The pH of the fermentation broth after acid extraction is adjusted to 4-5, and purified acid solution is obtained by microfiltration, ultrafiltration and nanofiltration;

[0012] 4) Two-phase extraction: Add a water-insoluble crystalline solvent to the purified acid solution, and add sodium hydroxide solution to the system while stirring to adjust the pH to 9-10, and extract by stirring at 50-60℃;

[0013] 5) Evaporation crystallization: After two-phase separation, the crystallization solution is concentrated by evaporation, and then slowly cooled to 0-5℃ to grow crystals;

[0014] 6) Centrifugation and drying: After centrifuging the crystallization solution, spinosad wet crystals are obtained, and then dried to obtain the product.

[0015] The bio-enzyme mentioned in step 1) is selected from one or more of peptidoglycanase, protease, and lysozyme. However, in order to better break the cell wall and better release spinosad into the fermentation broth, the bio-enzyme is a mixed enzyme of peptidoglycanase, protease, and lysozyme, with each enzyme added at a rate of 0.1-0.5 g / L; but for the best effect, preferably, the amount of each enzyme added is 0.2 g / L.

[0016] Preferably, the optimal enzymatic hydrolysis time in step 1) is 1 hour.

[0017] Preferably, the acid used to adjust the pH in step 2) is tartaric acid, and the optimal extraction time is 1 hour.

[0018] Preferably, the alkaline solution used to adjust the pH in step 3) is a 0.1 mol / L to 0.5 mol / L sodium hydroxide solution.

[0019] Preferably, the microfiltration operating pressure in step 3) is 0.3MPa-0.5MPa, and the operating temperature is 25℃-35℃.

[0020] Preferably, the ultrafiltration in step 3) uses a spiral wound ultrafiltration membrane with a molecular weight cutoff of 1000-5000 Da, an operating pressure of 0.1-0.7 MPa, and an operating temperature of 25-45°C. More preferably, the optimal molecular weight cutoff is 1000 Da, and the optimal operating pressure is 0.5 MPa.

[0021] Preferably, the nanofiltration process in step 3) uses a spiral wound nanofiltration membrane with a molecular weight cutoff of 200-500 Da, an operating pressure of 2-5 MPa, and an operating temperature of 25-45°C. More preferably, the optimal molecular weight cutoff is 400 Da, and the optimal operating pressure is 4 MPa.

[0022] Preferably, the crystallization solvent in step 4) is selected from one of n-butyl acetate, sec-butyl acetate, isopropyl acetate, and dichloroethane. The four crystallization solvents of this invention have significantly different boiling points from spinosad, making subsequent evaporation crystallization easier. Dichloroethane, due to its low boiling point, increases the difficulty of controlling the evaporation crystallization process compared to the other three. Therefore, more preferably, the crystallization solvent is selected from one of n-butyl acetate, sec-butyl acetate, and isopropyl acetate. Most preferably, the crystallization solvent is sec-butyl acetate.

[0023] Preferably, the volume ratio of the purified acid solution to the crystallization solvent in step 4) is (2-20):1; more preferably (2-10):1, and most preferably 4:1.

[0024] Preferably, the concentration of the sodium hydroxide solution used in step 4) is 0.1 mol / L to 0.5 mol / L.

[0025] Preferably, in step 5), the evaporation temperature is 40-50℃, the vacuum degree is 0.01MPa-0.08MPa, the solution is concentrated under vacuum to 1 / 25-1 / 5 of its original volume, the cooling rate is 10℃ / min, and the crystal growth time is 1-4h. More preferably, the optimal evaporation temperature is 45℃, the solution is concentrated to 1 / 20 of its original volume, and the crystal growth time is 2h.

[0026] Preferably, the drying temperature in step 6) is 70°C.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The key point of this invention is that, in the separation and purification process, a membrane separation method is used. Through a three-stage membrane filtration process (microfiltration, ultrafiltration, and nanofiltration), macromolecular impurities such as bacterial cells, insoluble proteins, colloids, and pigments, as well as water-soluble small molecule impurities, are removed from the solution. This achieves preliminary purification of the spinosad extract, making the solution ready for subsequent two-phase extraction and evaporation crystallization processes. Furthermore, this invention optimizes the crystallization solvent in the two-phase extraction process. Compared to existing extraction solvents, this allows for a larger boiling point difference with spinosad during the evaporation crystallization process, facilitating crystallization and ensuring stability and controllability. This optimized process route lays the foundation for a simplified process, simplifying or omitting steps such as extraction, back-extraction, fine filtration, and crystallization in existing technologies. Compared to adsorption and solvent methods, this simplifies the process, shortens the production cycle, and reduces process complexity and energy consumption.

[0029] In addition, the separation and purification process employs a combination of two-phase extraction and evaporation crystallization. This method achieves the purification of spinosad while using a single solvent, effectively reducing solvent consumption and wastewater generation. This results in a low-toxicity, high-efficiency, and environmentally friendly process that is suitable for industrial production.

[0030] In addition, this invention employs enzymatic hydrolysis to pretreat the fermentation broth, optimizing the enzymes used and their dosage to more rapidly and effectively disrupt the cell structure of *Saccharomyces cerevisiae*, maximizing the release of spinosad into the fermentation broth. Simultaneously, acid extraction is used to improve the extraction rate of spinosad. Compared to organic solvent extraction, this process reduces solvent consumption and environmental pollution. Furthermore, the pretreatment avoids the introduction of flocculants and filter aids, effectively reducing the difficulty of wastewater treatment and saving production costs.

[0031] In summary, the process of this invention is simpler and easier to implement for continuous industrial production compared to existing technologies. It reduces operational difficulty, shortens the production cycle, lowers production costs, and reduces wastewater discharge; it is also more environmentally friendly and efficient. Attached Figure Description

[0032] Figure 1 This is the HPLC chromatogram of the spinosad product in Example 1 of the present invention;

[0033] Figure 2 To compare the HPLC chromatogram of the spinosad product in Example 1;

[0034] Figure 3 To compare the HPLC chromatogram of the spinosad product in Example 2;

[0035] Figure 4 The HPLC chromatogram of spinosad product in Example 3 is used for comparison. Implementation

[0036] The following detailed embodiments further illustrate the above-described content of the present invention, but should not be construed as limiting the scope of the invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Unless otherwise specified, conventional techniques are used in the following embodiments.

[0037] The formula for calculating the yield of spinosad is as follows:

[0038] Spinosad yield = mass of spinosad in the product (g) ÷ mass of spinosad in the fermentation broth (g) × 100%.

[0039] The method for determining the concentration / content of spinosad in this invention is as follows:

[0040] High-performance liquid chromatography (HPLC) was used to quantitatively determine the content of spinosad in the samples. Chromatographic conditions: column: C18 (5 μm, 250 mm × 4.0 mm); mobile phase: methanol-acetonitrile-0.05% ammonium acetate aqueous solution (V(methanol):V(acetonitrile):V(0.05% ammonium acetate aqueous solution)) = 45:45:10; flow rate: 1.0 mL / min; injection volume: 10 μL; detection wavelength: 250 nm; column temperature: 35℃. Example 1

[0041] 1) Transfer 30L of fermentation broth (containing 4.84g / L spinosad) into a 50L reactor, add 18g of compound enzyme (6g peptidoglycanase, 6g protease, and 6g lysozyme), and enzymatically hydrolyze at 50℃ for 1h.

[0042] 2) After the enzymatic hydrolysis is complete, add a certain amount of tartaric acid to the reaction vessel, adjust its pH to 2-3, and stir and extract at room temperature for 1 hour.

[0043] 3) Adjust the pH of the acid-extracted fermentation broth to 4 using 0.5 mol / L sodium hydroxide solution, and perform microfiltration at an operating pressure of 0.3 MPa and an operating temperature of 30°C. Then, pass the filtrate through a spiral-wound ultrafiltration membrane with a molecular weight cutoff of 1000 Da or higher at an operating pressure of 0.5 MPa and an operating temperature of 36°C. Finally, pass the ultrafiltration filtrate through a spiral-wound nanofiltration membrane with a molecular weight cutoff of 400 Da or higher at an operating pressure of 4 MPa and an operating temperature of 37°C.

[0044] 4) Add 1 / 3 volume of sec-butyl acetate to the concentrated acid solution, and while stirring, add 0.5 mol / L sodium hydroxide solution to the system to adjust the pH to 9-10, and then stir and extract at 50℃ for 30 min.

[0045] 5) After two-phase separation, the sec-butyl acetate phase was vacuum concentrated to 1 / 20 of the original volume at a vacuum degree of 0.01 MPa and a temperature of 45℃, and then cooled to 5℃ at a cooling rate of 10℃ / min, and crystallized at 5℃ for 2 hours.

[0046] 6) After centrifugation and drying at 70°C, 140.33g of spinosad product was obtained with a purity of 92.15% and a yield of 89.06%. Example 2

[0047] 1) Transfer 30L of fermentation broth (containing 4.65g / L spinosad) into a 50L reactor, add 36g of compound enzyme (12g peptidoglycanase, 12g protease, and 12g lysozyme), and enzymatically hydrolyze at 45℃ for 2 hours.

[0048] 2) After the enzymatic hydrolysis is complete, add a certain amount of tartaric acid to the reaction vessel, adjust its pH to 2-3, and stir and extract at room temperature for 2 hours.

[0049] 3) Adjust the pH of the acid-extracted fermentation broth to 4 using 0.5 mol / L sodium hydroxide, and perform microfiltration at an operating pressure of 0.3 MPa and an operating temperature of 30°C. Then, pass the filtrate through a spiral-wound ultrafiltration membrane with a molecular weight cutoff of 3000 Da or higher at an operating pressure of 0.7 MPa and an operating temperature of 41°C. Finally, pass the ultrafiltration filtrate through a spiral-wound nanofiltration membrane with a molecular weight cutoff of 500 Da or higher at an operating pressure of 5 MPa and an operating temperature of 39°C.

[0050] 4) Add 1 / 5 volume of sec-butyl acetate to the concentrated acid solution, and while stirring, add 0.5 mol / L sodium hydroxide solution to the system to adjust the pH to 9-10, and then stir and extract at 60℃ for 30 min.

[0051] 5) After two-phase separation, the sec-butyl acetate phase was vacuum concentrated to 1 / 25 of the original volume under vacuum of 0.04 MPa and 50 °C, and then cooled to 5 °C at a cooling rate of 10 °C / min, and crystallized at 5 °C for 3 hours.

[0052] 6) After centrifugation and drying at 70°C, 135.49g of spinosad product was obtained with a purity of 90.15% and a yield of 87.56%. Example 3

[0053] 1) Transfer 30L of fermentation broth (containing 4.78g / L spinosad) into a 50L reactor, add 9g of compound enzyme (3g peptidoglycanase, 3g protease, 3g lysozyme), and enzymatically hydrolyze at 40℃ for 0.5h.

[0054] 2) After the enzymatic hydrolysis is complete, add a certain amount of tartaric acid to the reaction vessel, adjust its pH to 2-3, and stir and extract at room temperature for 0.5 hours.

[0055] 3) Adjust the pH of the acid-extracted fermentation broth to 4 using 0.5 mol / L sodium hydroxide, and perform microfiltration at an operating pressure of 0.3 MPa and an operating temperature of 33°C. Then, pass the filtrate through a spiral-wound ultrafiltration membrane with a molecular weight cutoff of 5000 Da or higher at an operating pressure of 0.2 MPa and an operating temperature of 35°C. Finally, pass the ultrafiltration filtrate through a spiral-wound nanofiltration membrane with a molecular weight cutoff of 200 Da or higher at an operating pressure of 3 MPa and an operating temperature of 41°C.

[0056] 4) Add 1 / 10 volume of sec-butyl acetate to the concentrated acid solution, and while stirring, add 0.5 mol / L sodium hydroxide solution to the system to adjust the pH to 9-10, and then stir and extract at 50℃ for 30 min.

[0057] 5) After two-phase separation, the sec-butyl acetate phase is vacuum concentrated to 1 / 10 of the original volume under vacuum of 0.08 MPa and 40℃. Then, the temperature is reduced to 5℃ at a cooling rate of 10℃ / min, and crystallization is carried out at 5℃ for 3 hours.

[0058] 6) After centrifugation and drying at 70°C, 134.83g of spinosad product was obtained with a purity of 90.53% and a yield of 85.12%. Example 4

[0059] A method for extracting and purifying spinosad, differing from Example 1, is described in that the spinosad content in the fermentation broth is 4.72 g / L in step (1), and isopropyl acetate (1 / 3 the volume of the concentrated acid solution) is added in step (3). The final yield is 137.77 g of spinosad product with a purity of 90.23% and a yield of 87.79%. Example 5

[0060] A method for extracting and purifying spinosad, differing from Example 1, is described in that the spinosad content in the fermentation broth is 4.58 g / L in step (1), and 1 / 3 volume of n-butyl acetate is added to the concentrated acid solution in step (3). The final yield is 130.09 g of spinosad product with a purity of 90.02% and a yield of 85.23%. Example 6

[0061] A method for extracting and purifying spinosad is provided, differing from Example 1 only in that the spinosad content in the fermentation broth in step (1) is 4.74 g / L, and the crystallization temperature in step (3) is 0°C. A total of 138.79 g of spinosad product was obtained, with a purity of 91.43% and a yield of 89.24%. Comparative Example 1

[0062] A method for extracting and purifying spinosad is presented, differing from Example 1 only in that the spinosad content in the fermentation broth in step (1) is 4.76 g / L, and the membrane separation in step (3) is replaced with an activated carbon column with a packing volume of 1.5 L. A total of 137.06 g of spinosad product was obtained, with a purity of 70.16% and a yield of 67.34%.

[0063] Furthermore, in this process, although the acid solution becomes noticeably clear after activated carbon filtration, its color is brown. During two-phase extraction, a large amount of pigments and impurities enter the sec-butyl acetate phase, increasing the difficulty of phase separation and causing an emulsion layer to appear at the phase interface. During evaporation and crystallization, the high impurity content causes system instability, making it prone to boiling over and difficult to crystallize. During cooling, crystallization begins; the crystals are brown solids that easily adhere to the walls, making the overall operation quite challenging. Comparative Example 2

[0064] A method for extracting and purifying spinosad is provided, differing from Example 1 only in that the spinosad content in the fermentation broth in step (1) is 4.56 g / L, and sec-butyl acetate is replaced with ethyl acetate in step (3). A total of 123.03 g of spinosad product was obtained, with a purity of 87.13% and a yield of 78.36%.

[0065] In this process, during the two-phase extraction, ethyl acetate has a higher polarity than sec-butyl acetate, resulting in a larger residual amount in water and incomplete extraction, leading to a lower yield. During the evaporation and crystallization process, the lower boiling point of ethyl acetate makes it difficult to control the evaporation rate, causing a large amount of crystals to precipitate and clump together, resulting in a decrease in product purity. Comparative Example 3

[0066] 1) Pretreatment: Adjust the pH of 30L of fermentation broth (containing 4.78g / L spinosad) to 8.0 with 0.5mol / L NaOH aqueous solution, add the pretreatment agent, and stir at room temperature for 30min. The volume ratio of fermentation broth to pretreatment agent is 1:0.01. The pretreatment agent consists of 25% zinc sulfate, 25% gypsum, and 50% diatomaceous earth. After centrifugation, spinosad cells precipitate.

[0067] 2) Extraction: Add extraction solvent (methanol) to the precipitate collected in step 1), with a mass ratio of precipitate to added extraction solvent of 1:4. Stir and extract at room temperature for 1.5 hours, filter to obtain the extract, and repeat once.

[0068] 3) Concentration: After mixing the two extracts, concentrate them under vacuum at 60°C to obtain an extract.

[0069] 4) Extraction: Add extraction solvent to the extract and stir at 65°C for 1 hour. Separate the extract to obtain a polysaccharide extract phase. The extraction solvent is ethyl acetate, and the mass ratio of extract to ethyl acetate is 1:2.

[0070] 4) Back-extraction: Add an equal volume of 0.5 mol / L tartaric acid solution to the extract phase and back-extract at room temperature for 1 h. Separate the extract phase to obtain the back-extracted phase.

[0071] 5) Alkali crystallization: Adjust the pH of the back-extraction phase to about 10 and stir for 1 hour. Filter to obtain spinosad wet product.

[0072] 6) Recrystallization: A certain volume of acetone was added to the wet spinosad product at a material-to-liquid ratio (g / ml) of 1:4, and dissolved at 65℃ for 1 hour. Then, distilled water (1 / 3 volume of acetone) was added at 65℃, and the temperature was lowered to 5℃ at a rate of 10℃ / h, allowing crystals to grow for 2 hours. After centrifugation and drying, 128.33 g of spinosad product was obtained with a purity of 89.53% and a yield of 80.12%.

[0073] It is evident that the traditional process (Comparative Example 3) involves numerous steps, is complex to operate, and has insufficient yield and purity.

[0074] In summary, compared with existing technologies, the technical solution of this application simplifies the process steps, reduces operational difficulty, and significantly improves the yield and purity of spinosad.

Claims

1. A method for extracting and purifying a spigginin, characterized by, Including the following steps: 1) Enzymatic hydrolysis: Add biological enzymes to the fermentation broth and hydrolyze at 40℃-50℃ for 0.5-2 hours; 2) Acid extraction: Adjust the pH of the fermentation broth after enzymatic hydrolysis to 2-3, and extract by stirring at room temperature for 0.5-2 hours; 3) Membrane separation: The pH of the fermentation broth after acid extraction is adjusted to 4-5, and purified acid solution is obtained by microfiltration, ultrafiltration and nanofiltration; 4) Two-phase extraction: Add a water-insoluble crystalline solvent to the purified acid solution, and add sodium hydroxide solution to the system while stirring to adjust the pH to 9-10, and extract by stirring at 50-60℃; 5) Evaporation crystallization: After two-phase separation, the crystallization solution is concentrated by evaporation, and then slowly cooled to 0-5℃ to grow crystals; 6) Centrifugation and drying: After centrifuging the crystallization solution, wet spinosad crystals are obtained, and then dried to obtain the product; The bio-enzyme mentioned in step 1) is selected from one or more of peptidoglycanase, protease, and lysozyme; The microfiltration operating pressure mentioned in step 3) is 0.3MPa-0.5MPa, and the operating temperature is 25℃-35℃; The ultrafiltration process described in step 3) uses spiral wound ultrafiltration membranes with a molecular weight cutoff of 1000-5000 Da, an operating pressure of 0.1-0.7 MPa, and an operating temperature of 25-45℃. The nanofiltration process described in step 3) uses spiral wound nanofiltration membranes with a molecular weight cutoff of 200-500 Da, an operating pressure of 2-5 MPa, and an operating temperature of 25-45℃. The crystallization solvent mentioned in step 4) is selected from one of n-butyl acetate, sec-butyl acetate, and isopropyl acetate.

2. The method of claim 1, wherein the method is a method for purifying spinosad. The bioenzyme is a mixture of peptidoglycanase, protease, and lysozyme, with each enzyme added at a rate of 0.1-0.5 g / L.

3. The method of claim 1, wherein the method is a method for purifying spinosad. The volume ratio of the purified acid solution to the crystallization solvent in step 4) is (2-20):

1.

4. The method of claim 1, wherein the method is a method for purifying spinosad. In step 5), the evaporation temperature is 40-50℃, the vacuum degree is 0.01MPa-0.08MPa, the solution is concentrated under vacuum to 1 / 25-1 / 5 of its original volume, the cooling rate is 10℃ / min, and the crystal growth time is 1-4h.

5. The method for extracting and purifying spinosad according to claim 1, characterized in that, In step 2), the acid used to adjust the pH is tartaric acid; in step 3), the alkaline solution used to adjust the pH is a 0.1 mol / L-0.5 mol / L sodium hydroxide solution; in step 4), the concentration of the sodium hydroxide solution used is 0.1 mol / L-0.5 mol / L.

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