A simple extraction method for spinosad

Abstract

The invention relates to a simple method for extracting spinosad, comprising the following steps: 1. pretreatment: adjusting the pH value of a Saccharopolysaccharides spinosa fermentation broth to 9-11, stirring at 75-85 DEG C for 3-5 hours, cooling, and setting aside; 2. extraction: adjusting the pH value of the pretreated fermentation broth to 7.5-8.0, adding an organic solvent, soaking the broth 2-3 times, and collecting an organic phase to obtain an extract; 3. concentration: step 4. alkali washing: alkali-washing the concentrated broth twice, and collecting an organic phase; 5. back-extraction: back-extracting the organic phase collected in step 4 with acid water 4-5 times to obtain an acid-water phase; 6. crystallization: concentrating the collected acid-water phase to 50-70% of the original volume, adjusting the pH value to 9-11, and generating crystals; and 7. filtering, washing, and drying. The invention provides a simple method for extracting spinosad, which has fewer process steps, uses less organic solvent, has low energy consumption, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of spinosad extraction, and in particular to a simple method for extracting spinosad. Background Art

[0002] Spinosad, also known as spinosad, is a highly effective, pollution-free macrolide biopesticide extracted from the fermentation broth of Saccharopolysporaspinosa. It is widely used to control Lepidoptera, Diptera, and Thysanoptera pests.

[0003] Spinosad is a secondary metabolite of Saccharopolyspora spinosa, and its preparation is usually obtained by extraction and separation from the fermentation broth of Saccharopolyspora spinosa. Currently, there are two main methods for extracting and separating spinosad from the fermentation broth of Saccharopolyspora spinosa. One is CN 101560231 discloses a macroporous resin adsorption extraction process. The macroporous resin adsorption extraction process currently has the defects of small adsorption capacity, difficult desorption, small processing capacity, high equipment requirements, multiple processes, long time consumption, and high cost, which is difficult to adapt to the requirements of large-scale industrial production; the other is a method disclosed in a Chinese patent application number CN201010159985.4 for extracting polysaccharides from a fermentation broth of Saccharopolyspora spinosa. The problem is that the entire process route is relatively long, resulting in a low yield. In addition, during the extraction process, not only a high dielectric constant polar organic solvent is required for soaking, but also a low node constant or high carbon alcohol organic solvent is required for extraction, resulting in a large amount of organic solvent used, which not only consumes a lot of energy during the concentration process, but also consumes a long time during the concentration process, especially the high dielectric constant polar organic solvent, which makes this problem more prominent. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a simple extraction method of spinosad, which has fewer process steps, less organic solvent usage, low energy consumption, and is suitable for industrial production.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] Technical solution 1:

[0007] A simple method for extracting spinosad comprises:

[0008] Step 1, pretreatment: adjust the pH of the Saccharopolysaccharides spinosa fermentation broth to 9-11, then stir at 75-85°C for 3-5 hours, and then cool the fermentation broth to below 40°C for use;

[0009] Step 2, extraction: After adjusting the pH of the pretreated fermentation broth to 7.5-8.0, an organic solvent is added and the broth is immersed for 2-3 times at 10-40°C with stirring until the residual spinosad in the fermentation broth is less than 100 mg / L. The organic phase is collected as the extract;

[0010] Step 3, concentration: Concentrate the extract to 50-60% of the original volume;

[0011] Step 4, alkali washing: using alkali washing solution A to wash the concentrate twice at 10-40°C with stirring, and collecting the organic phase;

[0012] Step 5, back extraction: at 20-40 degrees Celsius, use acid water to back extract the organic phase collected in step 4 4-5 times, each time for 5-15 minutes to obtain an acid-water phase;

[0013] Step 6, crystallization: the collected acid-water phase is concentrated to 50-70% of the original volume, and no organic solvent is left in the acid-water phase; then, after the acid-water phase is cooled to below 40°C, the feed liquid is maintained at 30-40°C and the alkaline solution A is added while stirring until the solution pH reaches 9-11, resulting in crystals and obtaining a crystallization liquid;

[0014] Step 7, filtering, washing, and drying: After filtering the crystallization liquid to obtain wet crystals, wash the wet crystals with alkaline washing solution B for 2-3 times, and then dry them at 70°C.

[0015] Furthermore, in step 2, the organic solvent is ethyl acetate or butyl acetate, and the stirring speed is 300-400 r / min.

[0016] Furthermore, in step 2, for the first soaking, the volume ratio of the fermentation liquid to the organic solvent is 1:0.5-1, and the soaking time is 5-6 hours; for the other soakings, the volume ratio of the fermentation liquid to the organic solvent is 1:0.3-0.5, and the soaking time is 2-3 hours.

[0017] Furthermore, in step 4, the alkaline washing solution is a 1-1.5% sodium bicarbonate solution, and the stirring speed is 100-200 r / min.

[0018] Furthermore, in step 4, during each alkaline washing, the volume ratio of the concentrated solution to the alkaline washing solution is 1:0.3-0.5.

[0019] Furthermore, in step 5, the acid water is 0.15-0.2 mol / L tartaric acid solution.

[0020] Furthermore, in step 5, during the first two back extractions, the volume ratio of the organic phase to the acid water is 1:0.45-0.55; during the other back extractions, the volume ratio of the organic phase to the acid water is 1:0.25-0.35.

[0021] Furthermore, in step 6, when adjusting the pH, the alkaline solution A is a sodium carbonate solution with a mass concentration of 30%, and the alkaline solution A is added slowly and drippingly at a speed of 2-3 L / min.

[0022] Furthermore, in step 7, the alkaline washing solution B is a sodium carbonate solution with a mass concentration of 10%.

[0023] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0024] 1. The present invention pre-treats the fermentation broth at a high temperature of 75-85°C under alkaline conditions of pH 9-11, which can change the cell membrane structure of Saccharopolyspora spinosa, stimulate cell autolysis, cause intracellular substances to flow out of the cells, and increase the conversion rate of the secondary metabolite spinosad.

[0025] 2. The present invention directly performs the extraction process of the fermentation broth after pretreatment of the fermentation broth. Compared with the traditional technology, it saves the process of leaching with a high dielectric constant organic solvent and concentrating and removing the organic solvent after leaching, thereby simplifying the production process.

[0026] 3. In the extraction process of the fermentation broth, the pH of the fermentation broth is adjusted to 7.5-8.0, which is conducive to the transfer of spinosad into the organic phase and improves the extraction effect of spinosad.

[0027] 4. Since the present invention uses a higher temperature during the fermentation liquid pretreatment process, which leads to more impurities in the spinosad extract, the present invention adds an alkaline washing process after the fermentation liquid extraction, thereby removing some compatible substances and oils in the extract and improving the product purity.

[0028] 5. The present invention adds a concentration process after extraction, thereby reducing the original volume of the feed liquid during subsequent process treatment, reducing the amount of alkaline washing solution and acid water used in the subsequent process, and reducing the treatment cost of the waste liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 HPLC chart of the spinosad standard sample of the present invention; DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the embodiments.

[0031] In the present invention,

[0032] Method for detecting the concentration of spinosad: HPLC method is used, specifically:

[0033] The extraction test of spinosad was carried out according to the volume ratio of sample to methanol of 1:4. After oscillation and mixing, ultrasonication was carried out for 30 minutes at 3500r / min and centrifuged for 10 minutes. The supernatant was filtered through an organic filter membrane and then detected by HPLC. Chromatographic conditions: Column: Shimadzu Shim-pack VP-ODS 150×4.6mm; Mobile phase: V 甲醇 :V 乙腈 :V 水 (containing 0.05% ammonium acetate) = 45:45:10; injection volume 20 μL; flow rate 1.0 mL / min; column temperature: 28°C; detection wavelength: 245 nm. Calculate the spinosad content in the sample based on the peak areas of the spinosad A and spinosad D components using the spinosad standard curve.

[0034] HPLC chart of spinosad standard sample Figure 1 As shown in the figure, the peak time of Spinosyn A is about 8.5min, and the peak time of Spinosyn D is about 10.1min. The standard curve is drawn with the concentration of spinosad as the horizontal axis and the sum of the peak areas of Spinosyn A and Spinosyn D as the vertical axis. The linear regression equation is Y=13.891X+35.461, and the correlation coefficient R 2 =0.9997, indicating that when the spinosad concentration in the test solution is between 40 and 200 mg / L, there is a good linear relationship between the concentration and the peak area, and this method can be used to detect the spinosad content in the sample.

[0035] Example 1

[0036] Step 1, pretreatment: 1000L of Saccharopolysaccharides spinosa fermentation broth (fermentation broth titer 687μg / ml) was adjusted to pH 10.2 with 10% sodium hydroxide solution, then stirred at 75-85°C for 5 hours, and then cooled to 22°C for standby use;

[0037] Step 2, extraction: the pretreated fermentation broth was adjusted to pH 7.88, ethyl acetate was added as an organic solvent, and the mixture was immersed twice at 10-40°C and stirred (at a stirring speed of 350 r / min) until the residual spinosad in the fermentation broth was less than 100 mg / L, and the organic phase was collected as the extract; the first immersion was performed at a volume ratio of 1:1 between the fermentation liquid and the organic solvent, and the immersion time was 6 hours. The titer of the fermentation broth was detected to be 127 μg / ml; the second immersion was performed at a volume ratio of 1:0.3 between the fermentation liquid and the organic solvent, and the immersion time was 2 hours. The titer of the fermentation broth was detected to be 28 μg / ml, and the extraction yield reached 98.32%;

[0038] Step 3, concentration: using a thin film evaporator to concentrate the extract to 55% of the original volume;

[0039] Step 4, alkali washing: the concentrate was alkali washed twice with alkali washing solution A at 30° C. and under stirring (stirring speed of 180 r / min), and the organic phase was collected; the alkali washing solution A was 1-1.5% sodium bicarbonate solution (pH=8); the volume ratio of the concentrate to the alkali washing solution A was 1:0.3 during each alkali washing; the yield after alkali washing was 97.5%;

[0040] Step 5, back extraction: the organic phase collected in step 4 was back extracted 5 times with acid water at 30°C, each time for 10 minutes to obtain an acid-water phase; the acid water was 0.15 mol / L tartaric acid solution;

[0041] The volume ratio of the organic phase to the acid water was 1:0.5 for the first two back extractions; the volume ratio of the organic phase to the acid water was 1:0.3 for the other back extractions; the back extraction yield was 95.32%.

[0042] Step 6, crystallization: the collected acid-water phase is concentrated to 60% of the original volume, and no organic solvent is left in the acid-water phase; then the acid-water phase is cooled to 40°C, and a 30% sodium carbonate solution is added to the feed liquid at 35°C-40°C while stirring (the sodium carbonate solution is added at a rate of 2-3 L / min), and the pH is adjusted to 10.2 to produce a large amount of crystals to obtain a crystallization solution;

[0043] Step 7, filtration, washing, and drying: After filtering the crystallization liquid to obtain wet crystals, the wet crystals were washed 2-3 times with alkaline washing solution B, and then dried at 70°C to obtain 620g of product; the alkaline washing solution B was 10% sodium carbonate solution, and the crystallization yield was 88.20%.

[0044] After testing: the product content is 98.16%, the product concentration is 893 mg / g, and the total yield is 80.59%.

[0045] Example 2

[0046] Step 1, pretreatment: 2000L of Saccharopolysaccharides spinosa fermentation broth (fermentation broth titer 829 μg / ml) was adjusted to pH 10.61 with 10% sodium hydroxide solution, then stirred at 75-85°C for 5 hours, and then cooled to 28°C for use;

[0047] Step 2, extraction: the pretreated fermentation broth was adjusted to pH 7.52, ethyl acetate was added as an organic solvent, and the mixture was immersed twice at 10-40°C and stirred (at a stirring speed of 350 r / min) until the spinosad residue in the fermentation broth was less than 100 mg / L, and the organic phase was collected as the extract; the first immersion was performed at a volume ratio of 1:1 between the fermentation liquid and the organic solvent, and the immersion time was 6 hours. The titer of the fermentation broth was detected to be 163 μg / ml; the second immersion was performed at a volume ratio of 1:0.29 between the fermentation liquid and the organic solvent, and the immersion time was 3 hours. The titer of the fermentation broth was detected to be 18 μg / ml, and the extraction yield reached 98.48%;

[0048] Step 3, concentration: using a thin film evaporator to concentrate the extract to 58% of the original volume;

[0049] Step 4, alkali washing: using alkali washing solution A to wash the concentrate twice at 30° C. and under stirring conditions (stirring speed of 180 r / min), and collecting the organic phase; the alkali washing solution A is 1-1.5% sodium bicarbonate solution (pH = 8); during each alkali washing, the volume ratio of the concentrate to the alkali washing solution A is 1:0.3, and the alkali washing yield is 97.85%;

[0050] Step 5, back extraction: the organic phase collected in step 4 was back extracted 5 times with acid water at 30 degrees Celsius, each time for 10 minutes to obtain an acid-water phase; the acid water was 0.15 mol / L tartaric acid solution;

[0051] In the first two back extractions, the volume ratio of the organic phase to the acid water was 1:0.5; in the other back extractions, the volume ratio of the organic phase to the acid water was 1:0.3, and the back extraction yield was 97.12%;

[0052] Step 6, crystallization: the collected acid-water phase is concentrated to less than 80% of the original volume, and no organic solvent is left in the acid-water phase; then, after the acid-water phase is cooled to 40°C, the feed liquid is maintained at 33-38°C and sodium carbonate solution is added while stirring (the sodium carbonate solution is added at a rate of 2-3 L / min), and the pH is adjusted to 10.5 to produce a large amount of crystals to obtain a crystallization solution;

[0053] Step 7, filtration, washing, and drying: After filtering the crystallization liquid to obtain wet crystals, the wet crystals were washed 2-3 times with alkaline washing solution B, and then dried at 70°C to obtain 1.39 kg of product; the alkaline washing solution B was 10% sodium carbonate solution; the crystallization yield was 87.22%

[0054] After testing: the product content is 97.37%, the product concentration is 973.763 mg / g, and the total yield is 81.63%.

[0055] Example 3

[0056] Step 1, pretreatment: 2000L of Saccharopolysaccharides spinosa fermentation broth (fermentation broth titer 847 μg / ml) was adjusted to pH 10.12 with 10% sodium hydroxide solution and then stirred at 100° C. for 5 hours, and then the fermentation broth was cooled to 20° C. and set aside;

[0057] Step 2, extraction: the pretreated fermentation broth was adjusted to pH 7.85, and an organic solvent, ethyl acetate, was added, and the mixture was immersed twice at 10-40° C. and under stirring conditions (stirring speed of 380 r / min) until the residual spinosad in the fermentation broth was less than 100 mg / L, and the organic phase was collected to obtain the extract; the first immersion was performed at a volume ratio of fermentation liquid to organic solvent of 1:0.7, and the immersion time was 6 hours. The titer of the fermentation broth was detected to be 375 μg / ml; the second immersion was performed at a volume ratio of fermentation liquid to organic solvent of 1:0.4, and the immersion time was 3 hours. The titer of the fermentation broth was detected to be 85 μg / ml, and the extraction yield reached 98.15%;

[0058] Step 3, concentration: using a thin film evaporator to concentrate the extract to 50-60% of the original volume;

[0059] Step 4, alkali washing: the concentrate was alkali washed twice with alkali washing solution A at 30° C. and under stirring conditions (stirring speed of 180 r / min), and the organic phase was collected; the alkali washing solution A was 1-1.5% sodium bicarbonate solution (pH=8); the volume ratio of the concentrate to the alkali washing solution A was 1:0.4 during each alkali washing; the alkali washing yield was 97.45%;

[0060] Step 5, back extraction: the organic phase collected in step 4 was back extracted 5 times with acid water at 30 degrees Celsius, each time for 10 minutes to obtain an acid-water phase; the acid water was 0.15 mol / L tartaric acid solution;

[0061] During the first two back extractions, the volume ratio of the organic phase to the acid water was 1:0.5; during the other back extractions, the volume ratio of the organic phase to the acid water was 1:0.3; the back extraction yield was 97.25%;

[0062] Step 6, crystallization: the collected acid-water phase is concentrated to 63% of the original volume, and no organic solvent is left in the acid-water phase; then, the acid-water phase is cooled to 40°C, the feed liquid is maintained at 34-39°C, and a 30% sodium carbonate solution is added while stirring (the sodium carbonate solution is added at a rate of 2-3 L / min), and the pH is adjusted to 9.98 to produce a large amount of crystals to obtain a crystallization solution;

[0063] Step 7, filtration, washing, and drying: After filtering the crystallization liquid to obtain wet crystals, the wet crystals are washed 2-3 times with alkaline washing solution B, and then dried at 70°C to obtain 1.4 kg of the product; the alkaline washing solution B is 10% sodium carbonate solution, and the crystallization yield is 87.06%;

[0064] After testing: the product content is 98.72%, the product concentration is 987.26 mg / g, and the total yield is 81.65%.

[0065] The above-described embodiments are only preferred embodiments of the present invention and are not exhaustive of all feasible implementations of the present invention. For those skilled in the art, any obvious modifications made thereto without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A simple method for extracting spinosad, characterized in that: include: Step 1, pretreatment: adjust the pH of the Saccharopolysaccharides spinosa fermentation broth to 9-11, then stir at 75-85°C for 3-5 hours, and then cool the fermentation broth to below 40°C for use; Step 2, extraction: After adjusting the pH of the pretreated fermentation broth to 7.5-8.0, an organic solvent is added and the broth is immersed for 2-3 times at 10-40°C with stirring until the residual spinosad in the fermentation broth is less than 100 mg / L. The organic phase is collected as the extract; In step 2, the organic solvent is ethyl acetate or butyl acetate, and the stirring speed is 300-400 r / min; Step 3, concentration: Concentrate the extract to 50-60% of the original volume; Step 4, alkali washing: using alkali washing solution A to wash the concentrate twice at 10-40°C with stirring, and collecting the organic phase; Step 5, back extraction: at 20-40 degrees Celsius, use acid water to back extract the organic phase collected in step 4 4-5 times, each time for 5-15 minutes to obtain an acid-water phase; Step 6, crystallization: the collected acid-water phase is concentrated to 50-70% of the original volume, and no organic solvent is left in the acid-water phase; then, after the acid-water phase is cooled to below 40°C, the feed liquid is maintained at 30-40°C and the alkaline solution A is added while stirring until the solution pH reaches 9-11, resulting in crystals and obtaining a crystallization liquid; Step 7, filtering, washing, and drying: After filtering the crystallization liquid to obtain wet crystals, wash the wet crystals with alkaline washing solution B for 2-3 times, and then dry them at 70°C.

2. The simple method for extracting spinosad according to claim 1, characterized in that: In step 2, the first soaking: the volume ratio of the fermentation liquid to the organic solvent is 1:0.5-1, and the soaking time is 5-6 hours; the other soaking: the volume ratio of the fermentation liquid to the organic solvent is 1:0.3-0.5, and the soaking time is 2-3 hours.

3. The simple method for extracting spinosad according to claim 1, characterized in that: In step 4, the alkaline washing solution is a 1-1.5% sodium bicarbonate solution, and the stirring speed is 100-200 r / min.

4. The simple method for extracting spinosad according to claim 1, characterized in that: In step 4, during each alkaline washing, the volume ratio of the concentrated solution to the alkaline washing solution is 1:0.3-0.

5.

5. The simple method for extracting spinosad according to claim 1, characterized in that: In step 5, the acid water is 0.15-0.2 mol / L tartaric acid solution.

6. The simple method for extracting spinosad according to claim 1, characterized in that: In step 5, during the first two back extractions, the volume ratio of the organic phase to the acid water is 1:0.45-0.55; during the other back extractions, the volume ratio of the organic phase to the acid water is 1:0.25-0.

35.

7. The simple method for extracting spinosad according to claim 1, characterized in that: In step 6, when adjusting the pH, the alkaline solution A is a sodium carbonate solution with a mass concentration of 30%, and the alkaline solution A is added dropwise at a low speed and a speed of 2-3 L / min.

8. The simple method for extracting spinosad according to claim 1, characterized in that: In step 7, the alkaline washing solution B adopts a sodium carbonate solution with a mass concentration of 10%.

Images