Method for deglycosylation and purification of avermectins

Abstract

The present application relates to a kind of methods for deglucosylation and purification of abamectin, abamectin paste is washed with hot water 2-3 times to remove sugar, the hot water washing method is as follows: then according to the mass volume ratio of abamectin paste and hot water 1g: 1-1.5mL, hot water with temperature of 75-80 ℃ is added to abamectin paste, stir for 1h, start cooling when temperature drops to T1, stop stirring, stratify after standing, then continue to cool to T2, the upper water layer is extracted, and it is finished.The present application replaces traditional deglucosylation solvent toluene with hot water to remove sugar and impurities of abamectin, which is environmentally friendly, avoids a series of problems caused by toluene residues, and reduces the cost of deglucosylation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of avermectin extraction, in particular to a method for deglucosylation and purification of avermectin. BACKGROUND

[0002] Avermectin is a kind of macrolide antibiotic produced by fermentation of Streptomyces avermitilis, which has strong insecticidal activity and is widely used as agricultural or veterinary insecticide and acaricide. It has a broad insecticidal spectrum and has strong insecticidal activity on almost all insects and mites and animal parasites. At the same time, this kind of avermectin is easily decomposed by microorganisms in the soil and has no accumulation and persistence in the body, so it will not pollute the environment, and therefore has great potential for development.

[0003] In the current extraction process of avermectin, the mycelium is usually soaked with methanol, then the soaking liquid is concentrated to obtain a paste, and then deglucosylation is carried out. In the material, 6-8 times of toluene and appropriate hot water at 80 DEG C are added, stirred for 1 h, and then the lower layer water is discharged. After the deglucosylation water is collected, it is transferred to the incinerator, and the toluene is recovered under vacuum. The clear water and toluene are separated and then reused. When the material is in paste form, methanol is added for crystallization, and the dried avermectin product is obtained.

[0004] However, in the deglucosylation process, the material cannot be completely concentrated to remove toluene (when the toluene content of the deglucosylation end material is 0.3%, the evaporation time and temperature no longer increase, and the toluene content detected by liquid chromatography no longer decreases). When methanol is added for crystallization, toluene enters the crystallization process and is mixed into methanol, which enters the soaking process again after being recycled, resulting in that many impurities dissolved in toluene in the mycelium enter the soaking liquid, affecting the quality of crystallization, causing the quality of the avermectin product to be unstable, and thus affecting the reaction of downstream products. SUMMARY

[0005] The present application aims to overcome the defects of the prior art and provide a method for purifying and deglucosylating avermectin, which uses hot water instead of the traditional deglucosylation solvent toluene to deglucosylate and remove impurities from avermectin. The process is environmentally friendly, avoids a series of problems caused by toluene residues, and reduces the cost of deglucosylation.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] Technical scheme one:

[0008] A method for deglucosylating avermectin, wherein the paste of avermectin is washed with hot water for 2-3 times for deglucosylation, and the hot water washing method is as follows:

[0009] The hot water is added to the abamectin paste at a mass-volume ratio of 1g:1-1.5mL, and stirring is performed for 0.5-1.5h, then temperature reduction is started, when the temperature is reduced to T1, stirring is stopped, and then the upper water layer is extracted when the temperature is reduced to T2.

[0010] Further, the temperature of the hot water is 75-80℃.

[0011] Before the hot water is added to the abamectin paste, the abamectin paste is heated to 70-80℃.

[0012] Further, the content of abamectin B1a in the abamectin paste is greater than or equal to 20%.

[0013] T1=60℃, and T2=35-40℃.

[0014] Further, the hot water washing method comprises the following steps: the hot water is added to the abamectin paste at a mass-volume ratio of 1g:1-1.5mL, and the temperature of the hot water is 75-80℃, stirring is performed for 0.5-1.5h, then temperature reduction is started, when the temperature is reduced to 60℃, stirring is stopped, and then the upper water layer is extracted when the temperature is reduced to 35-40℃.

[0015] Further, the abamectin paste is prepared from the mycelium of Streptomyces avermitilis by methanol extraction, desolventization and concentration.

[0016] Further, the mycelium of Streptomyces avermitilis is obtained from the fermentation broth of Streptomyces avermitilis fermentation, and the titer of the fermentation broth is greater than or equal to 7000μg / mL.

[0017] Further, the Streptomyces avermitilis is the strain FT26-9, the strain FT26-9 has the preservation number of CGMCC No.4341, and was preserved on November 12, 2010 in the China General Microbiological Culture Collection Center, and the strain FT26-9 belongs to the prior art.

[0018] Further, the preparation method of the abamectin paste is as follows:

[0019] The mycelium of Streptomyces avermitilis after drying is extracted by methanol for at least 3 times, the mass ratio of the mycelium to methanol is 1:10-12, and the extraction time is 4h each time, the extraction liquid is combined, filtered, and evaporated to desolventize in an evaporation tank, when the temperature in the evaporation tank is increased to 80-82℃ and the vacuum degree reaches-0.08MPa to-0.082MPa, the abamectin paste is obtained.

[0020] Technical solution two:

[0021] A method for purifying abamectin, wherein the abamectin paste is desugared by the desugaring method, and then crystallized 1-3 times, filtered, and dried to obtain abamectin.

[0022] Further, the first crystallization comprises the following steps: adding a crystallization solvent to the desugared abamectin paste, heating to 70-77℃, stirring for 30-40 minutes, then cooling crystallization, when the temperature drops to 25-30℃, keeping for 2 hours, filtering, obtaining abamectin crystals and crystallization mother liquor, and washing the abamectin crystals with the crystallization solvent for standby use.

[0023] The second and subsequent crystallization comprises the following steps: adding a crystallization solvent and a decolorizing agent to the abamectin crystals obtained by the previous crystallization, heating to 70-77℃, stirring for 30-40 minutes, then decarbonizing filtration, cooling crystallization of the filtrate, when the temperature drops to 25-30℃, keeping for 2 hours, filtering, obtaining abamectin crystals and crystallization mother liquor.

[0024] The crystallization solvent is methanol, and the decolorizing agent is activated carbon.

[0025] Technical solution three:

[0026] A method for purifying abamectin, comprising the following steps:

[0027] Step 1: desugaring the abamectin paste by washing with hot water 2-3 times;

[0028] Step 2: crystallizing the desugared abamectin paste 1-3 times, drying the wet crystals, and obtaining abamectin.

[0029] The first crystallization comprises the following steps: adding a crystallization solvent to the desugared abamectin paste, heating to 70-77℃, stirring for 30-40 minutes, then cooling crystallization, when the temperature drops to 25-30℃, keeping for 2 hours, filtering, obtaining abamectin crystals and crystallization mother liquor, and washing the abamectin crystals with the crystallization solvent for standby use.

[0030] The second and subsequent crystallization comprises the following steps: adding a crystallization solvent and a decolorizing agent to the abamectin crystals obtained by the previous crystallization, heating to 70-77℃, stirring for 30-40 minutes, then decarbonizing filtration, cooling crystallization of the filtrate, when the temperature drops to 25-30℃, keeping for 2 hours, filtering, obtaining abamectin crystals and crystallization mother liquor.

[0031] Technical solution four:

[0032] Abamectin prepared by the method for purifying abamectin.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The present application uses fermentation broth with a titer of greater than 7000 μg / mL to extract abamectin by replacing the fermentation strain of Streptomyces avermitilis. Compared with the traditional strain, the content of reducing sugar and total sugar in the fermentation broth is significantly reduced. Therefore, in the desugaring process, only hot water is used to remove the sugar impurities. Compared with the traditional desugaring process, toluene is no longer added, which not only reduces the production cost and protects the environment, but also completely solves the problems of deviation of leaching effective components and reduction of the purity of crystallization caused by the mixing of toluene in the extraction solvent during the production process in the extraction workshop, greatly improving the product quality.

[0035] The present application is simple to operate, has high purity of extracted abamectin, and is suitable for industrialized production. DETAILED DESCRIPTION

[0036] The present application will be further described in detail below in combination with examples.

[0037] In the present application,

[0038] The preparation method of the abamectin paste is as follows:

[0039] Step a, filtration: the fermentation broth of Streptomyces avermitilis (titer 7892 μg / mL) is heated to 80-90℃, and plate and frame pressure filtration is performed, and hot air drying is performed for 20-30 min to obtain mycelium; the Streptomyces avermitilis uses Streptomyces avermitilis FT26-9 strain;

[0040] Step b, drying: the mycelium is dried using a flash drying machine, the mycelium enters the stirring, crushing, drying chamber, and is crushed and hot air dried at the same time, until the moisture content of the mycelium is ≤40%, to obtain dry mycelium.

[0041] Step c, leaching: methanol is added to the dry mycelium, and leaching is performed at least 3 times, the mass ratio of mycelium to methanol is 1:11 each time, and the leaching time is 4 h each time, and the leaching liquid is combined;

[0042] Step d, evaporation and concentration: the collected leaching liquid is filtered and placed in an evaporation tank for evaporation and desolventization until the temperature in the evaporation tank is raised to 80-82℃ and the vacuum degree reaches -0.08 to -0.082 MPa, to obtain abamectin paste;

[0043] Example 1

[0044] A method for purifying abamectin, comprising the following steps:

[0045] Step 1, desugaring:

[0046] The 3000 KG of abamectin paste (containing 21.3% of B1a) was desugared by hot water washing twice, and 2480 KG of desugared abamectin paste was obtained, and the B1a content was 25.6% by detection. The yield was 99.4%;

[0047] The first hot water washing method is as follows:

[0048] First, the abamectin paste was heated to 80℃, then 3500L of hot water at 80℃ was added to the 3000KG of abamectin paste (containing 21.3% of B1a), after stirring for 1h, the temperature began to drop, when the temperature dropped to 60℃, the stirring was stopped, and the layers were separated by standing, then the temperature continued to drop to 35℃, and the upper water layer was extracted;

[0049] The second hot water washing method is as follows:

[0050] The abamectin paste after the first hot water washing was heated to 80℃, then 3500L of hot water at 80℃ was added, after stirring for 1h, the temperature began to drop, when the temperature dropped to 60℃, the stirring was stopped, and the layers were separated by standing, then the temperature continued to drop to 35℃, and the upper water layer was extracted;

[0051] Step 2, crystallization:

[0052] First crystallization: 2480 KG of desugared abamectin paste was added to methanol, heated to 73℃, stirred for 35min, then cooled to crystallize, when the temperature dropped to 25℃, the temperature was kept for 2h, and then filtered, to obtain the first crystal and the crystallization mother liquor; the first crystal was washed with methanol and prepared for use;

[0053] Second crystallization: methanol and activated carbon were added to the first crystal, heated to 73℃, stirred for 35min, then decarbonized and filtered, the filtrate was cooled to crystallize in a crystallization tank, when the temperature dropped to 25℃, the temperature was kept for 2h, and then filtered, to obtain the second crystal and the crystallization mother liquor; the second crystallization was used to crystallize again to obtain the third crystal.

[0054] The third crystal was washed with methanol and dried, to obtain 484.73KG of abamectin B1 product (containing 98.2% of B1a), and the crystallization mother liquor of the second crystallization was combined, to obtain 16300L of crystallization mother liquor (containing 9289ug / ml of B1a).

[0055] The abamectin B1 product of this example meets the relevant provisions of the national standard GB / T 19336-2017, and the yield of abamectin B1 (crystallization mother liquor and abamectin B1 product) is 98.19%.

[0056] Example 2

[0057] A method for purifying abamectin, comprising the following steps:

[0058] Step 1, desugaring:

[0059] 3500 kg of abamectin paste (containing 20.5% of B1a) is desugared by hot water washing twice, obtaining 2840 kg of desugared abamectin paste, and the content of B1a is 25.1% by detection. The yield is 99.4%;

[0060] The first hot water washing method is as follows:

[0061] First, the abamectin paste is heated to 76℃, then 3500L of hot water at 80℃ is added to 3500 kg of abamectin paste, and after stirring for 1h, the temperature begins to drop, and when the temperature drops to 60℃, the stirring is stopped, and the layers are separated, then the temperature continues to drop to 37℃, and the upper water layer is extracted;

[0062] The second hot water washing method is as follows:

[0063] The abamectin paste after the first hot water washing is heated to 78℃, then 4500L of hot water at 80℃ is added, and after stirring for 1h, the temperature begins to drop, and when the temperature drops to 60℃, the stirring is stopped, and the layers are separated, then the temperature continues to drop to 37℃, and the upper water layer is extracted;

[0064] Step 2, crystallization:

[0065] First crystallization: 2840 kg of desugared abamectin paste is added with methanol, heated to 65℃, stirred for 38 min, then cooled and crystallized, when the temperature drops to 25℃, keep for 2h, and then filter, obtaining the first crystal and the crystallization mother liquor; the first crystal is washed with methanol and reserved;

[0066] Second crystallization: methanol and activated carbon are added to the first crystal, heated to 65℃, stirred for 35 min, then decarbonized and filtered, the filtrate is cooled and crystallized in a crystallization tank, when the temperature drops to 25℃, keep for 2h, and then filter, obtaining the second crystal and the crystallization mother liquor; the second crystallization is used to crystallize again to obtain the third crystal.

[0067] The third crystal is washed with methanol and dried, obtaining 543.24 kg of abamectin B1 finished product (containing 98.30% of B1a), and the crystallization mother liquor of the second crystallization is combined, obtaining 19700L of crystallization mother liquor (containing 8768ug / ml of B1a).

[0068] The abamectin B1 product of the embodiment meets the relevant provisions of the national standard GB / T 19336-2017, and the yield of abamectin B1 (crystallization mother liquor and abamectin B1 product) is 98.5%.

[0069] Comparative Example 1

[0070] A purification method of abamectin, comprising the following steps:

[0071] Step 1, desugaring:

[0072] To 3000KG of abamectin paste (B1a content of 20.8%) add 4000L of toluene, heat to completely dissolve, then add 3000L of 80℃ hot water, continue to stir for 1 hour, stand for 1 hour, then discharge the lower water layer, and then evaporate and desolventize in an evaporator, until the temperature of the evaporator is raised to 80℃, and the vacuum degree reaches-0.08MPa, to obtain 2460KG of desugared abamectin paste, wherein the B1a content is 23.9%, and the yield is 94.3%;

[0073] Step 2, crystallization

[0074] First crystallization: to 2460KG of desugared abamectin paste, add methanol, heat to 65℃, stir for 35min, then perform temperature reduction crystallization, when the temperature is reduced to 25℃, keep for 2h, then perform suction filtration, to obtain first crystals and crystallization mother liquor; the first crystals are washed with methanol and reserved;

[0075] Second crystallization: to the first crystals, add methanol and activated carbon, heat to 65℃, stir for 35min, then perform decarbonization filtration, the filtrate is reduced in temperature in a crystallization tank, when the temperature is reduced to 25℃, keep for 2h, then perform suction filtration, to obtain second crystals and crystallization mother liquor; the second crystals are crystallized again to obtain third crystals.

[0076] The third crystals are washed with methanol and dried, to obtain 464.55KG of abamectin B1 product (B1a purity of 94.5%), and 15500L of crystallization mother liquor (crystallization mother liquor with a titer of 9439ug / ml) is also obtained.

[0077] The abamectin B1 product of the embodiment meets the relevant provisions of the national standard GB / T 19336-2017, and the yield of abamectin B1 (crystallization mother liquor and abamectin B1 product) is 98.5%.

[0078] The above-described embodiments are merely preferred embodiments of the present application, but are not exhaustive. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present application should be considered to be within the scope of the claims of the present application.

Claims

1. A method for purifying avermectin, characterized in that, After desugaring the avermectin paste, crystallize it 1-3 times, and dry the wet crystals to obtain avermectin. The avermectin paste is prepared from Streptomyces avermectin mycelium through methanol extraction, solvent removal and concentration processes. Streptomyces avermitus mycelium was obtained by solid-liquid separation of the fermentation broth from Streptomyces avermitus fermentation. The potency of the fermentation broth is greater than or equal to 7000 μg / mL; The sugar removal method includes the following steps: washing the abamectin paste with hot water 2-3 times to remove sugar; The hot water washing method is as follows: First, heat the abamectin paste to 70℃-80℃, then add hot water at 75℃-80℃ to the abamectin paste at a mass-volume ratio of 1g:1-1.5mL. Stir for 0.5-1.5 hours, then start cooling. When the temperature drops to 60℃, stop stirring, let it stand and separate into layers, and then continue cooling to 35℃-40℃. Then, remove the upper water layer. The first crystallization includes the following steps: add crystallization solvent to the desugared avermectin paste, heat to 70℃-77℃, stir for 30 min-40 min, and then perform cooling crystallization. When the temperature drops to 25℃-30℃, keep it at that temperature for 2 h, filter, and obtain avermectin crystals and crystallization mother liquor. The avermectin crystals are washed with crystallization solvent and set aside for later use. The second and subsequent crystallization processes include the following steps: adding crystallization solvent and decolorizing agent to the avermectin crystals obtained from the first crystallization, heating to 70℃-77℃, stirring for 30 min to 40 min, decarbonizing and filtering, and then cooling the filtrate for crystallization. When the temperature drops to 25℃ to 30℃, it is kept at that temperature for 2 h, and then filtered to obtain avermectin crystals and crystallization mother liquor.

2. The purification method for avermectin according to claim 1, characterized in that, The content of abamectin B1a in the abamectin paste is greater than or equal to 20%.

3. The purification method for avermectin according to claim 1, characterized in that, The Streptomyces avermectin used is strain FT26-9.

4. The purification method for avermectin according to claim 1, characterized in that, The preparation method of the avermectin paste is as follows: The mycelium of dried Streptomyces avermectin was extracted with methanol at least three times, with a mass ratio of mycelium to methanol of 1:10 to 12 for each extraction, and an extraction time of 4 hours each time. The extracts were combined, filtered, and the filtrate was placed in an evaporator to evaporate and desolvent until the temperature inside the evaporator reached 80℃ to 82℃ and the vacuum degree reached -0.08MPa to -0.082MPa, to obtain an avermectin paste.

5. The purification method for avermectin according to claim 1, characterized in that, The decolorizing agent is activated carbon.