A method for extracting and purifying avermectin B1a using avermectin fermentation broth

Abstract

The present invention relates to a method for extracting and purifying avermectin B1a using avermectin fermentation broth, comprising a fermentation broth active enzyme pretreatment step, a heat reflux circulation extraction step, a desugaring treatment step and a gradient crystallization step. The method can greatly shorten the extraction cycle, reduce energy consumption and reduce production costs, effectively improve the quality and yield of the finished product, and is environmentally friendly and can be applied in continuous large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of antibiotic extraction and purification, and specifically relates to a method for extracting and purifying avermectin B1a by utilizing avermectin fermentation broth. Background Art

[0002] Avermectin, also known as avermectin, is a new type of agricultural and livestock dual-purpose antibiotic. It belongs to the macrolide antibiotic class of insecticides and acaricides. It is a fermentation metabolite of the soil microorganism Streptomyces avermitilis. The chemical structure of avermectin is a macrolide with disaccharide branches. It is composed of 8 components, including A1a, A1b, A2a, A2b, Bla, B1b, B2a and B2b. Among the 8 components, the biological activity of component B is better than that of component A, and component B1a has the strongest activity.

[0003] However, in the process of realizing the technical solutions of the invention in the embodiments of the present application, the inventors of the present application found that the prior art has at least the following technical problems: the avermectin extraction method has a long extraction cycle, consumes a lot of solvents and heat sources, and has complicated procedures. In addition, the residual potency in the mushroom residue after extraction cannot be completely extracted into the extract, resulting in a large unit loss. The extraction rate of avermectin B1a is relatively low, the impurity content is high, and the clarity is poor. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a method for extracting avermectin B1a which can greatly shorten the extraction cycle, reduce energy consumption and production costs, and significantly improve the yield and purity of the finished product.

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

[0006] A method for extracting and purifying avermectin B1a using avermectin fermentation broth comprises the following steps:

[0007] (1) fermentation broth pretreatment step: adding active enzyme to the avermectin fermentation broth and filtering by plate and frame to obtain a filter cake;

[0008] (2) subjecting the filter cake from step (1) to a first solvent hot reflux cycle leaching to obtain a paste;

[0009] (3) desugaring the paste obtained in step (2) with a second solvent to obtain a crude avermectin product;

[0010] (4) Recrystallizing the crude avermectin product from step (3) to obtain a refined avermectin product.

[0011] In some embodiments of the present invention, the fermentation broth in step (1) is a fermentation broth of Streptomyces avermitilis.

[0012] In some embodiments of the present invention, the active enzyme in step (1) is peptidoglycan hydrolase.

[0013] In some embodiments of the present invention, the amount of active enzyme used in step (1) is 2-10‰ (V / V) of the fermentation broth.

[0014] In some embodiments of the present invention, the first solvent in step (2) is methanol.

[0015] In some embodiments of the present invention, in step (2), the concentration of the first solvent is above 95%.

[0016] In some embodiments of the present invention, the second solvent in step (3) is 1,1,1-trichloroethane.

[0017] In some embodiments of the present invention, the amount of the second solvent in step (3) is 5-6 times the mass ratio of avermectin mycelium B1a per billion.

[0018] In some embodiments of the present invention, the recrystallization in step (4) is gradient crystallization.

[0019] In some embodiments of the present invention, step (4) is as follows: after the gradient crystallization temperature is raised to 60-80°C, it is lowered from 75°C to 40°C, and then from 40°C to 10-15°C, and the cooling rate is controlled at 2-6°C / h.

[0020] The more specific implementation of the present invention is as follows:

[0021] A method for extracting and purifying avermectin B1a using avermectin fermentation broth comprises the following steps:

[0022] 1. Filter

[0023] Add 2-10‰ peptidoglycan hydrolase to the avermectin fermentation broth, stir evenly for 30 minutes, filter through a plate and frame filter press for 30 minutes, press the filter cake dry, and then transport the filter cake to a crusher for crushing.

[0024] 2. Extraction and concentration

[0025] The crushed filter cake is then placed in an extraction tank, and the amount of extract added at one time is 80 times the total amount of avermectin mycelium B1a, and methanol with a concentration of more than 95% is added from the storage tank to the extraction tank for stirring for 30-60 minutes at a temperature of 40-60°C. The extract after solid-liquid separation enters the evaporation tank when it fills the siphon and is heated and fully stirred until it evaporates. The generated solvent vapor enters the condenser, and after condensation, it is returned to the storage tank and flows into the extraction tank again. This cycle is repeated. After evaporation until there is no methanol, a paste is obtained in the tank. Then 1,1,1-trichloroethane with a concentration of 5-6 times the total amount of avermectin mycelium B1a is added to the paste, and the temperature is raised to 70°C before entering the desugaring step. The fungus residue after solid-liquid separation can be made into biological fertilizer for recycling.

[0026] 3. Sugar removal

[0027] Add 1,1,1-trichloroethane 5 times the total amount of avermectin B1a to the paste, then add 500-1000L of hot water to the desugaring tank, heat the desugaring tank to 60-70°C, continue stirring for 20-30 minutes, stop, let stand for 20-30 minutes, separate the liquid and release the upper water, repeat the washing once, and evaporate it with water to evaporate all the remaining 1,1,1-trichloroethane, add methanol 0.5-1.5 times the weight of the end material, heat to 70°C and stir for 20-30 minutes.

[0028] 4. Gradient crystallization

[0029] After the crystallization temperature rises to 60-80°C, it drops from 75°C to 40°C, and the cooling rate is controlled at 2-3°C / h, and then drops from 40°C to 10-15°C, and the cooling rate is 5°C / h. When the temperature is maintained at around 10-15°C and the temperature does not drop any more, stirring is maintained for 1h and then centrifuged to obtain a single crystal; similarly, after three crystallizations, the product is packaged and mixed.

[0030] The technical solution of the present invention has the following beneficial technical effects:

[0031] 1. In the present invention, peptidoglycan hydrolase is added to the avermectin fermentation broth and filtered through a plate and frame to obtain a filter cake. The peptidoglycan hydrolase can quickly destroy the dense cell wall membrane, and the avermectin B1a is effectively retained in the filter cake, which greatly shortens the leaching cycle. In addition, the impurity content in the leaching solution is low and the color is clear, which reduces the activated carbon decolorization step in the subsequent process.

[0032] 2. The filter cake is leached by hot reflux circulation to obtain the leaching solution. The hot reflux circulation leaching process adopts dynamic extraction. There is a high solute gradient between the filter cake and the solvent, which increases the leaching driving force, thereby improving the paste yield. This leaching process adopts a fully enclosed closed-loop cycle, which can extract 5-20% more than the conventional method, and can save 30-50% of the solvent, improve the quality of the leaching solution, and reduce energy consumption. Because secondary steam is used as the heat source, extraction and concentration are carried out simultaneously, and the temperature of the reflux condensate is close to the boiling temperature in the extraction tank, which saves about 55% of steam compared with the original process. The overall step does not produce odor, creating a good working environment for the operator and reducing environmental pressure;

[0033] 3. Use 1,1,1-trichloroethane for dissolution and extraction, which is low-cost and truly low-toxic and environmentally friendly.

[0034] This method does not involve heating the fermentation liquid, does not destroy the active ingredient of avermectin B1a, and greatly shortens the extraction cycle, reduces energy consumption and the amount of organic solvents and activated carbon, reduces production costs, and improves the quality and yield of finished products. The greatest advantage is that there are almost no avermectin B1a units remaining in the fungus residue, and reduces wastewater generation during the desugaring process. It is environmentally friendly and can be used in continuous large-scale production. DETAILED DESCRIPTION

[0035] The embodiments of the present invention will be described clearly and completely below in conjunction with the examples. Obviously, the described examples are only some examples of the present invention, rather than all examples. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0036] The avermectin fermentation liquid in the following examples is obtained by fermentation of Streptomyces avermitilis, and the fermentation process is a conventional process. The commonly used carbon sources in the fermentation medium are mainly soybean meal and starch; the commonly used nitrogen sources are mainly corn steep liquor, ammonium sulfate, etc. The fermentation cycle is generally about 300 to 380 hours, and the fermentation culture is completed with a titer of 4500 to 6500 U / ml.

[0037] Example 1

[0038] 1. Filter

[0039] Add 400 kg of peptidoglycan hydrolase to 100 m2 of avermectin fermentation liquid with a potency of 5500 U / ml, stir evenly for 30 minutes, pump it into a plate and frame filter with a feed pump for filtration, and press for 30 minutes.

[0040] 2. Extraction and concentration

[0041] The crushed filter cake is then placed in an extraction tank, and the amount of extract added at one time is 80 times the amount of avermectin mycelium B1a per billion. Methanol with a concentration of more than 95% is added from the storage tank to the extraction tank, and the stirring time is 45 minutes at a temperature of 50°C, and then solid-liquid separation is performed. After the extract is evaporated until there is no methanol, a paste is obtained, and 1,1,1-trichloroethane with an amount of 5 times the amount of avermectin mycelium B1a per billion is added to the paste. After the temperature rises to 70°C, the sugar removal step is performed. The residual potency of avermectin B1a in the filter residue is 23U / ml, and the potency of the concentrated solution in the tank is 12759U / ml.

[0042] 3. Sugar removal

[0043] Add 1,1,1-trichloroethane (5 times the total amount of Avermectin B1a) to the paste, then add 650L of hot water to each desugaring tank, raise the temperature of the desugaring tank to 68°C, continue stirring for 25 minutes, then stop stirring, let it stand for 30 minutes, separate the liquid and release the upper layer of water, repeat the washing once, and evaporate it with water to evaporate all the remaining 1,1,1-trichloroethane, add methanol (0.8 times the weight of the end material), raise the temperature to 70°C and stir for 30 minutes.

[0044] 4. Gradient crystallization

[0045] During the gradient crystallization, the temperature was raised to 60-80°C, then dropped from 75°C to 45°C, and then from 45°C to 12°C, and the cooling rate was controlled at 5°C / h. When the temperature was maintained at about 10-15°C and the temperature did not drop any more, the mixture was stirred for 1h and then centrifuged to obtain a primary crystal. Similarly, after three crystallizations, the mixture was mixed and packaged to obtain the finished product. The content of avermectin B1a was measured to be 95.2%, and the total yield was 97.19%.

[0046] Example 2

[0047] 1. Filter

[0048] 100㎡ of avermectin fermentation liquid with a potency of 6500U / ml was added with 600kg of peptidoglycan hydrolase, stirred evenly for 30min, pumped into a plate and frame filter with a feed pump for filtration, and pressed for 30min.

[0049] 2. Extraction and concentration

[0050] The crushed filter cake is then placed in an extraction tank, and the amount of extract added at one time is 80 times the amount of avermectin mycelium B1a per billion. Methanol with a concentration of more than 95% is added from the storage tank to the extraction tank, and the stirring time is 50 minutes at a temperature of 55°C, and then solid-liquid separation is performed. After the extract is evaporated until there is no methanol, a paste is obtained, and then 1,1,1-trichloroethane with an amount of 5 times the amount of avermectin mycelium B1a per billion is added to the paste. After the temperature rises to 70°C, the sugar removal step is performed. The residual potency of avermectin B1a in the filter residue is 21U / ml, and the potency of the concentrated solution in the tank is 13127U / ml.

[0051] 3. Sugar removal

[0052] Add 1,1,1-trichloroethane (5 times the total amount of Avermectin B1a) to the paste, then add 780L of hot water to each desugaring tank, heat the desugaring tank to 70°C, continue stirring for 25 minutes, then stop stirring, let it stand for 30 minutes, separate the liquid and release the upper layer of water, repeat the washing once, and evaporate it azeotropically with water to evaporate all the remaining 1,1,1-trichloroethane, add methanol (1.1 times the weight of the end material), heat to 70°C and stir for 25 minutes.

[0053] 4. Gradient crystallization

[0054] During the gradient crystallization, the temperature was raised to 60-80°C, then decreased from 75°C to 45°C, and then from 45°C to 14°C, and the cooling rate was controlled at 5°C / h. When the temperature was maintained at about 10-15°C and the temperature did not drop any more, the mixture was stirred for 1h and then centrifuged to obtain a primary crystal. Similarly, after three crystallizations, the mixture was mixed and packaged to obtain the finished product. The content of avermectin B1a was measured to be 95.7%, and the total yield was 96.26%.

[0055] Example 3

[0056] 1. Filter

[0057] 100㎡ of avermectin fermentation liquid with a potency of 6200U / ml was added with 830kg of peptidoglycan hydrolase, stirred evenly for 30min, pumped into a plate and frame filter with a feed pump for filtration, and pressed for 30min.

[0058] 2. Extraction and concentration

[0059] The crushed filter cake is then placed in an extraction tank, and the amount of extract added at one time is 80 times the amount of avermectin mycelium B1a per billion. Methanol with a concentration of more than 95% is added from the storage tank to the extraction tank, and the stirring time is 52 minutes at a temperature of 60°C, and then solid-liquid separation is performed. After the extract is evaporated until there is no methanol, a paste is obtained, and 1,1,1-trichloroethane with an amount of 5 times the amount of avermectin mycelium B1a per billion is added to the paste. After the temperature rises to 70°C, the sugar removal step is performed. The residual potency of avermectin B1a in the filter residue is 30U / ml, and the potency of the concentrated solution in the tank is 12991U / ml.

[0060] 3. Sugar removal

[0061] Add 1,1,1-trichloroethane (5 times the total amount of Avermectin B1a) to the paste, then add 810L of hot water to each desugaring tank, raise the temperature of the desugaring tank to 63°C, continue stirring for 28 minutes, then stop stirring, let it stand for 30 minutes, separate the liquid and release the upper layer of water, repeat the washing once, and evaporate it azeotropically with water to evaporate all the remaining 1,1,1-trichloroethane, add methanol (1 times the weight of the end material), heat to 65°C and stir for 30 minutes.

[0062] 4. Gradient crystallization

[0063] During the gradient crystallization, the temperature was raised to 60-80°C, then dropped from 75°C to 45°C, and then from 45°C to 15°C, and the cooling rate was controlled at 5°C / h. When the temperature was maintained at about 10-15°C and the temperature did not drop any more, the mixture was stirred for 1h and then centrifuged to obtain the first crystal. Similarly, after three crystallizations, the mixture was mixed and packaged to obtain the finished product. The content of avermectin B1a was measured to be 95.4%, and the total yield was 96.91%.

[0064] Example 4

[0065] 1. Filter

[0066] 100㎡ of avermectin fermentation liquid with a potency of 5800U / ml was added with 650kg of peptidoglycan hydrolase, stirred evenly for 30min, pumped into a plate and frame filter with a feed pump for filtration, and pressed for 30min.

[0067] 2. Extraction and concentration

[0068] The crushed filter cake is then placed in an extraction tank, and the amount of extract added at one time is 80 times the amount of avermectin mycelium B1a per billion. Methanol with a concentration of more than 95% is added from the storage tank to the extraction tank, and the stirring time is 35 minutes at a temperature of 55°C, and then solid-liquid separation is performed. After the extract is evaporated until there is no methanol, a paste is obtained, and 1,1,1-trichloroethane with an amount of 5 times the amount of avermectin mycelium B1a per billion is added to the paste. After the temperature rises to 70°C, the sugar removal step is performed. The residual potency of avermectin B1a in the filter residue is 74U / ml, and the potency of the concentrated solution in the tank is 12119U / ml.

[0069] 3. Sugar removal

[0070] Add 1,1,1-trichloroethane (5 times the total amount of Avermectin B1a) to the paste, then add 700L of hot water to each desugaring tank, raise the temperature of the desugaring tank to 68°C, continue stirring for 30 minutes, then stop stirring, let it stand for 30 minutes, separate the liquid and release the upper layer of water, repeat the washing once, and evaporate it azeotropically with water to evaporate all the remaining 1,1,1-trichloroethane, add methanol (1.2 times the weight of the end material), raise the temperature to 70°C and stir for 30 minutes.

[0071] 4. Gradient crystallization

[0072] During the gradient crystallization, the temperature was raised to 60-80°C, then dropped from 75°C to 45°C, and then from 45°C to 13°C, and the cooling rate was controlled at 5°C / h. When the temperature was maintained at about 10-15°C and the temperature did not drop any more, the mixture was stirred for 1h and then centrifuged to obtain a primary crystal. Similarly, after three crystallizations, the mixture was mixed and packaged to obtain the finished product. The content of avermectin B1a was measured to be 94.9%, and the total yield was 96.37%.

[0073] Comparative Example 1

[0074] 1. Filter

[0075] 100㎡ of avermectin fermentation liquid with a potency of 5800U / ml was added with 650kg of peptidoglycan hydrolase, stirred evenly for 30min, pumped into a plate and frame filter with a feed pump for filtration, and pressed for 30min.

[0076] 2. Extraction

[0077] Add 100 times the total amount of methanol per billion of avermectin mycelium B1a to the filter cake at one time; extract and stir for 3 hours, and let stand for 2 hours; obtain a primary extract after solid-liquid separation, add 100 times the total amount of methanol per billion of avermectin mycelium B1a to the filter residue, and obtain secondary liquids after one extraction and filtration for repeated use.

[0078] 3. Concentration

[0079] The primary solution was heated to 80°C until all the methanol evaporated and then 5 ml3 After stirring with toluene, the mixture was transferred to the desugaring process. The residual potency of avermectin B1a in the filter residue was 108U / ml, and the potency of the concentrated liquid in the tank was 12363U / ml.

[0080] 4. Sugar removal

[0081] After adding 10 times the total amount of toluene of the extract Avermectin B1a, add 2000L of hot water, heat up and stir, release the waste water and evaporate until there is no toluene.

[0082] 5. Gradient crystallization

[0083] The temperature was raised to 70°C, kept warm for 1.5 hours, and 15 kg of activated carbon was added after the gradient crystallization for the second time, and finally the third crystallization was performed, and the mixture was mixed and packaged to obtain the finished product after drying. The avermectin B1a content was measured to be 94.2%, and the total yield was 93.47%.

[0084] Comparative Example 2

[0085] 1. Filter

[0086] 100 m2 of avermectin fermentation liquid with a potency of 6300 U / ml was added with 1000 kg of filter aid, stirred and heated to 85°C for 30 minutes, and then pumped into a plate and frame filter with a feed pump for filtration and pressed for 30 minutes.

[0087] 2. Extraction and concentration

[0088] The crushed filter cake is then placed in an extraction tank, and the amount of extracting liquid added at one time is 80 times of the total amount of avermectin mycelium B1a by hot reflux circulation extraction technology. Methanol with a concentration of more than 95% is added from the storage tank to the extraction tank, and the stirring time is 40 minutes at a temperature of 55°C, and then solid-liquid separation is performed. After the extracting liquid evaporates until there is no methanol, a paste is obtained, and then 5 times of toluene of the total amount of avermectin mycelium B1a is added to the paste. After the temperature rises to 70°C, it is transferred to the desugaring step. The residual potency of avermectin B1a in the filter residue is 216U / ml, and the potency of the concentrated liquid in the tank is 10019U / ml.

[0089] 3. Sugar removal

[0090] After adding 10 times the total amount of toluene of the extract Avermectin B1a, add 1500L of hot water, heat up and stir, release the waste water and evaporate until there is no toluene.

[0091] 4. Gradient crystallization

[0092] The temperature was raised to 70°C and kept for 1.5 hours, and the mixture was mixed and packaged after three crystallizations and drying to obtain the finished product. The content of avermectin B1a was measured to be 90.9%, and the total yield was 94.28%.

[0093] Comparative Example 3

[0094] 1. Filter

[0095] 100 m2 of avermectin fermentation liquid with a potency of 6000 U / ml was added with 1000 kg of filter aid, stirred evenly, heated to 85°C and stirred for 30 minutes, and then pumped into a plate and frame filter with a feed pump for filtration and pressed for 30 minutes.

[0096] 2. Extraction

[0097] Add 100 times the total amount of methanol of avermectin mycelium B1a to the filter cake at one time; extract and stir for 3 hours, and let stand for 2 hours; obtain a primary extract after solid-liquid separation, and repeatedly extract the filter residue with methanol twice to obtain a secondary liquid and a tertiary liquid for repeated use.

[0098] 3. Concentration

[0099] The primary solution was heated to 80°C until all the methanol evaporated and then 5 ml 3 After stirring with toluene, the mixture was transferred to the desugaring process. The residual potency of avermectin B1a in the filter residue was 538U / ml, and the potency of the concentrated liquid in the tank was 10363U / ml.

[0100] 4. Sugar removal

[0101] After adding 10 times the total amount of toluene of the extract Avermectin B1a, add 2000L of hot water, heat up and stir, release the waste water and evaporate until there is no toluene.

[0102] 5. Gradient crystallization

[0103] The temperature was raised to 70°C, kept warm for 1.5 hours, and 13 kg of activated carbon was added after the gradient crystallization for the second time, and finally the third crystallization was performed, and the mixture was mixed and packaged to obtain the finished product. The avermectin B1a content was measured to be 90.9%, and the total yield was 91.28%.

[0104] Table 1 Comparison of main raw material consumption and yield in Example 3 of the present invention with that in conventional comparative example 3

[0105] Serial number project Invention process Traditional crafts 1 Total yield (%) 96.91 91.28 2 Methanol consumption (kg / kg) 0.9 3.4 3 Activated carbon unit consumption (kg / kg) 0 0.2 4 Heat energy consumption (T / h) 0.2 0.7 5 Extraction time (h) 1 8 6 Avermectin B1a content (%) 95.4 90.9 7 Avermectin B1a residue in filter cake (%) 30 538

Claims

1. A method for extracting and purifying avermectin B1a using avermectin fermentation broth, comprising the following steps: (1) adding peptidoglycan hydrolase to the avermectin fermentation broth and filtering through a plate and frame to obtain a filter cake; (2) subjecting the filter cake from step (1) to methanol hot reflux cycle extraction to obtain a paste; (3) treating the paste obtained in step (2) with 1,1,1-trichloroethane to remove sugars to obtain a crude product of avermectin; (4) subjecting the crude avermectin product of step (3) to gradient crystallization to obtain a fine avermectin product, wherein the gradient crystallization temperature is raised to 60-80° C., then lowered from 75° C. to 40° C., and then lowered from 40° C. to 10-15° C., and the cooling rate is controlled at 4-7° C. / h.

2. The method according to claim 1, wherein the fermentation broth in step (1) is a fermentation broth of Streptomyces avermitilis.

3. according to the method described in claim 1, wherein the peptidoglycan hydrolase consumption described in step (1) is 2-10‰ V / V of fermentation liquid by volume.

4. The method according to claim 1, wherein the methanol concentration in step (2) is above 95%.

5. The method according to claim 1, wherein the amount of 1,1,1-trichloroethane used in step (3) is 5-6 times the total amount of mycelium by mass.