阅读说明：本技术 采用树脂从刺糖多孢菌发酵液中提取分离多杀菌素的方法 (Method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquor by adopting resin ) 是由 律恩法 朱明贺 赵康 王玲 *** 朴哲 于 2019-09-09 设计创作，主要内容包括：本发明公开了一种采用树脂从刺糖多孢菌发酵液中提取分离多杀菌素的方法,其特征在于包括如下步骤：发酵液预处理、选用大孔吸附树脂对上清液进行初步提取、采用阴离子交换树脂对解析液进行脱色处理、用阳离子交换树脂进行进一步精致、减压脱溶,萃取,调节pH值,旋转蒸馏,结晶；上清液中还加入0.5-5%盐类和5-50%水,本发明的有益效果是：采用大孔吸附树脂、阴离子交换树脂和阳离子交换树脂相结合的处理方法,对多杀菌素发酵液进行粗提,脱色和精提；多杀菌素发酵液预处理时,加入了0.5%-5%盐和5-50%水以至于使浸提液能够被树脂直接吸附,传统工艺需要加入三倍以上体积的水,减少了水污染。(The invention discloses a method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquor by adopting resin, which is characterized by comprising the following steps: pretreating fermentation liquor, selecting macroporous adsorption resin to primarily extract supernatant, adopting anion exchange resin to decolorize analysis liquid, further refining by cation exchange resin, performing desolventizing under reduced pressure, extracting, adjusting pH value, performing rotary distillation, and crystallizing; 0.5-5% of salt and 5-50% of water are also added into the supernatant, and the invention has the beneficial effects that: adopting a treatment method combining macroporous adsorption resin, anion exchange resin and cation exchange resin to carry out crude extraction, decolorization and fine extraction on spinosad fermentation liquor; when the spinosad fermentation liquor is pretreated, 0.5-5% of salt and 5-50% of water are added, so that the leaching liquor can be directly adsorbed by resin, and the traditional process needs to add more than three times of water, thereby reducing water pollution.)

1. The method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquor by adopting resin is characterized by comprising the following steps:

(1) pretreatment of fermentation liquor: firstly adjusting pH of fermentation liquor, then centrifuging at high speed to obtain mycelium, adding organic solvent, leaching for a period of time, centrifuging at high speed again to obtain supernatant,

(2) selecting macroporous adsorption resin to primarily extract the supernatant;

(3) carrying out decoloring treatment on the desorption solution by adopting anion exchange resin;

(4) further refinement is carried out by using cation exchange resin;

(5) decompression desolventizing, extracting, pH value regulating, rotary distilling and crystallizing.

2. The method of claim 1, wherein the supernatant is further added with 0.5-5% of salt and 5-50% of water, and the supernatant is left to stand.

3. The method for extracting and separating spinosad from saccharopolyspora spinosa fermentation broth by using resin according to claim 2, wherein the step (1): the pH value of the fermentation liquor pretreatment is adjusted to be 10-12 alkaline, the first centrifugal rotating speed is 8000-8000 and the second centrifugal rotating speed is 12000, the leaching time is 8-16 hours, and 0.5-5 percent of salt and 5-50 percent of water are added into the supernatant.

4. The method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquor by using resin according to claim 2, wherein the organic solvent is methanol or ethyl acetate, and the salt substance is sodium chloride or sodium carbonate or sodium bicarbonate or ammonium sulfate.

5. The method for extracting and separating spinosad from a saccharopolyspora spinosa fermentation broth by using resin according to claim 2, wherein the step (2): the model of the macroporous adsorption resin is LK20, the adsorption amount is 25mg/mL, the adsorption flow rate is 0.5-1.5BV/h, the pre-decomposition rate is 30%, 50%, and 70% methanol is eluted in a gradient manner, the pre-decomposition amounts are respectively 2-6BV, the flow rate is 1-3BV/h, the resolving agent is 80-100% methanol or acetone and 0-20% sulfuric acid with the concentration of 1mol/L, the resolving amount is 4-8BV, and the resolving flow rate is 0.2-3 BV/h.

6. The method for extracting and separating spinosad from a saccharopolyspora spinosa fermentation broth by using resin according to claim 2, wherein the step (3): the anion exchange resin is LK30 and D941, the upper column liquid is the analytic liquid of macroporous adsorption resin, the adsorption flow rate is 1-3BV/h, the decolorization rate is 85%, and the pleocidin adsorption rate is lower than 5%.

7. The method of claim 2, wherein the type of cation exchange tree is LK150, 001 x 16, AMA36, the upper column liquid is mixed anion exchange resin adsorption liquid, the adsorption flow rate is 1-3BV/h, the pre-solution is 0.1-1% salt solution for elution, the pre-solution amount is 1-3BV, the flow rate is 0.3-1BV/h, the resolving agent is 50% methanol salt solution, and the resolving flow rate is 0.2-3 BV/h.

8. The method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquid by using resin according to claim 2, wherein in the step (5), the extraction solvent can be ethyl acetate, petroleum ether, n-hexane and the like, the crystal is filtered, and the extraction solvent can be rinsed by alkaline aqueous solution.

The technical field is as follows:

the invention belongs to the technical field of extraction of bioactive components, and particularly relates to a method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquor by using resin.

Background art:

spinosad, also known as spinosad, is a macrolide nuisance-free high-efficiency biopesticide extracted from saccharopolyspora spinosa fermentation broth. Parent Strain which produces spinosad the soil actinomycete, Saccharopolyspora spinosa, was originally isolated from a abandoned brewery site of the Callerian. Is a secondary metabolite produced by aerobic fermentation of soil actinomycete Saccharum polycephalum in culture medium. The main effective components are spinosad A and spinosad D, which respectively account for 85-90% and 10-15% of all the components. The spinosad serving as a novel microbial source insecticide has the characteristics of high toxicity, wide insecticidal spectrum, unique action mode, low residue, environmental friendliness, no harm to human and livestock and the like, so that the spinosad is regarded as a green biological pesticide with very wide application prospect.

Although the spinosad is an environment-friendly biological pesticide, the main problems of high production cost, complex fermentation process, low fermentation efficiency, incomplete extraction process, low product purity and the like are the current limitations on the realization of industrialization and wide application of spinosad.

In recent years, many reports on spinosad separation and purification processes are available, and adsorption methods and solvent extraction methods are mainly focused on. First, there are international reports of Baker P J et al [ US5227295, 1993-07-13] of Yinong Dou, USA, using HP-20ss adsorbent resin, 0% -95% methanol: acetonitrile 1: 1 (containing 0.1% of sodium acetate) solution is used for gradient elution of spinosad A and D components, HPLC is used for tracking detection, eluent is collected in sections, the spinosad eluent is diluted by petroleum ether after being concentrated, the diluent is put on a silica gel chromatographic column, petroleum ether and methanol are used for gradient elution, HPLC tracking detection is used for tracking detection, the eluent is collected in sections, and the eluent of spinosad A and D is respectively obtained; secondly, in China, the years also show that the DM11 resin used for beautiful jade and the like adsorbs pleocidin when the pH is 9.5, and is desorbed by acetone, and the recovery rate is as high as 85.8%. The extraction of the spinosad is carried out by using a technical route of combining extraction and back extraction in summer, autumn and the like, and the total yield reaches more than 80%. Chinese patent CN101560231 discloses an extraction process of macroporous resin gradient elution, the yield reaches 68.2%, and the purity reaches 95.3%.

However, the process research of the prior resin adsorption method is not complete, and the problems of high leakage rate and low product yield and purity generally exist in the reports of the prior resin adsorption method. The principle of the solvent extraction method is that spinosad and impurities have different solubilities in a solvent, and the spinosad is selectively transferred from one solvent to another solvent, so that the aim of concentration and impurity removal is achieved. The field urgently needs to deeply research and optimize the fermentation process and the separation and purification process of the spinosad, effectively improve the yield of the spinosad and reduce the production cost

The invention content is as follows:

in order to solve the problems and overcome the defects of the prior art, the invention provides a method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquor by using resin;

the first technical problem to be solved is that: the existing method for extracting and separating pleocidin from saccharopolyspora spinosa fermentation liquor has the advantages of low decolorization rate, low yield and low product purity;

the second technical problem to be solved is that: in order to enable the resin to achieve a better adsorption effect, more than three times of volume of water is required to be added during resin adsorption in the traditional process, so that a large amount of water containing a solvent is generated, the content of the solvent is low, the environment is polluted by discharge, and the recovery cost is high and the difficulty is high;

the third technical problem to be solved is: in the traditional preparation method of spinosad, the leaching liquor is mainly acetone, but the interference peak value of acetone is too large during liquid phase detection, so that the purity of spinosad in the solution is difficult to directly see.

The specific technical scheme for solving the technical problems comprises the following steps: the method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquor by adopting resin is characterized by comprising the following steps:

(1) pretreatment of fermentation liquor: firstly adjusting pH of fermentation liquor, then centrifuging at high speed to obtain mycelium, adding organic solvent, leaching for a period of time, centrifuging at high speed again to obtain supernatant,

(2) selecting macroporous adsorption resin to primarily extract the supernatant;

(3) carrying out decoloring treatment on the desorption solution by adopting anion exchange resin;

(4) further refinement is carried out by using cation exchange resin;

(5) decompression desolventizing, extracting, pH value regulating, rotary distilling and crystallizing.

Further, 0.5-5% of salt and 5-50% of water are added into the supernatant liquid and are placed for standby.

Further, the step (1): the pH value of the fermentation liquor pretreatment is adjusted to be 10-12 alkaline, the first centrifugal rotating speed is 8000-8000 and the second centrifugal rotating speed is 12000, the leaching time is 8-16 hours, and 0.5-5 percent of salt and 5-50 percent of water are added into the supernatant.

Further, the organic solvent is methanol or ethyl acetate, and the salt substance is sodium chloride or sodium carbonate or sodium bicarbonate or ammonium sulfate.

Further, the step (2): the model of the macroporous adsorption resin is LK20, the adsorption amount is 25mg/mL, the adsorption flow rate is 0.5-1.5BV/h, the pre-decomposition rate is 30%, 50%, and 70% methanol is eluted in a gradient manner, the pre-decomposition amounts are respectively 2-6BV, the flow rate is 1-3BV/h, the resolving agent is 80-100% methanol or acetone and 0-20% sulfuric acid with the concentration of 1mol/L, the resolving amount is 4-8BV, and the resolving flow rate is 0.2-3 BV/h.

Further, the step (3): the anion exchange resin is LK30 and D941, the upper column liquid is the analytic liquid of macroporous adsorption resin, the adsorption flow rate is 1-3BV/h, the decolorization rate is 85%, and the pleocidin adsorption rate is lower than 5%.

Furthermore, in the step (4), the types of the cation exchange trees are LK150, 001 × 16 and AMA36, the upper column liquid is anion exchange resin adsorption mixed liquid, the adsorption flow rate is 1-3BV/h, the pre-solution is eluted by 0.1-1% saline solution, the pre-solution amount is 1-3BV, the flow rate is 0.3-1BV/h, the resolving agent is 50% methanol saline solution, and the resolving flow rate is 0.2-3 BV/h.

Further, in the step (5), the extraction solvent can be ethyl acetate, petroleum ether, n-hexane and the like, the crystals are filtered, and the crystals can be rinsed by alkaline aqueous solution.

The invention has the beneficial effects that:

firstly, the invention adopts a treatment method combining macroporous adsorption resin, anion exchange resin and cation exchange resin to carry out crude extraction, decolorization and fine extraction on spinosad fermentation liquor, the resin can be used for many times, the environment is protected, and the process operation is simple;

when the spinosad fermentation liquor is pretreated, most of water is removed through centrifugation, a small amount of organic solvent is added for extraction, the use of the organic solvent is reduced, 0.5-5% of salt and 5-50% of water are added so that the extract can be directly adsorbed by resin, more than three times of water is added in the traditional process, and the water pollution is reduced;

the traditional leaching liquor is mainly acetone, but the interference peak value of the acetone is too large during liquid phase detection, so that the purity of the spinosad in the solution is difficult to directly see;

fourthly, the purified product has high spinosad content, and the mass content is 97 to 99 percent.

Description of the drawings:

FIG. 1: traditional acetone is used as a liquid phase detection map of a leaching solution;

FIG. 2 is a drawing: the methanol is used as a liquid phase detection map of a leaching liquor.

The specific implementation mode is as follows:

in the description of the invention, specific details are given only to enable a full understanding of the embodiments of the invention, but it should be understood by those skilled in the art that the invention is not limited to these details for the implementation. In other instances, well-known structures and functions have not been described or shown in detail to avoid obscuring the points of the embodiments of the invention. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

The specific implementation mode of the invention is as follows: in the following case, the resin volume of the resin column was 20 mL.

The detection method is high performance liquid chromatography, the chromatographic column is a C18 silica gel column, a mobile phase, acetonitrile: methanol: 2% ammonium acetate 46: 46: 8, flow rate: 1.0mL/min, detection wavelength: 250 nm;