CN119241353A - A method for extracting alpha-ketoglutaric acid from fermentation broth - Google Patents

Abstract

The invention provides a method for extracting alpha-ketoglutaric acid from fermentation liquor. The method comprises the steps of carrying out multistage fractional extraction on the pretreated fermentation liquor, wherein an extracting agent for multistage fractional extraction comprises an oxygen-containing organic solvent with certain hydrogen bond alkalinity, preferably, the fermentation liquor is obtained by producing alpha-ketoglutaric acid and pyruvic acid by a microbial fermentation method, more preferably, the fermentation liquor is produced by taking yarrowia lipolytica as a raw material, preferably, the concentration of the alpha-ketoglutaric acid in the fermentation liquor is 0.5g/L-200g/L, and the oxygen-containing organic solvent used by the method has higher separation selectivity, lower water solubility and toxicity on the alpha-ketoglutaric acid, and is simple in process, safe and environment-friendly.

Description

Method for extracting alpha-ketoglutaric acid from fermentation liquor

Technical Field

The invention relates to the technical field of chemical separation, in particular to a method for extracting alpha-ketoglutaric acid from fermentation liquor.

Background

Alpha-ketoglutaric acid is white fine crystalline powder at normal temperature, participates in tricarboxylic acid circulation and amino acid metabolism in microorganisms, is important dicarboxylic acid, and plays a vital role in regulating carbon-nitrogen balance intermediate metabolism. In the field of food health, a mixture of alpha-ketoglutaric acid and L-arginine is called a seminal ketone mixture, can be added into functional beverages as a nutrition enhancer, and can also be used for tissue engineering, drug delivery and the like in biomedicine. The biological fermentation method and the enzyme conversion method are main methods for producing alpha-ketoglutarate, glycerol is used as raw material, and the products prepared by the method also contain impurities such as pyruvic acid, malic acid, citric acid and the like. The alpha-ketoglutaric acid and the impurity acid have similar structure and physical and chemical properties, have better water solubility and larger polarity, and have larger difficulty in separating the alpha-ketoglutaric acid from fermentation liquor.

The existing method for separating alpha-ketoglutaric acid from fermentation liquor is less, and mainly comprises a calcium salt precipitation method, a membrane separation method and a crystallization method.

The calcium salt precipitation method is based on that most of calcium salt of carboxylic acid has small solubility in water, and alpha-ketoglutarate in fermentation liquor is converted into calcium salt precipitation at a certain temperature and pH value so as to be separated. Patent CN105198732A discloses a method for separating alpha-ketoglutaric acid from fermentation liquor by utilizing calcium salt, which comprises the steps of adding excessive calcium hydroxide into the fermentation liquor, centrifugally separating out the alpha-ketoglutaric acid calcium and calcium hydroxide, adding sulfuric acid for acidification to obtain alpha-ketoglutaric acid solution, decompressing and concentrating to obtain alpha-ketoglutaric acid crude product, and washing with methanol to obtain the alpha-ketoglutaric acid. The process does not take into account that the impurity acid in the fermentation broth can also form calcium salt precipitates, which cause the subsequent difficulty in separating the alpha-ketoglutarate calcium.And PROCHASKA [ Biochemical Engineering Journal,2021,166:107883] a multistage membrane process was devised, assisted by vacuum evaporation and crystallization, to recover alpha-ketoglutarate from the fermentation broth. The pretreated fermentation broth is subjected to electrodialysis separation of alpha-ketoglutarate by using a commercial ceramic membrane and a nanofiltration ceramic membrane and is converted into an acid form, the migration of other acids in the fermentation broth is limited, and the purity of the final product is 94.8%, but the membrane is expensive and is easy to pollute. Crystallization is generally carried out as a step in the process, on the basis of extraction or pretreatment.

In summary, the method for extracting α -ketoglutaric acid in the prior art has the problems of complicated process, high cost and poor separation selectivity, and a method capable of efficiently extracting α -ketoglutaric acid from fermentation broth needs to be developed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for extracting alpha-ketoglutaric acid from fermentation liquor. The method adopted by the invention has simple flow, low cost, good stability and higher separation selectivity of the used extractant, safe and easy amplification of the multistage fractional extraction process, and is beneficial to realizing industrial production.

The invention provides a method for extracting alpha-ketoglutaric acid from fermentation liquor, which comprises the following steps of carrying out multistage fractional extraction on the pretreated fermentation liquor, wherein an extractant of the multistage fractional extraction comprises an oxygen-containing organic solvent with hydrogen bond alkalinity.

Alpha-ketoglutaric acid is a dicarboxylic acid containing carbonyl, its pK _a1 value is 2.47, the acidity and hydrogen bond acidity are both stronger, both carboxyl hydrogens can be used as hydrogen bond donor. Through researches, an extractant with a certain hydrogen bond alkalinity is adopted, and the alpha-ketoglutarate in the fermentation broth can be selectively extracted and separated through the action force of the hydrogen bond formed by solute molecules and the extractant. Furthermore, the efficiency of extracting and separating the alpha-ketoglutaric acid from the fermentation liquor after pretreatment is higher.

In some embodiments, the source of the fermentation broth is a fermentation broth that produces alpha-ketoglutarate and pyruvic acid by a microbial fermentation process.

In some embodiments, the fermentation broth is a fermentation broth that produces alpha-ketoglutarate and pyruvic acid from yarrowia lipolytica.

In some embodiments, the fermentation broth comprises α -ketoglutarate and pyruvic acid.

In some embodiments, the concentration of alpha-ketoglutaric acid in the fermentation broth is from 0.5g/L to 200g/L, for example 1g/L, 2g/L, 5g/L, 8g/L, 10g/L, 12g/L, 14g/L, 16g/L, 18g/L, 20g/L, 30g/L, 50g/L, 80g/L, 110g/L, 150g/L, 190g/L or any value therebetween.

In some embodiments, the concentration of alpha-ketoglutaric acid in the fermentation broth is from 5g/L to 20g/L.

In some embodiments, the oxygen-containing organic solvent having hydrogen bond basicity includes at least one of an alcohol solvent, an ester solvent, an ether solvent, an amide solvent, or an organic phosphorus solvent. The invention selects the oxygen-containing organic solvent with hydrogen bond alkalinity as the extractant, and has higher separation selectivity.

In some embodiments, the alcoholic solvent comprises a C1-C10 straight or branched chain alkyl substituted alcoholic solvent, preferably one or more of n-butanol, n-octanol, or 2-ethyl-1-hexanol.

In some embodiments, the ester solvent comprises an ester compound having a carbon number greater than 6, preferably one or more of amyl acetate, butyl butyrate, butyl valerate.

In some embodiments, the ethereal solvent comprises methyl tert-butyl ether.

In some embodiments, the amide-based solvent comprises one or more of N, N-di-N-butyl formamide, N-di-N-butyl acetamide, N-di-N-butyl butyramide.

In some embodiments, the organophosphorus solvent includes an alkyl phosphate and/or an alkyl phosphine oxide.

In some embodiments, the alkyl groups in the alkyl phosphate and the alkyl phosphine oxide are selected from substituted or unsubstituted C3-C8 straight or branched alkyl groups.

In some embodiments, at least one hydrogen atom of the above-described C3-C8 straight or branched alkyl group is substituted with at least one of a carboxyl group, an amino group, a carbonyl group, an alkylene group, a hydroxyl group, a halogen atom, a sulfonyl group, and a cyano group. The substitution may be mono-substitution, di-substitution, tri-substitution or poly-substitution, and the types and the numbers of the substituents at different positions may be the same or different in the case of multi-substitution.

In some embodiments, the organophosphorus solvent is selected from one or more of tributyl phosphate, trioctyl phosphate, tri-n-octylphosphine oxide, tri (2-ethylhexyl) phosphine oxide, and tricyclohexylphosphine oxide.

In some embodiments, the oxygen-containing organic solvent having hydrogen bond basicity is selected from one or more of amyl acetate, butyl butyrate, butyl valerate, N-dibutyl formamide, N-dibutyl acetamide, N-di-N-butyl butyramide, N-hexanol, N-octanol, 2-ethyl-1-hexanol, tributyl phosphate, trioctyl phosphate, tri-N-octylphosphine oxide, di (ethylhexyl) phosphate, tri (2-ethylhexyl) phosphine oxide, tricyclohexylphosphine oxide, or a mixture of trioctylphosphine oxide and di (ethylhexyl) phosphate.

In some embodiments, the oxygen-containing organic solvent having hydrogen bond basicity is selected from one or more of butyl butyrate, butyl valerate, N-dibutyl formamide, tributyl phosphate, tri-N-octyl phosphine oxide, di (ethylhexyl) phosphate, tricyclohexyl phosphine oxide, or a mixture of trioctylphosphine oxide and di (ethylhexyl) phosphate.

In some embodiments, the extractant further comprises an optional diluent. The oxygen-containing organic solvent may be used alone or in a diluent.

In some embodiments, the diluent comprises one or more of an alkane organic solvent, an aromatic hydrocarbon organic solvent, a halogenated hydrocarbon organic solvent, an alcohol organic solvent, a ketone organic solvent, an ether organic solvent, and an ester organic solvent.

In some embodiments, the diluent comprises at least one of petroleum ether, n-hexane, n-dodecane, toluene, o-xylene, methylene chloride, chloroform, kerosene, n-butanol, n-hexanol, n-octanol, methyl isobutyl ketone, methyl tert-butyl ether, isopropyl ether, ethyl acetate, butyl acetate, amyl acetate, preferably one or more of n-butanol, ethyl acetate, butyl acetate, amyl acetate, methylene chloride, n-hexanol, methyl isobutyl ketone, methyl tert-butyl ether, and isopropyl ether.

In some embodiments, the diluent is n-butanol and/or ethyl acetate.

In some embodiments, the extractant includes an oxygen-containing organic solvent and a diluent that have hydrogen bond basicity.

In some embodiments, the extractant is selected from trioctylphosphine oxide and N-butanol, tricyclohexylphosphine oxide and ethyl acetate, N-di-N-butylformamide and ethyl acetate.

In some embodiments, the mass fraction of the oxygen-containing organic solvent in the extractant is 0.1% -99.99%, such as 0.1%, 1%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 23%, 26%, 29%, 33%, 36%, 39%, 40%, 45%, 50%, 70%, 80%, 99.9%, or any value therebetween.

In some embodiments, the oxygen-containing organic solvent is present in an amount of 1% to 50% by mass.

In some embodiments, the oxygen-containing organic solvent is present in an amount of 5% to 50% by mass.

In some embodiments, the oxygen-containing organic solvent is present in an amount of 10% to 40% by mass.

In some embodiments, the multi-stage fractional extraction further comprises a detergent.

In some embodiments, the detergent is water.

In the multi-stage extraction, the factors such as product quality, production cost and the like are comprehensively considered, and the ratio of the volume flow rates of the extractant, the detergent and the pretreated fermentation liquor is 1 (0.1-20): 0.1-20, for example 1:1:(0.2-8)、1:3:(0.2-8)、1:5:(0.2-8)、1:9:(0.2-8)、1:13:(0.2-8)、1:17:(0.2-8)、1:(0.2-8):1、1:(0.2-8):3、1:(0.2-8):5、1:(0.2-8):9、1:(0.2-8):6、1:(0.2-8):7、1:(0.2-8):8 or any value between the extractant and the detergent.

In some embodiments, the ratio of the volumetric flow rates of the extractant, the detergent, and the pretreated broth is 1 (0.1-10): 0.1-10.

In some embodiments, the ratio of the volumetric flow rates of the extractant, the detergent, and the pretreated broth is 1 (0.2-8): 0.2-8.

In some embodiments, the multistage fractional extraction is operated at a temperature of 15 ℃ to 60 ℃, such as 20 ℃, 25 ℃,30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, or any value therebetween. If the temperature is too low, the viscosity of the extractant will be high, the mass transfer rate will be low, the throughput will be low, which is unfavorable for production operation, and if the temperature is too high, on the one hand, the volatilization rate of the diluent or solvent will be high, on the other hand, the distribution coefficient and selectivity of the alpha-ketoglutarate will be low.

In some embodiments, the pretreatment includes removal of insoluble and macromolecular impurities from the fermentation broth, decolorization, and desalting.

In some embodiments, the macromolecular impurities have a molecular weight greater than 1000Da. The macromolecular impurities can be biomass macromolecules such as cell fragments, lipids, polysaccharides, proteins and the like.

In some embodiments, the method of removing insoluble impurities and macromolecular impurities from a fermentation broth comprises one or more of centrifugation, ultrafiltration, microfiltration, and plate and frame filtration.

In some embodiments, the method of removing insoluble impurities and macromolecular impurities from a fermentation broth comprises:

(1) Centrifuging the fermentation liquor, and collecting supernatant;

(2) Performing first filtration on the supernatant to obtain a first filtrate;

(3) Performing second filtration on the first filtrate to obtain second filtrate;

the centrifugation and filtration processes are safe and environment-friendly, and can remove most macromolecular impurities.

In some embodiments, the first filtration employs a ceramic membrane.

In some embodiments, the ceramic membrane module is an external pressure type structural membrane module of PVDF hollow fiber membrane, and can intercept impurities with molecular weight greater than 1000 Da.

In some embodiments, the first filtration is operated at a pressure of 0.1MPa to 0.5MPa, such as 0.15MPa, 0.2MPa, 0.25MPa, 0.3MPa, 0.35MPa, 0.4MPa, 0.45MPa, 0.5MPa, or any value therebetween.

In some embodiments, the ceramic membrane has a pore size of 0.1-1 μm, for example, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, or any value therebetween.

In some embodiments, the second filtration employs an ultrafiltration membrane.

In some embodiments, the ultrafiltration membrane is a hollow fiber membrane made from polyvinylidene fluoride.

In some embodiments, the second filtration is operated at a pressure of 0.1MPa to 0.7MPa, such as 0.15MPa, 0.2MPa, 0.25MPa, 0.3MPa, 0.35MPa, 0.4MPa, 0.45MPa, 0.5MPa, 0.55MPa, 0.6MPa, 0.65MPa, or any value therebetween.

In some embodiments, the ultrafiltration membrane has a pore size of 0.01 to 0.1 μm, for example, 0.02 μm, 0.03 μm, 0.04 μm, 0.05 μm, 0.06 μm, 0.07 μm, 0.08 μm, 0.09 μm, or any value therebetween.

In some embodiments, the method of decolorizing comprises adsorption chromatography column.

In some embodiments, the decolorizing employs macroporous adsorbent resins and/or decolorizing activated carbon.

In some embodiments, the macroporous adsorbent resin is selected from one or more of HPD-750、HPD-722、AB-8、ADS-7、SD200、SD300、SD331、SD333、LSA-210、LSA-220、LSA-230、Amberlite XAD-4、Amberlite XAD-16 and Amberlite XAD-18.

In some embodiments, the macroporous adsorbent resin is selected from one or more of HPD-750, AB-8, ADS-7, SD300, amberlite XAD-4, amberlite XAD-16, and Amberlite XAD-18.

The macroporous adsorption resin has large decoloring capacity and is easy to regenerate.

In some embodiments, the method of desalting comprises ion exchange chromatography.

In some embodiments, the desalting is with a cation exchange resin.

The column chromatography for decoloring and desalting is simple and easy to implement, and has good cycle performance.

In some embodiments, the cation exchange resin is selected from one or more of the cation exchange resins selected from ZG CER50、ZG ER 8420、ZG ER 8410、D851、Amberlite HPR1200 H、Amberlite HPR 2900H、Amberlite IRC 120H、001×7、001×8、D001、LX-160、LSI-010.

In some embodiments, the cation exchange resin is selected from the group consisting of ZG CER50, amberlite IR 120H, FPC, H, HPR 2900H, 001 x 7, and 001 x 8.

In some embodiments, the method further comprises post-processing.

In some embodiments, the post-treatment method comprises crystallization after distillation under reduced pressure or crystallization after back extraction acidification.

In some embodiments, the α -ketoglutaric acid is enriched in the extract phase or raffinate phase, depending on the extractant used. If the alpha-ketoglutaric acid is enriched in the extraction phase, the low boiling point oxygen-containing organic solvent is distilled under reduced pressure to recover the solvent and solute, if the extractant is organophosphorus solvent or oxygen-containing organic solvent with boiling point higher than 100 ℃, the alkali liquor is used for back extraction, acidification, crystallization and vacuum drying to obtain the high purity alpha-ketoglutaric acid product, if the alpha-ketoglutaric acid is enriched in the raffinate phase, the raffinate is concentrated under reduced pressure and then crystallized to obtain the alpha-ketoglutaric acid product.

In some embodiments, the low boiling point oxygenated organic solvent is a solvent having a boiling point below 100 ℃.

In some embodiments, the crystallization is a cooling crystallization.

In some embodiments, the crystallization temperature is 4 ℃ to 20 ℃, e.g., 5 ℃, 8 ℃, 11 ℃, 14 ℃, 17 ℃, 20 ℃, or any value therebetween.

In some embodiments, the crystallization time is 8-24h, e.g., 9h, 12h, 15h, 18h, 21h, 24h, or any value therebetween.

In some embodiments, the reduced pressure distillation temperature is from 30 ℃ to 60 ℃, such as 35 ℃, 40 ℃,45 ℃, 50 ℃, 55 ℃,60 ℃, or any value therebetween.

In some embodiments, the reduced pressure distillation pressure is from-100 to 100kPa (absolute), such as-100 kPa, -50kPa, 0kPa, 50kPa, 100kPa, or any value therebetween.

The fractional extraction comprises an extraction section and a washing section. The extractant enters the system from the first stage of the extraction section, after the stages of the extraction section and the washing section are determined, raw material liquid enters the system from the feeding stage, the detergent and the raw material liquid are mixed in the feeding stage, the extraction phase and the washing phase are subjected to multistage countercurrent contact, the extraction liquid flows out from the first stage of the washing section, and the raffinate flows out from the first stage of the extraction section.

In some embodiments, the method comprises the steps of:

(1) Pretreatment, namely, taking fermentation liquor produced by a microbial fermentation method to produce alpha-ketoglutaric acid as raw material liquor, centrifuging and filtering, and then carrying out resin decolorization and desalination treatment;

(2) Fractional extraction, namely, fractional extraction by taking an oxygen-containing organic solvent and an optional diluent as an extractant and water as a detergent through multistage countercurrent contact;

(3) And (3) obtaining an alpha-ketoglutaric acid product, namely collecting the alpha-ketoglutaric acid through back extraction or direct evaporation of the solvent after fractional extraction.

In some embodiments, the alpha-ketoglutarate content (HPLC) extracted from the fermentation broth is greater than or equal to 98wt% with a yield of greater than or equal to 90%.

Compared with the prior art, the invention has the following beneficial effects:

1. the pretreatment method adopted by the invention is safe and environment-friendly, has simple process and low energy consumption, and the resin used in the decoloring and desalting processes has low cost and is easy to regenerate and can be recycled;

2. the multistage fractional extraction process is safe, easy to amplify and beneficial to realizing industrial production;

3. the oxygen-containing organic solvent with hydrogen bond alkalinity used in the invention has higher separation selectivity, lower water solubility and toxicity to alpha-ketoglutaric acid, and the extractant can be recovered and recycled after reduced pressure distillation or strong alkali back extraction;

4. The multistage fractional extraction process adopted by the invention can give consideration to both the purity and the yield of the product, and the purity of the alpha-ketoglutaric acid product reaches more than 98wt% by adopting optimized conditions, and the recovery rate is more than 90%.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The reagents used in the following examples are all conventional biochemical reagents unless otherwise specified, the raw materials, instruments, equipment and the like used in the following examples are all commercially available or available by the existing methods, the amounts of the reagents are all the amounts of the reagents used in conventional experimental operations unless otherwise specified, and the experimental methods are all conventional methods unless otherwise specified.

The fractional extraction in the invention means that water phase feed liquid (F) containing components to be separated flows in from a certain stage in the middle of the cascade extractor, and washing liquid (W) and organic phase (S) without the components to be separated flow in from two ends of the cascade extractor according to a constant flow ratio.

"Hydrogen bond basicity" in the present invention refers to the ability of a substance to form hydrogen bonds as a hydrogen bond acceptor, also known as hydrogen bond basicity or hydrogen bond acceptor ability. The more basic the hydrogen bond of a substance, the more capable it will be of hydrogen bond interaction with other hydrogen bond donor substances as hydrogen bond acceptors, forming intermolecular hydrogen bonds.

The following examples are methods for extracting alpha-ketoglutarate from a microbial fermentation broth containing alpha-ketoglutarate, and are exemplified by fermentation broths obtained by using yarrowia lipolytica as a fermentation strain. Other similar microbial fermentation broths or enzymatic conversion broths containing alpha-ketoglutarate may be processed according to embodiments of the invention to produce alpha-ketoglutarate products having higher purity.

Example 1

Extracting alpha-ketoglutaric acid from fermentation liquor obtained by using yarrowia lipolytica as a fermentation strain, wherein the initial concentration of the alpha-ketoglutaric acid is 10.3g/L, and the method comprises the following steps:

(1) And (3) centrifuging and filtering, namely pumping the fermentation liquor into an excessively high-speed centrifugal machine, wherein the pump flow rate is 100mL/min, the centrifugal machine rotation speed is 8000rpm, the centrifugal time is 5min, and filtering is carried out after centrifugation to remove thalli and other visible solids, so as to obtain a filtered liquid.

(2) Membrane filtration, namely filtering the filtering liquid obtained by centrifugal filtration through a ceramic membrane with the pore diameter of 0.1-1 mu m, removing bacterial fragments, lipid, polysaccharide, protein and other biological macromolecules in the filtering liquid, and finally washing the filtering residual liquid with a small amount of water to ensure that the Tao Lvguo-pass yield is close to 100%. Tao Lv the permeate passes through an ultrafiltration membrane with a membrane pore size of 0.01-0.1 μm, and finally the filtered residue is washed with a small amount of water. The ceramic membrane component is an external pressure type structural membrane component of PVDF hollow fiber membrane, can intercept impurities with molecular weight larger than 1000Da, has operation pressure of 0.1MPa, and the ultrafiltration membrane is a hollow fiber membrane prepared from polyvinylidene fluoride serving as a raw material, and has operation pressure of 0.1MPa.

(3) And (3) decoloring with macroporous adsorption resin, namely, processing the fermentation liquor containing alpha-ketoglutaric acid after centrifugation and membrane filtration with macroporous decoloring resin HPD 750, and adsorbing to remove most of pigments. The loading flow rate is 1.5BV/h, and the water of 1.5BV is used for washing the feed liquid in the column, and the effluent liquid is collected. The column passing temperature was 25℃and operated at normal pressure.

(4) And (3) treating the decolored fermentation liquor by using a ZG CER50 resin layer to remove metal cations in the decolored fermentation liquor and convert alpha-ketoglutarate into alpha-ketoglutarate. And finally, eluting the feed liquid in the column by using 1BV water at the sample loading flow rate of 1.2BV/h, and collecting effluent liquid to obtain pretreated fermentation liquor. The column passing temperature was 25℃and operated at normal pressure.

(5) And (3) fractional extraction, namely taking the pretreated fermentation liquor as raw material liquid, taking di (ethylhexyl) phosphate as an extractant, taking water as a detergent, and carrying out fractional extraction at the temperature of 30 ℃ with the flow ratio of the extractant to the detergent to the pretreated fermentation liquor being 1:2:1. The fractionation extraction is divided into an extraction section and a washing section (the extraction section is 8 stages and the washing section is 5 stages), an extract enriched in alpha-ketoglutaric acid flows out from the first stage of the washing section, and the extract is collected.

(6) Back extraction, namely back extraction of the extract liquid by using 1M NaOH aqueous solution. Adding the extract into a stirring tank, and slowly dripping the back-extraction agent dropwise while mechanically stirring until back-extraction is complete. The molar ratio of NaOH in the stripping agent to alpha-ketoglutaric acid in the extract is 1:1, and the operating temperature is 30 ℃. Acidifying the back extraction phase by cation exchange resin ZG CER50, cooling, crystallizing, and vacuum drying to obtain alpha-ketoglutaric acid product. The purity of the alpha-ketoglutarate is 99.57 percent and the yield is 94.08 percent.

Example 2

The method is characterized in that the decolorization pretreatment in the step (3) is not carried out, 36.5v/v% hydrochloric acid solution is added into the deionized fermentation liquor after the desalination in the step (4) for continuous stirring, di (ethylhexyl) phosphate is used as an extractant, water is used as a detergent, and the flow ratio of the extractant to the detergent to the pretreated fermentation liquor is 1:2:1, so that the fractionation extraction is carried out at 40 ℃. Acidifying the back extraction phase by cation exchange resin Amberlite IR120H, cooling, crystallizing, and vacuum drying to obtain alpha-ketoglutaric acid product. The purity of the alpha-ketoglutarate is 98.97%, and the yield is 95.25%.

Example 3

The fermentation liquor obtained by taking yarrowia lipolytica as a fermentation strain is taken as a raw material liquid, the initial concentration of alpha-ketoglutaric acid is 40.2g/L, and the implementation mode is the same as that of example 2, wherein the pretreated fermentation liquor takes tributyl phosphate as an extractant and water as a detergent, the flow ratio of the extractant, the detergent and the raw material liquid is 1:2:1, and the fractional extraction is carried out at 40 ℃. The purity of the alpha-ketoglutarate was 99.06% and the yield was 96.31%.

Example 4

The fermentation liquor obtained by taking yarrowia lipolytica as a fermentation strain is taken as a raw material liquid, the initial concentration of alpha-ketoglutaric acid is 60.3g/L, the implementation mode is the same as that of example 1, and the difference is that the fermentation liquor after centrifugal filtration, decolorization and desalination treatment is subjected to fractional extraction by using a mixture of trioctylphosphine oxide (TOPO) and di (ethylhexyl) phosphate at 30 ℃, the mass fraction of TOPO in the mixture is 10 percent, water is taken as a detergent, the flow ratio of the raw material liquid, the detergent and the extractant is 1:2:1, the extraction section is 5 stages, and the washing section is 8 stages. The extract enriched in alpha-ketoglutaric acid is discharged from the first stage of the washing section and the extract is collected.

The extract is acidified, crystallized and dried after being back extracted by NaOH, and the purity of the alpha-ketoglutaric acid product is 98.50%, and the yield is 96.17%.

Example 5

The pretreatment process and the back extraction process were the same as in example 1 except that SD300 was used for the decolorizing resin and IR 120H was used for the desalting resin. The extraction process is characterized in that butyl valerate is used as an extractant, water is used as a detergent, the volume flow ratio of raw material liquid to the detergent to the extractant is 1:6:8.3, fractional extraction is carried out at 30 ℃ (10 stages are used in an extraction section and 8 stages are used in a washing section), raffinate enriched in alpha-ketoglutaric acid flows out in the first stage of the extraction section, and the raffinate and the extract are collected.

The purity of the alpha-ketoglutarate is 98.09 percent and the yield is 95.40 percent.

Example 6

Separating and extracting alpha-ketoglutaric acid from fermentation liquor obtained by using yarrowia lipolytica as a fermentation strain, wherein the initial concentration of the alpha-ketoglutaric acid is 50.6g/L, and the method comprises the following steps:

(1) And (3) centrifuging and filtering, namely pumping the fermentation liquor into a high-rotation-speed centrifuge, wherein the pump flow rate is 50mL/min, the centrifuge rotation speed is 5000rpm, centrifuging for 10min, and filtering to remove thalli and other visible solids, thereby obtaining filtered liquid.

(2) Membrane filtration, in which the filtered liquid obtained by centrifugal filtration is filtered by ultrafiltration membrane with pore diameter of 0.01-0.1 μm, and finally the filtered residual liquid is washed with a small amount of water. The ultrafiltration membrane is a hollow fiber membrane prepared from polyvinylidene fluoride as a raw material, and the operating pressure is 1.5MPa.

(3) And (3) decoloring with macroporous adsorption resin, namely, processing the fermentation liquor containing alpha-ketoglutaric acid after centrifugation and membrane filtration on ion exchange resin Amberlite XAD-16, and removing most of pigments. The loading flow rate was 1.5BV/h. The column passing temperature was 25℃and operated at normal pressure.

(4) Desalting with cation exchange resin, namely treating the decolorized fermentation liquor with FPC 22H resin layer, removing metal cations in the decolorized fermentation liquor, and converting alpha-ketoglutarate into alpha-ketoglutarate. The loading flow rate is 1.25BV/h, and finally the feed liquid in the column is washed out by 1.2BV water, and the effluent liquid is collected. The column passing temperature was 25℃and operated at normal pressure.

(5) And (3) fractional extraction, namely taking the pretreated fermentation liquor as a raw material liquid, taking an extracting agent which is a binary mixed solvent consisting of TOPO and n-butanol serving as a diluent, wherein the mass fraction of the TOPO in the extracting agent is 30%, the flow ratio of the extracting agent to the washing agent to the pretreated fermentation liquor is 1.1:1.9:1, and performing fractional extraction at 30 ℃ (the extraction section is 6-level and the washing section is 5-level), and collecting the extracting liquor and raffinate.

(6) Back extraction

The extract is back extracted by using 1M NaOH aqueous solution, the mol ratio of NaOH in the back extractant and alpha-ketoglutaric acid in the extract is 1.5:1, and the operation temperature is 30 ℃. Acidifying the back extraction phase by cation exchange resin ZG CER50, cooling, crystallizing for 24h, and vacuum drying to obtain alpha-ketoglutaric acid product. The purity of the product is 98.56 percent, and the yield is 95.02 percent.

Example 7

The method is characterized in that the extracting agent is a binary mixed solvent consisting of tricyclohexylphosphine oxide and ethyl acetate serving as a diluent, the mass fraction of the tricyclohexylphosphine oxide in the extracting agent is 40%, the flow ratio of the extracting agent, the washing agent and the raw material liquid is 4:4:1, the operating temperature is 15 ℃, and the fractional extraction is carried out (the total 5 stages of the extracting section and the total 5 stages of the washing section). The purity of the alpha-ketoglutarate product is 98.68 percent, and the yield is 95.69 percent.

Example 8

The method is characterized in that the extracting agent is a binary mixed solvent consisting of N, N-di-N-butyl formamide and ethyl acetate serving as a diluent, the mass fraction of the N, N-di-N-butyl formamide in the extracting agent is 25%, the flow ratio of the extracting agent, the detergent and the pretreated fermentation liquor is 2.5:3.3:1, and the fractional extraction is carried out at 30 ℃ (the extraction section is 10 levels, and the washing section is 7 levels). The purity of the alpha-ketoglutarate product is 98.12 percent and the yield is 94.58 percent.

Example 9

The steps of pretreatment, extraction and post-treatment were the same as in example 1 except that butyl butyrate was used as the extractant, and alpha-ketoglutarate was extracted from a fermentation broth obtained by using yarrowia lipolytica as a fermentation strain, wherein the initial concentration of alpha-ketoglutarate was 10.6 g/L. The purity of the obtained alpha-ketoglutarate is 98.70%, and the yield is 94.14%.

Example 10

The procedure of example 1 was repeated except that N, N-di-N-butyl formamide was used as the extractant, in which alpha-ketoglutaric acid was extracted from the fermentation broth obtained by using Saccharomyces cerevisiae as the fermentation medium, and the initial concentration of alpha-ketoglutaric acid was 10.6 g/L. The purity of the alpha-ketoglutarate in the obtained product is 99.50%, and the yield is 95.27%.

Example 11

Extracting alpha-ketoglutaric acid from fermentation liquor obtained by using yarrowia lipolytica as fermentation strain, wherein the initial concentration of the alpha-ketoglutaric acid is 10.3g/L, and the steps of pretreatment, extraction and post-treatment are the same as in example 1, the difference from example 1 is that the extractant is a mixture of trioctylphosphine oxide (TOPO) and di (ethylhexyl) phosphate, the mass fraction of TOPO in the extractant is 10%. The purity of the obtained alpha-ketoglutarate is 99.56%, and the yield is 96.27%.

Example 12

Extracting alpha-ketoglutaric acid from fermentation liquor obtained by using yarrowia lipolytica as fermentation strain, wherein the initial concentration of the alpha-ketoglutaric acid is 10.3g/L, and the steps of pretreatment, extraction and post-treatment are the same as in example 1, the difference from example 1 is that the flow ratio of extractant, detergent and pretreated fermentation broth is 1:6:1 during fractional extraction. The purity of the alpha-ketoglutarate in the obtained product is 99.82%, and the yield is 93.68%.

Example 13

Extracting alpha-ketoglutaric acid from fermentation liquor obtained by using yarrowia lipolytica as fermentation strain, wherein the initial concentration of the alpha-ketoglutaric acid is 10.3g/L, and the steps of pretreatment, extraction and post-treatment are the same as in example 1, the difference from example 1 is that the flow ratio of extractant, detergent and pretreated fermentation broth is 1:1:6 during fractional extraction. The purity of the obtained alpha-ketoglutarate is 99.59% and the yield is 91.53%.

Comparative example 1

The method is similar to example 1 in that the extractant is methyl tertiary butyl ether, the flow ratio of the extractant, the detergent and the pretreated fermentation liquor is 1:10:10, and fractional extraction is carried out at 30 ℃ (30 extraction sections and 1 washing section are all) under 30 ℃. The extraction liquid is distilled under reduced pressure to remove methyl tertiary butyl ether with low boiling point, and the methyl tertiary butyl ether is recovered and recycled. The raffinate was dried in vacuo after crystallization. The purity of the alpha-ketoglutarate product is 31.68 percent, and the yield is 3.25 percent.

Comparative example 2

The method is characterized in that alpha-ketoglutaric acid is separated and extracted from fermentation liquor obtained by taking yarrowia lipolytica as a fermentation strain, the initial concentration of the alpha-ketoglutaric acid is 100.4g/L, the method is similar to that of example 1, the method is characterized in that resin decolorization is carried out after centrifugation and membrane filtration, the cation exchange resin desalination treatment of step (4) is not carried out, the decolorized fermentation liquor is directly used for fractional extraction, the extractant is methyl isobutyl ketone, the flow ratio of the extractant, the detergent and the pretreated fermentation liquor is 1:10:10, and fractional extraction is carried out at 30 ℃ (the extraction section is 30 levels, and the washing section is 1 level). The purity of the alpha-ketoglutarate product is 33.93 percent, and the yield is 14.94 percent.

Comparative example 3

The method is characterized in that alpha-ketoglutaric acid is separated and extracted from fermentation liquor obtained by using yarrowia lipolytica as a fermentation strain, the initial concentration of the alpha-ketoglutaric acid is 10.3g/L, and the embodiment is the same as that of example 1, wherein the extractant is methyl tertiary butyl ether. The purity of the alpha-ketoglutarate product is 44.51%, and the yield is 32.41%.

Comparative example 4

The method is characterized in that alpha-ketoglutaric acid is separated and extracted from fermentation liquor obtained by using yarrowia lipolytica as a fermentation strain, the initial concentration of the alpha-ketoglutaric acid is 10.3g/L, and the embodiment is the same as that of the example 1, except that the extractant is methyl isobutyl ketone. The purity of the alpha-ketoglutarate product is 24.25%, and the yield is 19.76%.

Table 1 summary of operating conditions and results for examples and comparative examples

As can be seen from Table 1, the present invention uses an oxygen-containing organic solvent having hydrogen bond basicity as an extractant, and combines a multistage fractional extraction method, thereby achieving both product purity and yield.

Furthermore, the invention adopts a specific pretreatment method (decoloring by adsorption resin and acidifying and desalting by resin), a specific extractant and a specific operation mode of multistage fractional extraction, has higher separation selectivity on the alpha-ketoglutaric acid, can give consideration to the product purity and yield, ensures that the purity of the obtained alpha-ketoglutaric acid product reaches more than 98wt%, ensures that the recovery rate is more than 90%, does not need subsequent continuous purification, and has less extractant consumption and lower cost.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims (10)

1. A method for extracting alpha-ketoglutaric acid from fermentation broth comprises subjecting pretreated fermentation broth to multistage fractional extraction;

wherein the extractant of the multistage fractional extraction comprises an oxygen-containing organic solvent with hydrogen bond alkalinity;

Preferably, the fermentation liquor source is fermentation liquor obtained by producing alpha-ketoglutarate and pyruvic acid by a microbial fermentation method, more preferably fermentation liquor obtained by producing alpha-ketoglutarate and pyruvic acid by using yarrowia lipolytica as raw materials;

preferably, the concentration of alpha-ketoglutaric acid in the fermentation broth is between 0.5g/L and 200g/L.

2. The method according to claim 1, wherein the oxygen-containing organic solvent having hydrogen bond basicity comprises at least one of an alcohol solvent, an ester solvent, an ether solvent, an amide solvent, or an organic phosphorus solvent;

Preferably, the alcoholic solvent comprises a C1-C10 straight or branched chain alkyl substituted alcoholic solvent, preferably one or more of n-butanol, n-octanol or 2-ethyl-1-hexanol, and/or

The ester solvent comprises an ester compound with carbon number greater than 6, preferably one or more of amyl acetate, butyl butyrate or butyl valerate, and/or

The ether solvent comprises methyl tertiary butyl ether, and/or

The amide solvent comprises one or more of N, N-di-N-butyl formamide, N-di-N-butyl acetamide or N, N-di-N-butyl butyramide, and/or

The organic phosphorus solvent comprises alkyl phosphate and/or alkyl phosphine oxide;

Preferably, the alkyl group in the alkyl phosphate and the alkyl phosphine oxide is selected from a substituted or unsubstituted C3-C8 linear or branched alkyl group, preferably at least one hydrogen atom in the C3-C8 linear or branched alkyl group is substituted with at least one of a carboxyl group, an amino group, a carbonyl group, an alkylene group, a hydroxyl group, a halogen atom, a sulfonyl group and a cyano group;

preferably, the organophosphorus solvent is selected from one or more of tributyl phosphate, trioctyl phosphate, tri-n-octylphosphine oxide, tri (2-ethylhexyl) phosphine oxide and tricyclohexylphosphine oxide.

3. The process according to claim 1 or 2, wherein the oxygen-containing organic solvent having hydrogen bond basicity is selected from one or more of amyl acetate, butyl butyrate, butyl valerate, N-dibutylformamide, N-dibutylacetamide, N-di-N-butylbutanamide, N-hexanol, N-octanol, 2-ethyl-1-hexanol, tributyl phosphate, trioctyl phosphate, tri-N-octylphosphine oxide, di (ethylhexyl) phosphate, tri (2-ethylhexyl) phosphine oxide, tricyclohexylphosphine oxide or a mixture of trioctylphosphine oxide and di (ethylhexyl) phosphate,

Preferably, the oxygen-containing organic solvent having hydrogen bond basicity is selected from one or more of butyl butyrate, butyl valerate, N-dibutyl formamide, tributyl phosphate, tri-N-octyl phosphine oxide, di (ethylhexyl) phosphate, tricyclohexyl phosphine oxide or a mixture of trioctylphosphine oxide and di (ethylhexyl) phosphate.

4.A method according to any one of claims 1-3, wherein the extractant further comprises an optional diluent;

Preferably, the diluent comprises one or more of alkane organic solvents, aromatic hydrocarbon organic solvents, halogenated hydrocarbon organic solvents, alcohol organic solvents, ketone organic solvents, ether organic solvents and ester organic solvents;

Preferably, the diluent comprises at least one of n-hexane, n-dodecane, toluene, o-xylene, dichloromethane, chloroform, kerosene, n-butanol, n-hexanol, n-octanol, methyl isobutyl ketone, methyl tert-butyl ether, isopropyl ether, ethyl acetate, butyl acetate, amyl acetate, preferably one or more of n-butanol, ethyl acetate, butyl acetate, dichloromethane, n-hexanol, methyl isobutyl ketone, methyl tert-butyl ether and isopropyl ether, more preferably n-butanol and/or ethyl acetate.

5. The method of claim 4, wherein the extractant comprises an oxygen-containing organic solvent having hydrogen bond basicity and a diluent,

Preferably, the extractant is selected from trioctylphosphine oxide and N-butanol, tricyclohexylphosphine oxide and ethyl acetate, N-di-N-butylformamide and ethyl acetate;

Preferably, the mass content of the oxygen-containing organic solvent with hydrogen bond alkalinity in the extractant is 0.1% -99.99%, preferably 1% -50%, more preferably 5% -50%.

6. The process of any one of claims 1-5, wherein the multistage fractional extraction further comprises a detergent,

Preferably, the detergent comprises water, and/or

The ratio of the volume flows of the extractant, the detergent and the pretreated fermentation broth is 1 (0.1-20): 0.1-20, preferably 1 (0.2-10): 0.2-10, more preferably 1 (0.2-8): 0.2-8, and/or

The operating temperature of the multistage fractional extraction is 15-60 ℃.

7. The method according to any one of claims 1 to 5, wherein the pretreatment comprises removal of insoluble impurities and macromolecular impurities from the fermentation broth, decolorization and desalting treatment;

preferably, the macromolecular impurities have a molecular weight greater than 1000Da;

Preferably, the method for removing insoluble impurities and macromolecular impurities in the fermentation broth comprises one or more of centrifugation, ultrafiltration, microfiltration and plate and frame filtration;

Preferably, the method for removing insoluble impurities and macromolecular impurities from the fermentation broth comprises:

(1) Centrifuging the fermentation liquor, and collecting supernatant;

(2) Performing first filtration on the supernatant to obtain a first filtrate;

(3) Performing second filtration on the first filtrate to obtain second filtrate;

Preferably, the first filtration is performed using a ceramic membrane, preferably having a pore size of 0.1-1 μm, and/or

The second filtration adopts an ultrafiltration membrane, preferably, the pore size of the ultrafiltration membrane is 0.01-0.1 μm.

8. The method according to claim 7, wherein the method of decolorizing comprises adsorption chromatography column method, preferably, macroporous adsorption resin and/or decolorizing activated carbon is used for decolorizing;

Preferably, the macroporous adsorbent resin is selected from one or more of HPD-750、HPD-722、AB-8、ADS-7、SD200、SD300、SD331、SD333、LSA-210、LSA-220、LSA-230、Amberlite XAD-4、Amberlite XAD-16 and Amberlite XAD-18, and/or

The method of desalting includes ion exchange chromatography, preferably, the desalting is carried out by using cation exchange resin,

Preferably, the cation exchange resin is selected from one or more of ZG CER50、ZG ER 8420、ZG ER 8410、D851、Amberlite HPR1200 H、Amberlite HPR 2900H、Amberlite IRC 120H、001×7、001×8、D001、LX-160 or LSI-010.

9. The method according to any one of claims 1-8, further comprising post-treatment;

preferably, the post-treatment method comprises reduced pressure distillation, crystallization after reduced pressure distillation or crystallization after back extraction acidification;

preferably, the reduced pressure distillation is at a temperature of from 30 ℃ to 60 ℃, and/or

The absolute pressure of the reduced pressure distillation is-100 to 100kPa, and/or

The temperature of the crystallization is 4-20 ℃;

the crystallization time is 8-24h.

10. The process according to any one of claims 1 to 9, wherein the purity of the alpha-ketoglutarate extracted from the fermentation broth is greater than or equal to 98% by weight and the yield is greater than or equal to 90%.