Method for extracting alpha-ketoglutaric acid from fermentation liquor

Abstract

The invention provides a method for extracting alpha-ketoglutaric acid from fermentation liquor. The method comprises the following steps: carrying out multi-stage fractional extraction on pretreated fermentation liquor; wherein an extracting agent for multi-stage 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 through a microbial fermentation method; more preferably, fermentation liquor of alpha-ketoglutaric acid and pyruvic acid is produced by taking the Yarrowia lipolytica as a raw material; preferably, the concentration of the alpha-ketoglutaric acid in the fermentation liquor is 0.5 g / L-200 g / L. The oxygen-containing organic solvent used in the method has high separation selectivity and low water solubility and toxicity on the alpha-ketoglutaric acid, and the process is simple, safe and environmentally friendly.

Description

Technical Field

[0001] The invention relates to the technical field of chemical separation, and in particular to a method for extracting alpha-ketoglutaric acid from a fermentation broth. Background Art

[0002] α-Ketoglutaric acid is a white fine crystalline powder at room temperature. It participates in the tricarboxylic acid cycle and amino acid metabolism in microorganisms. It is an important dicarboxylic acid and plays a vital role in the intermediate metabolism process of regulating carbon-nitrogen balance. In the field of food health, the mixture of α-ketoglutaric acid and L-arginine is called arginine mixture, which can be added to functional beverages as a nutritional enhancer; it can also be used in biomedicine for tissue engineering and drug delivery. Biofermentation and enzyme conversion are the main methods for producing α-ketoglutaric acid, using glycerol as the raw material. The product prepared by this method also contains impurities such as pyruvic acid, malic acid, and citric acid. α-Ketoglutaric acid has similar structures and physicochemical properties to these impurity acids, and both have good water solubility and large polarity. It is very difficult to separate α-ketoglutaric acid from the fermentation broth.

[0003] There are few existing methods for separating α-ketoglutaric acid from fermentation broth, which mainly include calcium salt precipitation method, membrane separation method and crystallization method.

[0004] The calcium salt precipitation method is based on the fact that the calcium salts of most carboxylic acids have low solubility in water. At a certain temperature and pH value, the α-ketoglutaric acid in the fermentation broth is converted into a calcium salt precipitate and separated. Patent CN105198732A discloses a method for separating α-ketoglutaric acid from a fermentation broth using a calcium salt: adding an excess of calcium hydroxide to the fermentation broth, centrifuging to separate calcium α-ketoglutarate and calcium hydroxide, adding sulfuric acid to acidify to obtain an α-ketoglutaric acid solution, and then concentrating under reduced pressure to obtain a crude α-ketoglutaric acid product, and then washing with methanol to obtain α-ketoglutaric acid. This process does not take into account that the impurity acid in the fermentation broth will also form a calcium salt precipitate, making it difficult to separate it from calcium α-ketoglutarate in the future. and PROCHASKA [Biochemical Engineering Journal, 2021, 166: 107883] designed a multi-stage membrane process, assisted by vacuum evaporation and crystallization, to recover α-ketoglutaric acid from fermentation broth. The pretreated fermentation broth was electrodialyzed using commercial ceramic membranes and nanofiltration ceramic membranes to separate α-ketoglutarate and convert it into acid form. The migration of other acids in the fermentation broth was restricted, and the final product purity was 94.8%, but the membrane was expensive and easily contaminated. Crystallization is generally implemented as a step in the treatment, based on extraction or pretreatment.

[0005] In summary, the methods for extracting α-ketoglutaric acid in the prior art have the problems of complicated process, high cost and poor separation selectivity, and it is necessary to develop a method for efficiently extracting α-ketoglutaric acid from fermentation broth. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for extracting alpha-ketoglutaric acid from a fermentation broth. The method used in the present invention has a simple process and low cost, and the extractant used has good stability and higher separation selectivity. The multi-stage fractionation extraction process is safe and easy to scale up, which is conducive to industrial production.

[0007] The present invention provides a method for extracting alpha-ketoglutaric acid from a fermentation broth, comprising the following steps: subjecting the pretreated fermentation broth to multi-stage fractionation extraction; wherein the extractant of the multi-stage fractionation extraction comprises an oxygen-containing organic solvent with hydrogen bond alkalinity.

[0008] α-Ketoglutaric acid is a dicarboxylic acid containing a carbonyl group, and its pK a1 The value is 2.47, the acidity and hydrogen bond acidity are both strong, and the two carboxyl hydrogens can serve as hydrogen bond donors. After research, it was found that by using an extractant with a certain hydrogen bond alkalinity, the α-ketoglutaric acid in the fermentation broth can be selectively extracted and separated through the hydrogen bond force formed by the solute molecules and the extractant. Furthermore, the efficiency of extracting and separating α-ketoglutaric acid after pretreatment of the fermentation broth of the present invention is higher.

[0009] In some embodiments, the fermentation broth source is a fermentation broth produced by a microbial fermentation method to produce α-ketoglutarate and pyruvate.

[0010] In some embodiments, the fermentation broth is a fermentation broth for producing α-ketoglutarate and pyruvate using Yarrowia lipolytica as a raw material.

[0011] In some embodiments, the fermentation broth includes alpha-ketoglutarate and pyruvate.

[0012] In some embodiments, the concentration of α-ketoglutarate in the fermentation broth is 0.5 g / L-200 g / L, for example, 1 g / L, 2 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, 30 g / L, 50 g / L, 80 g / L, 110 g / L, 150 g / L, 190 g / L or any value therebetween.

[0013] In some embodiments, the concentration of α-ketoglutarate in the fermentation broth is 5 g / L-20 g / L.

[0014] In some embodiments, the oxygen-containing organic solvent with hydrogen bond alkalinity includes at least one of an alcohol solvent, an ester solvent, an ether solvent, an amide solvent or an organophosphorus solvent. The present invention selects the above oxygen-containing organic solvent with hydrogen bond alkalinity as an extractant, which has a higher separation selectivity.

[0015] In some embodiments, the alcohol solvent includes an alcohol solvent substituted with a C1-C10 straight-chain or branched alkyl group; preferably one or more of n-butanol, n-octanol or 2-ethyl-1-hexanol.

[0016] In some embodiments, the ester solvent includes an ester compound with a carbon number greater than 6, preferably one or more of amyl acetate, butyl butyrate, and butyl valerate.

[0017] In some embodiments, the ether solvent includes methyl tert-butyl ether.

[0018] In some embodiments, the amide solvent includes one or more of N,N-di-n-butylformamide, N,N-di-n-butylacetamide, and N,N-di-n-butylbutanamide.

[0019] In some embodiments, the organophosphorus solvent includes alkyl phosphate ester and / or alkyl phosphine oxide.

[0020] In some embodiments, the alkyl group in the alkyl phosphate and the alkyl phosphine oxide is selected from substituted or unsubstituted C3-C8 straight chain or branched alkyl groups.

[0021] In some embodiments, at least one hydrogen atom in the C3-C8 straight or branched alkyl group is substituted by at least one of a carboxyl group, an amino group, a carbonyl group, an olefin group, a hydroxyl group, a halogenated atom, a sulfonyl group, and a cyano group. The substitution may be mono-, di-, tri- or poly-substitution, and in the case of poly-substitution, the types and quantities of the substituents at different sites may be the same or different.

[0022] In some embodiments, the organophosphorus solvent is selected from one or more of tributyl phosphate, trioctyl phosphate, tri-n-octyl phosphine oxide, tri(2-ethylhexyl)phosphine oxide and tricyclohexylphosphine oxide.

[0023] In some embodiments, the oxygen-containing organic solvent having hydrogen bond alkalinity is selected from one or more of amyl acetate, butyl butyrate, butyl valerate, N,N-dibutylformamide, N,N-dibutylacetamide, N,N-di-n-butylbutyramide, n-hexanol, n-octanol, 2-ethyl-1-hexanol, tributyl phosphate, trioctyl phosphate, tri-n-octyl phosphine oxide, di(ethylhexyl) phosphate, tri(2-ethylhexyl) phosphine oxide, tricyclohexyl phosphine oxide, or a mixture of trioctyl phosphine oxide and di(ethylhexyl) phosphate.

[0024] In some embodiments, the oxygen-containing organic solvent having hydrogen bond alkalinity is selected from one or more of butyl butyrate, butyl valerate, N,N-dibutylformamide, tributyl phosphate, tri-n-octyl phosphine oxide, di(ethylhexyl) phosphate, tricyclohexyl phosphine oxide, or a mixture of trioctyl phosphine oxide and di(ethylhexyl) phosphate.

[0025] In some embodiments, the extractant further comprises an optional diluent. The above oxygen-containing organic solvent can be used alone or in a diluent.

[0026] In some embodiments, the diluent includes 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.

[0027] In some embodiments, the diluent includes at least one of petroleum ether, 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, and amyl acetate, preferably one or more of n-butanol, ethyl acetate, butyl acetate, amyl acetate, dichloromethane, n-hexanol, methyl isobutyl ketone, methyl tert-butyl ether, and isopropyl ether.

[0028] In some embodiments, the diluent is n-butanol and / or ethyl acetate.

[0029] In some embodiments, the extractant includes an oxygen-containing organic solvent having hydrogen bonding basicity and a diluent.

[0030] In some embodiments, the extractant is selected from trioctylphosphine oxide and n-butanol, tricyclohexylphosphine oxide and ethyl acetate, N,N-di-n-butylformamide and ethyl acetate.

[0031] In some embodiments, the mass fraction of the oxygen-containing organic solvent in the extractant is 0.1%-99.99%, for example, 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.

[0032] In some embodiments, the mass fraction of the oxygen-containing organic solvent is 1%-50%.

[0033] In some embodiments, the mass fraction of the oxygen-containing organic solvent is 5%-50%.

[0034] In some embodiments, the mass fraction of the oxygen-containing organic solvent is 10%-40%.

[0035] In some embodiments, the multi-stage fractionation extraction further comprises a detergent.

[0036] In some embodiments, the detergent is water.

[0037] When performing multi-stage extraction, taking into account factors such as product quality and production cost, the volume flow ratio of the extractant, detergent and pretreated fermentation liquid 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 therebetween.

[0038] In some embodiments, the ratio of the volume flow rates of the extractant, the detergent, and the pretreated fermentation broth is 1:(0.1-10):(0.1-10).

[0039] In some embodiments, the ratio of the volume flow rates of the extractant, the detergent, and the pretreated fermentation broth is 1:(0.2-8):(0.2-8).

[0040] In some embodiments, the operating temperature of the multi-stage fractionation extraction is 15°C-60°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or any value therebetween. If the temperature is too low, the viscosity of the extractant will be large, the mass transfer rate will be reduced, the processing volume will decrease, and it will be unfavorable for production operation; if the temperature is too high, on the one hand, the volatilization rate of the diluent or solvent will become faster, and on the other hand, the distribution coefficient and selectivity of α-ketoglutaric acid will be reduced.

[0041] In some embodiments, the pretreatment includes: removing insoluble impurities and macromolecular impurities in the fermentation broth, decolorizing and desalting.

[0042] In some embodiments, the molecular weight of the macromolecular impurities is greater than 1000 Da. The macromolecular impurities may be biological macromolecules such as bacterial fragments, lipids, polysaccharides and proteins.

[0043] In some embodiments, 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.

[0044] In some embodiments, the method for removing insoluble impurities and macromolecular impurities in a fermentation broth comprises:

[0045] (1) centrifuging the fermentation broth and collecting the supernatant;

[0046] (2) performing a first filtration on the supernatant to obtain a first filtrate;

[0047] (3) subjecting the first filtrate to a second filtration to obtain a second filtrate;

[0048] The centrifugation and filtration process is safe and environmentally friendly and can remove most macromolecular impurities.

[0049] In some embodiments, the first filtration uses a ceramic membrane.

[0050] In some embodiments, the ceramic membrane assembly is an external pressure structure membrane assembly of a PVDF hollow fiber membrane, which can intercept impurities with a molecular weight greater than 1000Da.

[0051] In some embodiments, the operating pressure of the first filtration is 0.1 MPa-0.5 MPa; for example, 0.15 MPa, 0.2 MPa, 0.25 MPa, 0.3 MPa, 0.35 MPa, 0.4 MPa, 0.45 MPa, 0.5 MPa or any value therebetween.

[0052] In some embodiments, the pore size of the ceramic membrane is 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.

[0053] In some embodiments, the second filtration uses an ultrafiltration membrane.

[0054] In some embodiments, the ultrafiltration membrane is a hollow fiber membrane made of polyvinylidene fluoride.

[0055] In some embodiments, the operating pressure of the second filtration is 0.1 MPa-0.7 MPa; for example, 0.15 MPa, 0.2 MPa, 0.25 MPa, 0.3 MPa, 0.35 MPa, 0.4 MPa, 0.45 MPa, 0.5 MPa, 0.55 MPa, 0.6 MPa, 0.65 MPa or any value therebetween.

[0056] In some embodiments, the pore size of the ultrafiltration membrane is 0.01-0.1 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.

[0057] In some embodiments, the decolorization method comprises adsorption chromatography.

[0058] In some embodiments, the decolorization uses macroporous adsorption resin and / or decolorization activated carbon.

[0059] In some embodiments, the macroporous adsorption 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.

[0060] In some embodiments, the macroporous adsorption 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.

[0061] The above macroporous adsorption resin has large decolorization capacity and is easy to regenerate.

[0062] In some embodiments, the desalting method comprises ion exchange chromatography.

[0063] In some embodiments, the desalination uses a cation exchange resin.

[0064] The column chromatography method used for decolorization and desalting is simple and easy to operate, and has good circulation performance.

[0065] In some embodiments, the cation exchange resin is selected from one or more of ZGCER50, ZG ER 8420, ZG ER 8410, D851, Amberlite HPR1200 H, Amberlite HPR 2900H, Amberlite IRC 120H, 001×7, 001×8, D001, LX-160, and LSI-010.

[0066] In some embodiments, the cation exchange resin is selected from the group consisting of ZG CER50, Amberlite IR 120H, FPC 22H, HPR 2900H, 001×7 and 001×8.

[0067] In some embodiments, the method further comprises post-processing.

[0068] In some embodiments, the post-treatment method includes: reduced pressure distillation followed by crystallization, or stripping acidification followed by crystallization.

[0069] In some embodiments, α-ketoglutaric acid is enriched in the extract phase or the raffinate phase, depending on the extractant used. If α-ketoglutaric acid is enriched in the extract phase, when the extractant is a low-boiling oxygen-containing organic solvent, the low-boiling oxygen-containing organic solvent is distilled under reduced pressure to recover the solvent and solute, and when the extractant is an organophosphorus solvent or an oxygen-containing organic solvent with a boiling point higher than 100° C., a high-purity α-ketoglutaric acid product is obtained by back extraction with alkaline solution, acidification, crystallization and vacuum drying; if α-ketoglutaric acid is enriched in the raffinate phase, the α-ketoglutaric acid product is obtained by concentrating the raffinate under reduced pressure and then crystallizing.

[0070] In some embodiments, the low boiling point oxygen-containing organic solvent is a solvent having a boiling point below 100°C.

[0071] In some embodiments, the crystallization is cooling crystallization.

[0072] In some embodiments, the crystallization temperature is 4°C-20°C, for example, 5°C, 8°C, 11°C, 14°C, 17°C, 20°C or any value therebetween.

[0073] In some embodiments, the crystallization time is 8-24 h, for example, 9 h, 12 h, 15 h, 18 h, 21 h, 24 h or any value therebetween.

[0074] In some embodiments, the reduced pressure distillation temperature is 30°C-60°C, for example, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or any value therebetween.

[0075] In some embodiments, the reduced pressure distillation pressure is -100-100 kPa (absolute pressure); for example, -100 kPa, -50 kPa, 0 kPa, 50 kPa, 100 kPa, or any value therebetween.

[0076] The fractional distillation extraction includes an extraction section and a washing section. The extractant enters the system from the first stage of the extraction section. After the number of stages of the extraction section and the washing section is determined, the raw liquid enters the system from the feed stage, the washing agent and the raw liquid are mixed at the feed stage, the extraction phase and the washing phase are in multi-stage countercurrent contact, the extract flows out from the first stage of the washing section, and the raffinate flows out from the first stage of the extraction section.

[0077] In some embodiments, the method comprises the following steps:

[0078] (1) Pretreatment: The fermentation liquid of α-ketoglutaric acid produced by microbial fermentation is used as the raw material liquid, which is centrifuged and filtered, and then subjected to resin decolorization and desalination treatment;

[0079] (2) Fractional extraction: fractional extraction with multi-stage countercurrent contact using an oxygen-containing organic solvent and an optional diluent as the extractant and water as the detergent;

[0080] (3) Obtaining α-ketoglutaric acid product: After fractional extraction, α-ketoglutaric acid is collected by back extraction or direct evaporation of the solvent.

[0081] In some embodiments, the content (HPLC) of α-ketoglutaric acid extracted from the fermentation broth is ≥98 wt %, and the yield is ≥90%.

[0082] Compared with the prior art, the present invention has the following beneficial effects:

[0083] 1. The pretreatment method adopted by the present invention is safe and environmentally friendly, has a simple process, low energy consumption, and the resin used in the decolorization and desalination process is low in cost, easy to regenerate, and can be recycled;

[0084] 2. The multi-stage fractionation extraction process of the present invention is safe, easy to scale up, and conducive to industrial production;

[0085] 3. The oxygen-containing organic solvent with hydrogen bond alkalinity used in the present invention has high separation selectivity, low water solubility and toxicity for α-ketoglutaric acid, and the extractant can be recovered and recycled after vacuum distillation or strong alkali stripping;

[0086] 4. The multi-stage fractionation extraction process adopted in the present invention can take into account both product purity and yield. Under optimized conditions, the purity of the α-ketoglutaric acid product reaches more than 98wt%, and the recovery rate is greater than 90%. DETAILED DESCRIPTION

[0087] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention.

[0088] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0089] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the raw materials, instruments and equipment used in the following examples, etc., can all be purchased on the market or can be obtained by existing methods; the reagent amounts, unless otherwise specified, are the reagent amounts used in conventional experimental operations; the experimental methods, unless otherwise specified, are all conventional methods.

[0090] The "fractionation extraction" in the present invention refers to a cascade extraction in which an aqueous phase feed liquid (F) containing the components to be separated flows into the cascade extractor from a middle stage, and a washing liquid (W) and an organic phase (S) not containing the components to be separated flow into the cascade extractor from both ends at a certain flow ratio.

[0091] The "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 capacity. The stronger the hydrogen bond basicity of a substance, the stronger its ability to interact with other hydrogen bond donor substances as a hydrogen bond acceptor and form intermolecular hydrogen bonds.

[0092] The following example is a method for extracting α-ketoglutaric acid from a microbial fermentation broth containing α-ketoglutaric acid, taking the fermentation broth obtained by using Yarrowia lipolytica as the fermentation strain as an example. Other similar microbial fermentation broths or enzyme conversion broths containing α-ketoglutaric acid can be processed according to the embodiments of the present invention to obtain an α-ketoglutaric acid product with a higher purity.

[0093] Example 1

[0094] α-ketoglutarate was extracted from the fermentation broth obtained from Yarrowia lipolytica, with an initial concentration of α-ketoglutarate of 10.3 g / L, according to the following steps:

[0095] (1) Centrifugation and filtration: The fermentation broth was pumped into a high-speed centrifuge at a pump flow rate of 100 mL / min and a centrifuge speed of 8000 rpm for 5 min. After centrifugation, the broth was filtered to remove the bacteria and other visible solids to obtain the filtrate.

[0096] (2) Membrane filtration: The filtrate obtained by centrifugal filtration is filtered through a ceramic membrane with a pore size of 0.1-1μm to remove biomacromolecules such as bacterial fragments, lipids, polysaccharides and proteins in the filtrate, and finally the residual filtrate is washed with a small amount of water to ensure that the yield of the ceramic filtration process is close to 100%. The ceramic filtration permeate is passed through an ultrafiltration membrane with a pore size of 0.01-0.1μm, and finally the residual filtrate is washed with a small amount of water. The ceramic membrane assembly used is an external pressure structure membrane assembly of a PVDF hollow fiber membrane, which can intercept impurities with a molecular weight greater than 1000Da, and the operating pressure is 0.1MPa; the ultrafiltration membrane is a hollow fiber membrane made of polyvinylidene fluoride as raw material, and the operating pressure is 0.1MPa.

[0097] (3) Macroporous adsorption resin decolorization: The fermentation broth containing α-ketoglutaric acid after centrifugation and membrane filtration was treated with macroporous decolorization resin HPD 750 to remove most of the pigments by adsorption. The sample flow rate was 1.5 BV / h, and the column was washed out with 1.5 BV of water, and the effluent was collected. The column temperature was 25°C and the operation was carried out under normal pressure.

[0098] (4) Treatment with strong acid cation exchange resin: The decolorized fermentation broth was treated with a ZG CER50 resin layer to remove metal cations in the decolorized fermentation broth and convert α-ketoglutarate into α-ketoglutaric acid. The sample flow rate was 1.2 BV / h, and finally 1 BV of water was used to wash out the feed liquid in the column, and the effluent was collected to obtain the pretreated fermentation broth. The column temperature was 25°C and the operation was carried out under normal pressure.

[0099] (5) Fractional extraction: The pretreated fermentation liquid is used as the raw material, di(ethylhexyl) phosphate is used as the extractant, water is used as the detergent, and the flow ratio of the extractant, the detergent, and the pretreated fermentation liquid is 1:2:1. Fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 8 stages and the washing section has 5 stages). The extract enriched in α-ketoglutaric acid flows out from the first stage of the washing section, and the extract is collected.

[0100] (6) Stripping: The extract is stripped using a 1M NaOH aqueous solution. The extract is added to a stirring tank, and the stripping agent is slowly added dropwise while mechanically stirring until the stripping is complete. The molar ratio of NaOH in the stripping agent to α-ketoglutaric acid in the extract is 1:1, and the operating temperature is 30°C. The stripping phase is acidified with a cation exchange resin ZG CER50, cooled and crystallized, and vacuum dried to obtain an α-ketoglutaric acid product. The purity of α-ketoglutaric acid is 99.57%, and the yield is 94.08%.

[0101] Example 2

[0102] The fermentation liquid obtained by using Yarrowia lipolytica as the fermentation strain is the raw material liquid, the initial concentration of α-ketoglutaric acid is 20.6 g / L, the implementation method is the same as that of Example 1, the difference being that the decolorization pretreatment of step (3) is not performed, and after desalting in step (4), a 36.5 v / v% hydrochloric acid solution is added dropwise to the fermentation liquid after deionization and stirred continuously; the fermentation liquid after the above treatment uses di(ethylhexyl) phosphate as an extractant and water as a detergent, and the flow ratio of the extractant, the detergent, and the pretreated fermentation liquid is 1:2:1, and fractional extraction is performed at 40°C. The back extraction phase is acidified by a cation exchange resin Amberlite IR120H, cooled and crystallized, and vacuum dried to obtain an α-ketoglutaric acid product. The purity of α-ketoglutaric acid is 98.97%, and the yield is 95.25%.

[0103] Example 3

[0104] The fermentation liquid obtained by using Yarrowia lipolytica as the fermentation strain is used as the raw material liquid, the initial concentration of α-ketoglutaric acid is 40.2 g / L, and the implementation method is the same as that of Example 2, except that the pretreated fermentation liquid uses tributyl phosphate as the extractant and water as the detergent, and the flow ratio of the extractant, the detergent and the raw material liquid is 1:2:1, and fractional extraction is performed at 40° C. The purity of α-ketoglutaric acid is 99.06%, and the yield is 96.31%.

[0105] Example 4

[0106] The fermentation liquid obtained by using Yarrowia lipolytica as the fermentation strain is used as the raw liquid, the initial concentration of α-ketoglutaric acid is 60.3 g / L, and the implementation method is the same as that of Example 1, except that the fermentation liquid after centrifugal filtration, decolorization and desalting is fractionated and extracted at 30°C using a mixture of trioctylphosphine oxide (TOPO) and di(ethylhexyl) phosphate, the mass fraction of TOPO in the mixture is 10%, water is used as the detergent, the flow ratio of the raw liquid, the detergent and the extractant is 1:2:1, the extraction section has 5 stages, and the washing section has 8 stages. The extract enriched in α-ketoglutaric acid flows out in the first stage of the washing section, and the extract is collected.

[0107] The extract was stripped with NaOH, then acidified, crystallized and dried to obtain an α-ketoglutaric acid product with a purity of 98.50% and a yield of 96.17%.

[0108] Example 5

[0109] The fermentation liquid obtained by using Yarrowia lipolytica as the fermentation strain is the raw material liquid, the initial concentration of α-ketoglutaric acid is 100.4 g / L, and the pretreatment process and the stripping process are the same as in Example 1, except that the decolorization resin uses SD300 and the desalting resin uses IR120 H. The difference in the extraction process is that butyl valerate is used as the extractant, water is used as the detergent, the volume flow ratio of the raw material liquid, the detergent and the extractant is 1:6:8.3, and fractional distillation extraction is performed at 30° C. (10 stages in the extraction section and 8 stages in the washing section), and the raffinate enriched in α-ketoglutaric acid flows out in the first stage of the extraction section, and the raffinate and the extract are collected.

[0110] The purity of α-ketoglutaric acid was 98.09%, and the yield was 95.40%.

[0111] Example 6

[0112] α-ketoglutarate was separated and extracted from the fermentation broth obtained by using Yarrowia lipolytica as the fermentation strain, with the initial concentration of α-ketoglutarate being 50.6 g / L, according to the following steps:

[0113] (1) Centrifugation and filtration: The fermentation broth was pumped into a high-speed centrifuge at a pump flow rate of 50 mL / min and a centrifuge speed of 5000 rpm. After centrifugation for 10 min, the broth was filtered to remove the bacteria and other visible solids to obtain the filtrate.

[0114] (2) Membrane filtration: The filtrate obtained by centrifugal filtration is filtered through an ultrafiltration membrane with a pore size of 0.01-0.1 μm, and the residual liquid is washed with a small amount of water. The ultrafiltration membrane used is a hollow fiber membrane made of polyvinylidene fluoride, and the operating pressure is 1.5 MPa.

[0115] (3) Macroporous adsorption resin decolorization: The fermentation broth containing α-ketoglutaric acid after centrifugation and membrane filtration was treated with ion exchange resin Amberlite XAD-16 to remove most of the pigment. The sample flow rate was 1.5 BV / h. The column temperature was 25°C and the operation was carried out under normal pressure.

[0116] (4) Cation exchange resin desalination: The decolorized fermentation broth is treated with an FPC 22H resin layer to remove metal cations in the decolorized fermentation broth and convert α-ketoglutarate into α-ketoglutaric acid. The sample flow rate is 1.25 BV / h, and finally 1.2 BV of water is used to wash out the feed liquid in the column and collect the effluent. The column temperature is 25°C and the operation is carried out under normal pressure.

[0117] (5) Fractional extraction: The fermentation broth after the above pretreatment was used as the raw material liquid, the extractant was a binary mixed solvent consisting of TOPO and a diluent n-butanol, the mass fraction of TOPO in the extractant was 30%, the flow ratio of the extractant, the detergent and the pretreated fermentation broth was 1.1:1.9:1, and fractional extraction was carried out at 30° C. (a total of 6 extraction stages and a total of 5 washing stages), and the extract and the raffinate were collected.

[0118] (6) Stripping

[0119] The extract was stripped with 1M NaOH aqueous solution, the molar ratio of NaOH in the stripping agent to α-ketoglutaric acid in the extract was 1.5:1, and the operating temperature was 30°C. The stripping phase was acidified with cation exchange resin ZG CER50, cooled and crystallized for 24 hours, and vacuum dried to obtain α-ketoglutaric acid product. The product purity was 98.56% and the yield was 95.02%.

[0120] Example 7

[0121] α-ketoglutaric acid was separated and extracted from the fermentation liquid obtained by fermenting Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 50.6 g / L, the pretreatment method and the stripping method were the same as those in Example 6, and the difference in the fractional extraction process was that the extractant was a binary mixed solvent composed of tricyclohexylphosphine oxide and diluent ethyl acetate, the mass fraction of tricyclohexylphosphine oxide in the extractant was 40%, the flow ratio of the extractant, the detergent and the raw material liquid was 4:4:1, the operating temperature was 15°C, and fractional extraction was performed (5 extraction stages and 5 washing stages). The purity of the α-ketoglutaric acid product was 98.68%, and the yield was 95.69%.

[0122] Example 8

[0123] α-ketoglutaric acid was separated and extracted from the fermentation broth obtained from the fermentation strain Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 50.6 g / L, the pretreatment method and the stripping method were the same as those in Example 6, and the difference in the fractional extraction process was that the extractant was a binary mixed solvent composed of N, N-di-n-butylformamide and a diluent ethyl acetate, the mass fraction of N, N-di-n-butylformamide in the extractant was 25%, the flow ratio of the extractant, the detergent, and the pretreated fermentation broth was 2.5:3.3:1, and the fractional extraction was performed at 30°C (10 extraction stages and 7 washing stages). The purity of the α-ketoglutaric acid product was 98.12%, and the yield was 94.58%.

[0124] Example 9

[0125] α-ketoglutaric acid was extracted from a fermentation broth obtained by fermenting Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 10.6 g / L, and the pretreatment, extraction and post-treatment steps were the same as those in Example 1, except that the extractant was butyl butyrate. The purity of α-ketoglutaric acid in the obtained product was 98.70%, and the yield was 94.14%.

[0126] Example 10

[0127] α-ketoglutaric acid was extracted from a fermentation broth obtained by fermenting Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 10.6 g / L, and the pretreatment, extraction and post-treatment steps were the same as those in Example 1, except that the extractant was N,N-di-n-butylformamide. The purity of α-ketoglutaric acid in the obtained product was 99.50%, and the yield was 95.27%.

[0128] Embodiment 11

[0129] α-ketoglutaric acid was extracted from a fermentation broth obtained by fermenting Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 10.3 g / L, and the pretreatment, extraction and post-treatment steps were the same as those in Example 1, except that the extractant was a mixture of trioctylphosphine oxide (TOPO) and di(ethylhexyl) phosphate, and the mass fraction of TOPO in the extractant was 10%. The purity of α-ketoglutaric acid in the obtained product was 99.56%, and the yield was 96.27%.

[0130] Example 12

[0131] α-ketoglutaric acid was extracted from a fermentation broth obtained by fermenting Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 10.3 g / L, and the pretreatment, extraction and post-treatment steps were the same as in Example 1, except that during fractional extraction, the flow ratio of the extractant, the detergent and the pretreated fermentation broth was 1:6:1. The purity of α-ketoglutaric acid in the obtained product was 99.82%, and the yield was 93.68%.

[0132] Embodiment 13

[0133] α-ketoglutaric acid was extracted from a fermentation broth obtained by fermenting Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 10.3 g / L, and the pretreatment, extraction and post-treatment steps were the same as in Example 1, except that during fractional extraction, the flow ratio of the extractant, the detergent and the pretreated fermentation broth was 1:1:6. The purity of α-ketoglutaric acid in the obtained product was 99.59%, and the yield was 91.53%.

[0134] Comparative Example 1

[0135] Alpha-ketoglutaric acid is separated and extracted from the fermentation liquid obtained from the fermentation strain of Yarrowia lipolytica. The initial concentration of alpha-ketoglutaric acid is 100.4 g / L. The pretreatment method is the same as that in Example 1, the extractant used is methyl tert-butyl ether, and the flow ratio of the extractant, the detergent, and the pretreated fermentation liquid is 1:10:10. Fractional extraction is performed at 30°C (30 levels in the extraction section and 1 level in the washing section). The extract is distilled under reduced pressure to remove the low-boiling methyl tert-butyl ether, and the methyl tert-butyl ether is recovered and recycled. The raffinate is vacuum dried after crystallization. The purity of the alpha-ketoglutaric acid product is 31.68%, and the yield is 3.25%.

[0136] Comparative Example 2

[0137] α-ketoglutaric acid was separated and extracted from a fermentation broth obtained from Yarrowia lipolytica, with an initial α-ketoglutaric acid concentration of 100.4 g / L. The implementation method was the same as that of Example 1, except that resin decolorization was performed after centrifugation and membrane filtration, and the cation exchange resin desalination treatment in step (4) was not performed. The decolorized fermentation broth was directly used for fractional extraction, and the extractant used was methyl isobutyl ketone. The flow ratio of the extractant, the detergent, and the pretreated fermentation broth was 1:10:10, and the fractional extraction was performed at 30° C. (30 extraction stages and 1 washing stage). The purity of the α-ketoglutaric acid product was 33.93%, and the yield was 14.94%.

[0138] Comparative Example 3

[0139] α-ketoglutaric acid was separated and extracted from the fermentation liquid obtained from Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 10.3 g / L, the implementation method was the same as Example 1, the difference being that the extractant used was methyl tert-butyl ether. The purity of the α-ketoglutaric acid product was 44.51%, and the yield was 32.41%.

[0140] Comparative Example 4

[0141] α-ketoglutaric acid was separated and extracted from the fermentation broth obtained from Yarrowia lipolytica, the initial concentration of α-ketoglutaric acid was 10.3 g / L, the implementation method was the same as Example 1, except that the extractant was methyl isobutyl ketone. The purity of the α-ketoglutaric acid product was 24.25%, and the yield was 19.76%.

[0142] Table 1 Summary of operating conditions and results of examples and comparative examples

[0143]

[0144]

[0145]

[0146] As can be seen from Table 1, the present invention uses an oxygen-containing organic solvent with hydrogen bond alkalinity as an extractant, and combines it with a multi-stage fractionation extraction method, which can take into account both product purity and yield.

[0147] Furthermore, the present invention adopts a specific pretreatment method (adsorption resin decolorization and resin acidification desalting), a specific extractant and a specific multi-stage fractionation extraction operation mode, which has a high separation selectivity for α-ketoglutaric acid and can take into account both product purity and yield. The purity of the obtained α-ketoglutaric acid product reaches more than 98wt%, the recovery rate is greater than 90%, and no subsequent purification is required, the amount of extractant used is less, and the cost is lower.

[0148] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for extracting α-ketoglutaric acid from a fermentation broth, comprising the following steps: subjecting the pretreated fermentation broth to multi-stage fractionation extraction; in, The extractant of the multi-stage fractionation extraction includes an oxygen-containing organic solvent having hydrogen bond alkalinity; Preferably, the fermentation broth is derived from a fermentation broth produced by a microbial fermentation method to produce α-ketoglutarate and pyruvate; more preferably, it is a fermentation broth produced by using Yarrowia lipolytica as a raw material to produce α-ketoglutarate and pyruvate; Preferably, the concentration of α-ketoglutaric acid in the fermentation broth is 0.5 g / L-200 g / L.

2. The method according to claim 1, characterized in that: The oxygen-containing organic solvent having hydrogen bond alkalinity includes at least one of an alcohol solvent, an ester solvent, an ether solvent, an amide solvent or an organophosphorus solvent; Preferably, the alcohol solvent comprises an alcohol solvent substituted with a C1-C10 straight-chain or branched alkyl group; preferably one or more of n-butanol, n-octanol or 2-ethyl-1-hexanol; and / or The ester solvent comprises an ester compound having a carbon number greater than 6; preferably one or more of amyl acetate, butyl butyrate or butyl valerate; and / or The ether solvent includes methyl tert-butyl ether; and / or The amide solvent includes one or more of N,N-di-n-butylformamide, N,N-di-n-butylacetamide or N,N-di-n-butylbutanamide; and / or The organophosphorus solvent includes 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 straight chain or branched alkyl group, and preferably at least one hydrogen atom in the C3-C8 straight chain or branched alkyl group is substituted by at least one of a carboxyl group, an amino group, a carbonyl group, an olefin group, a hydroxyl group, a halogenated 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-octyl phosphine oxide, tri(2-ethylhexyl)phosphine oxide and tricyclohexylphosphine oxide.

3. The method according to claim 1 or 2, characterized in that: The oxygen-containing organic solvent having hydrogen bond alkalinity is selected from one or more of amyl acetate, butyl butyrate, butyl valerate, N,N-dibutylformamide, N,N-dibutylacetamide, N,N-di-n-butylbutyramide, 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 alkalinity is selected from one or more of butyl butyrate, butyl valerate, N,N-dibutylformamide, tributyl phosphate, tri-n-octyl phosphine oxide, di(ethylhexyl) phosphate, tricyclohexyl phosphine oxide or a mixture of trioctyl phosphine oxide and di(ethylhexyl) phosphate.

4. The method according to any one of claims 1 to 3, characterized in that The extractant also includes an optional diluent; Preferably, the diluent includes 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; Preferably, the diluent includes 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, and 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 according to claim 4, characterized in that The extractant comprises an oxygen-containing organic solvent having hydrogen bond alkalinity and a diluent, Preferably, the extractant is selected from trioctylphosphine oxide and n-butanol, tricyclohexylphosphine oxide and ethyl acetate, N,N-di-n-butylformamide and ethyl acetate; Preferably, in the extractant, the mass content of the oxygen-containing organic solvent having hydrogen bond alkalinity is 0.1%-99.99%; preferably 1%-50%, more preferably 5%-50%.

6. The method according to any one of claims 1 to 5, characterized in that The multi-stage fractionation extraction further comprises a detergent, Preferably, the detergent comprises water; and / or The ratio of the volume flow 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 multi-stage fractionation extraction is 15°C-60°C.

7. The method according to any one of claims 1 to 5, characterized in that The pretreatment includes: removing insoluble impurities and macromolecular impurities in the fermentation liquid, decolorizing and desalting; Preferably, the molecular weight of the macromolecular impurity is greater than 1000Da; Preferably, the method for removing insoluble impurities and macromolecular impurities in the fermentation broth comprises one or more of centrifugal separation, ultrafiltration, microfiltration and plate and frame filtration; Preferably, the method for removing insoluble impurities and macromolecular impurities in the fermentation broth comprises: (1) centrifuging the fermentation broth and collecting the supernatant; (2) performing a first filtration on the supernatant to obtain a first filtrate; (3) subjecting the first filtrate to a second filtration to obtain a second filtrate; Preferably, the first filtration uses a ceramic membrane, preferably, the pore size of the ceramic membrane is 0.1-1 μm; and / or The second filtration uses an ultrafiltration membrane, and preferably, the pore size of the ultrafiltration membrane is 0.01-0.1 μm.

8. The method according to claim 7, characterized in that The decolorization method includes an adsorption chromatography column method. Preferably, the decolorization uses a macroporous adsorption resin and / or decolorization activated carbon; Preferably, the macroporous adsorption 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 desalination method includes an ion exchange chromatography column method. Preferably, the desalination uses a cation exchange resin. Preferably, the cation exchange resin is selected from one or more of ZG CER50, ZG ER 8420, ZGER 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 to 8, characterized in that The method also includes post-processing; Preferably, the post-treatment method includes: vacuum distillation, vacuum distillation followed by crystallization, or stripping acidification followed by crystallization; Preferably, the temperature of the reduced pressure distillation is 30°C-60°C; and / or The absolute pressure of the reduced pressure distillation is -100-100 kPa; and / or The crystallization temperature is 4°C-20°C; The crystallization time is 8-24h.

10. The method according to any one of claims 1 to 9, characterized in that The purity of α-ketoglutaric acid extracted from the fermentation broth is ≥98wt% and the yield is ≥90%.

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