CN107739308B - Method for simultaneously extracting alpha-ketoglutaric acid and pyruvic acid from microbial fermentation liquor or enzyme conversion liquor - Google Patents
Method for simultaneously extracting alpha-ketoglutaric acid and pyruvic acid from microbial fermentation liquor or enzyme conversion liquor Download PDFInfo
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- C07C51/42—Separation; Purification; Stabilisation; Use of additives
Abstract
The invention discloses a method for simultaneously extracting alpha-ketoglutaric acid and pyruvic acid from microbial fermentation liquor or enzyme conversion liquor, belonging to the technical field of biological separation and extraction. The invention centrifugalizes microbial fermentation liquor or enzyme conversion liquor containing alpha-ketoglutaric acid and pyruvic acid to remove thalli and other visible solid substances, ultrafilters to remove macromolecular impurities, decolors after acidification to remove metal ions, uses mixed organic solvent to carry out gradient elution to separate alpha-ketoglutaric acid and pyruvic acid, combines eluent obtained by staged collection containing alpha-ketoglutaric acid and carries out rotary evaporation together, and obtains alpha-ketoglutaric acid, and the alpha-ketoglutaric acid is put into an oven to be dried and crushed to obtain a qualified alpha-ketoglutaric acid product. And combining the eluate collected by stages and containing pyruvic acid, and performing rotary evaporation on the eluate to obtain a qualified pyruvic acid product. The method has the advantages of simple and efficient process operation, low cost, high product purity and the like, and is suitable for the co-extraction industry of alpha-ketoglutaric acid and pyruvic acid.
Description
Technical Field
The invention relates to a method for simultaneously extracting alpha-ketoglutaric acid and pyruvic acid from microbial fermentation liquor or enzyme conversion liquor, belonging to the technical field of biological separation and extraction.
Background
Alpha-ketoglutaric acid (KGA) and pyruvic acid (PYR) are important organic carboxylic acids and are widely applied to the food, agricultural and pharmaceutical industries. Alpha-ketoglutarate is an important precursor for amino acid metabolism and is also an important intermediate metabolite in the tricarboxylic acid cycle. Pyruvate plays an important pivotal role in the three major nutrient metabolic connections.
At present, there are two main methods for industrially synthesizing organic acids, i.e., chemical synthesis and biological synthesis. Biosynthesis mainly utilizes microbial fermentation to convert carbon source substances into target organic acids. The existing methods for extracting organic acid mainly comprise an ion exchange method, reduced pressure distillation, a double-membrane method, an organic solvent extraction method and a calcium salt precipitation method. Because of the similarity in chemistry between alpha-ketoglutarate and pyruvate, ion exchange methods cannot completely separate the two ketoacids. The components of the fermentation liquor or the enzyme liquid are complex, so the reduced pressure distillation method is not suitable for extracting and separating the alpha-ketoglutaric acid and the pyruvic acid. Organic solvent, double membrane method and calcium salt extraction method tend to obtain solid ketonate, sodium pyruvate can be changed into pyruvic acid liquid under alkaline condition and high temperature in later period, and pyruvic acid is unstable and easy to produce polymerization reaction, so that final purity and yield of pyruvic acid are low.
Disclosure of Invention
The invention aims to provide a method for simultaneously extracting alpha-ketoglutaric acid and pyruvic acid from microbial fermentation liquor or enzyme conversion liquor, which is to concentrate, acidify, decolor and desalt, chromatograph, distill and refine the microbial fermentation liquor or the enzyme conversion liquor containing the alpha-ketoglutaric acid and the pyruvic acid.
In one embodiment of the invention, the method comprises the steps of:
1) Pretreatment: separating the microbial fermentation liquor or enzyme conversion liquor containing alpha-ketoglutaric acid and pyruvic acid to remove thallus and other visible solid substances and macromolecular substances, and concentrating to make the concentrations of alpha-ketoglutaric acid and pyruvic acid be greater than 200 g/L; acidifying the fermentation liquor by concentrated hydrochloric acid, decolorizing by cation exchange resin, and removing high-valence metal ions to obtain a pretreatment solution containing alpha-ketoglutaric acid and pyruvic acid;
2) separation of alpha-ketoglutarate and pyruvate: filling the silica gel filler into the column by a wet method, and sampling the pretreatment solution by the wet method; specifically, the treated fermentation liquor is directly added into a silica gel filler glass chromatographic column packed by a wet method, and is kept stand, and then a mixed organic solvent is used for eluting and separating alpha-ketoglutaric acid and pyruvic acid; pyruvic acid is eluted before alpha-ketoglutaric acid, and when the elution solvent does not contain pyruvic acid, the polarity of the elution solvent is increased to elute the alpha-ketoglutaric acid;
3) Obtaining a refined product of alpha-ketoglutaric acid: distilling the collected alpha-ketoglutaric acid solution under reduced pressure, concentrating, drying and crushing to obtain an alpha-ketoglutaric acid refined product;
4) Obtaining a fine pyruvic acid product: and (4) carrying out rotary evaporation on the collected pyruvic acid crude solution to obtain a pyruvic acid refined product.
In one embodiment of the present invention, the separation and filtration in step (1) comprises one or more of centrifugation, ultrafiltration, microfiltration or plate-and-frame filtration.
In one embodiment of the present invention, the cation exchange resin for decoloring in step (1) is 732 cation exchange resin.
In one embodiment of the invention, the acidification is to adjust the pH of the solution to 1.5 or less.
In one embodiment of the present invention, the wet loading in step (2) refers to directly feeding the pretreated concentrated fermentation broth into a glass chromatography column.
In one embodiment of the present invention, the wet packing in step (2) is performed by weighing 200-mesh 300-mesh silica gel 15 times, 20 times, 25 times or 30 times the total amount of α -ketoglutaric acid and pyruvic acid, and adding a single solvent with the least polarity in the mixed elution solvents. The size of the column was 50mm by 500 mm.
In one embodiment of the present invention, the mixed organic solvent in step (2) is ethyl acetate and petroleum ether, or ethyl acetate and n-hexane, or ethyl acetate and dichloromethane. Elution of the pyruvate fraction was performed with ethyl acetate in a volume ratio between 2:3 and 1: a mixed solvent of petroleum ether; or a mixed solvent of ethyl acetate and n-hexane in a volume ratio of 4:7 to 5: 8; or a mixed solvent of ethyl acetate and dichloromethane in a volume ratio of 2:9 to 2: 7. The elution of the alpha-ketoglutarate fraction is carried out using a mixed solvent ratio greater than the maximum ratio of the pyruvic acid elution solvent ratios, or using ethyl acetate alone to elute the alpha-ketoglutarate.
In one embodiment of the present invention, the solvent in step (2) is eluted at a flow rate of 4 to 10mL/min, for example, 10 mL/min.
In one embodiment of the present invention, the silica gel filler in step (2) is a silica gel filler with a particle size of 100-400 mesh, for example, 300-400 mesh.
In one embodiment of the present invention, the aspect ratio of the chromatography column in step (2) is (1: 4) to (1: 10).
in an embodiment of the present invention, the step (3) of obtaining the refined α -ketoglutaric acid product is to perform rotary evaporation and concentration on the separated and collected α -ketoglutaric acid solution, dry and crush; the drying condition is 50-80 ℃.
In one embodiment of the present invention, the collected pyruvic acid solution is subjected to rotary evaporation in step (4), and the rotary evaporation conditions are as follows: 120rpm,40 ℃.
In one embodiment of the invention, the operation steps are as follows:
a: removing thalli and other visible solid matters from microbial fermentation liquor or enzyme conversion liquor containing alpha-ketoglutaric acid and pyruvic acid;
b: filtering the clear liquid from which the thalli and other visible solid matters are removed by a nanofiltration membrane to remove macromolecular substances such as thalli fragments, pigments, lipids, proteins, polysaccharides and the like, and washing and filtering the residual liquid by using a small amount of water; the ultrafiltration membrane component can be a roll-type membrane, a tubular membrane and a plate-type membrane, and the molecular weight cut-off of the membrane is 500-3000 Da;
c: concentrating the ultrafiltrate by rotary evaporation to ensure that the concentration of alpha-ketoglutaric acid and pyruvic acid is more than 200g/L, wherein the rotary evaporation conditions are as follows: 120rpm,40 ℃.
d: adding concentrated hydrochloric acid into the distilled concentrated solution while stirring to adjust the pH value to be less than or equal to 1.5;
e: removing metal ions and pigment in the fermentation liquor by passing the acidified fermentation liquor through 732 cation exchange resin, wherein the volume ratio of the resin to the concentrated liquor is about 10: 3.
f: concentrating the fermentation liquor treated by 732 cation exchange resin to 220g/L for later use; the evaporation conditions were 120rpm,40 ℃.
g: weighing 200-mesh 300-mesh silica gel 15 times, 20 times, 25 times or 30 times of the total amount of alpha-ketoglutaric acid and pyruvic acid, and adding a single solvent with the minimum polarity in the mixed elution solvents for wet column packing. The size of the column was 50mm by 500 mm.
h: slowly adding a sample mixed with silica gel into a glass chromatographic column, and then adding a certain amount of mixed elution solvent with lower polarity to elute a pyruvic acid part, wherein the mixed solvent for eluting the pyruvic acid part is ethyl acetate with the volume ratio of 2:3 to 1: petroleum ether, or ethyl acetate and n-hexane in a volume ratio of 4:7 to 5: 8; alternatively, ethyl acetate and dichloromethane in a volume ratio of between 2:9 and 2: 7. The mixed solvent ratio adopted for eluting the alpha-ketoglutaric acid part is larger than the maximum ratio in the ratio of pyruvic acid eluting solvents, and ethyl acetate can also be completely adopted for eluting.
i: evaporating the alpha-ketoglutaric acid solution collected in the h-1 by a rotary evaporator, and pumping by an oil pump for about 3 hours. The rotary evaporation conditions are as follows: 120rpm, 45 ℃. Then drying in a drying oven at a constant temperature of 50-80 ℃, and crushing to obtain a solid powdery alpha-ketoglutaric acid product;
j: and (3) removing the organic solvent and water from the pyruvic acid solution collected in the h-1 through rotary evaporation to obtain a pyruvic acid crude product, wherein the rotary evaporation conditions are as follows: 120rpm,40 ℃.
Has the advantages that: the extraction method of the invention effectively extracts and produces the alpha-ketoglutaric acid and the pyruvic acid from the microbial fermentation or the enzyme conversion liquid at the same time, the purity of the alpha-ketoglutaric acid and the pyruvic acid reaches more than 98 percent, the product quality reaches food grade, and the yield of the alpha-ketoglutaric acid and the pyruvic acid is between 70 and 90 percent. Compared with other separation and extraction methods, the method has the advantages of low cost, high efficiency, great industrial application potential and the like. For example, in the patent publication No. CN106496019A, the separation and extraction of alpha-ketoglutaric acid and pyruvic acid from microbial fermentation or enzyme conversion liquid by crystallization distillation method requires 3-5 times of ethyl acetate extraction of concentrated solution, but the process is not required in the present invention, which greatly reduces the production cost. Secondly, the crystallization process needs about 2 days, the whole refining process needs 60-72 hours, and the refining process only needs about 12-24 hours, so that the production efficiency is greatly improved. In general, the invention only needs one-step silica gel column chromatography to effectively separate the alpha-ketoglutaric acid and pyruvic acid. The present invention is a highly efficient method for separating alpha-ketoglutarate and pyruvate, either from a cost or efficiency standpoint.
Drawings
FIG. 1 is a flow chart of an extraction process according to one embodiment of the present invention.
Detailed Description
The method for measuring alpha-ketoglutaric acid and pyruvic acid comprises the following steps: high Performance Liquid Chromatography (HPLC)
the instrument is Agilent 1260 high performance liquid chromatograph (with ultraviolet visible detector and workstation), the chromatographic conditions are that a chromatographic column is Aminex HPX-87H ion exchange column, a mobile phase is 5mM H 2 SO 4, the flow rate is 0.6mL/min, the column temperature is 40 ℃, the sample injection amount is 10 mu L, and the wavelength of the ultraviolet detector is 210 nm.
The purity calculation method comprises the following steps:
f 1 peak area of standard solution detected by HPLC;
f 2 peak area of sample solution detected by HPLC;
m 1 -Standard article Mass in grams (g);
m 2 -sample mass in grams (g);
p-purity of the standard, values are in%.
the yield calculation method comprises the following steps:
m-extracting the total mass of the obtained refined product;
w is the purity of the obtained refined product;
C-concentration of sample in fermentation broth;
V-volume of fermentation liquor after removing thalli.
the following examples are methods for simultaneously extracting α -ketoglutarate and pyruvate from a microbial fermentation broth or an enzyme-converted solution containing α -ketoglutarate and pyruvate, and are exemplified by fermentation broths (α -ketoglutarate concentration: pyruvate concentration ═ 1: 1) obtained by using Yarrowia lipolytica WSH-Z06CCTCC M20714 as a fermentation strain, respectively. Other similar KGA and PYR-containing microbial fermentation broths or enzyme conversion broths can be carried out according to embodiments of the present invention, and the KGA and PYR products with higher purity can be obtained through simple modification.
The invention will be further described with reference to the accompanying drawings.
example 1
taking a fermentation liquid obtained by taking Y.lipolytica WSH-Z06 as a fermentation strain as an example (the concentration of alpha-ketoglutaric acid: the concentration of pyruvic acid is 1: 1), the extraction is carried out according to the following steps:
(1) Centrifugal filtration: and pumping the fermentation liquor containing alpha-ketoglutaric acid and pyruvic acid into a centrifugal machine, and centrifuging to remove thalli and other visible solid substances. Centrifugation conditions: normal temperature, 8000g of rotating speed.
(2) Membrane filtration: filtering out impurities by ultrafiltration, ultrafiltering the clear liquid after centrifugal removal of thalli and other visible solid matters to remove macromolecular substances such as thalli fragments, pigments, lipid, protein, polysaccharide and the like in the fermentation liquor, and washing the filtered residual liquor by a small amount of water to improve the benefit of the ultrafiltration process. The tubular membrane component of the ultrafiltration membrane component is characterized in that the membrane material is a polyethylene membrane with the molecular weight cutoff of 500-3000Da, and the ultrafiltration conditions are as follows: normal temperature and operation pressure of 4-10 Bar.
(3) concentration: distilling the ultrafiltration filtrate under reduced pressure of-0.08 Mpa at 65 deg.C to make the concentration of alpha-ketoglutaric acid and pyruvic acid above 200 g/L.
(4) Acidifying: adding concentrated hydrochloric acid into the concentrated solution treated by the ion exchange column under stirring at normal temperature, and adjusting the pH of the concentrated solution to be less than or equal to 1.5.
(5) Removing impurities by using ion exchange resin: treating 732 cation exchange resin with 1mol/LNaOH and 1mol/LHCl alternately, washing with deionized water to neutrality, pretreating with 1mol/LHCl, washing with deionized water to neutrality (sodium type resin is converted into hydrogen type resin), loading onto column, and treating the concentrated solution with exchange resin. This operation is aimed at removing the high-valence cations from the concentrated solution and at the same time, it can play the role of decolouring.
(6) Chromatographic separation: 120g of 200-300 mesh silica gel is added into a glass chromatographic column, and the silica gel is filled into the column by a wet method (50mm multiplied by 500mm), and the diameter-height ratio is 1: 4. then 20mL of the pretreated concentrated fermentation liquid is added to the surface layer of the chromatographic column and stands for 30 min. Equilibrated with 100mL of petroleum ether, followed by elution with 4 column volumes of ethyl acetate and petroleum ether organic solvent, the volume ratio of ethyl acetate to petroleum ether being 1:1, wherein pyruvic acid is mainly contained in the effluent eluent; when the solvent in the chromatographic column is close to the surface of silica gel, adding 2-3 times of column volume of ethyl acetate and petroleum ether, wherein the volume ratio of the ethyl acetate to the petroleum ether is 2: 1, or the more polar single solvent ethyl acetate. The elution flow rate was 7mL/min at room temperature.
(7) And (3) distillation: combining the eluates containing pyruvic acid part after chromatographic separation, performing rotary evaporation, and combining the eluates containing alpha-ketoglutaric acid part after chromatographic separation, performing rotary evaporation at 40 deg.C and 120 rpm.
(8) Refining alpha-ketoglutaric acid: pumping the alpha-ketoglutaric acid subjected to rotary evaporation for about 3 hours by an oil pump, drying in a constant-temperature drying box at 65 ℃, and crushing to obtain an alpha-KG fine product. The purity of the alpha-ketoglutaric acid is 98.7 percent and the yield is 86.7 percent through the quality detection.
(9) And (3) pyruvic acid detection: the purity of pyruvic acid is 99.1% by mass detection, and the yield is 75.9%.
Example 2
Taking a fermentation liquid obtained by taking Y.lipolytica WSH-Z06 as a fermentation strain as an example (the concentration of alpha-ketoglutaric acid: the concentration of pyruvic acid is 1: 1), the embodiment is the same as that of example 1, and the difference is that the elution solvents in the step (6) are ethyl acetate and n-hexane, the volume ratio is 2:3, detecting the content of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 98.5 percent, and the yield is 84.9 percent; pyruvic acid: the purity was 97.7% and the yield was 73.3%.
Example 3
Taking a fermentation liquid obtained by taking Y.lipolytica WSH-Z06 as a fermentation strain as an example (the concentration of alpha-ketoglutaric acid: the concentration of pyruvic acid is 1: 1), the embodiment is the same as that of example 1, and the difference is that the elution solvents in the step (6) are ethyl acetate and dichloromethane, the volume ratio is 1: 4. detecting the contents of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 99.0 percent, and the yield is 88.7 percent; pyruvic acid: the purity was 98.2% and the yield was 73.5%.
Example 4
The procedure was as in example 1, except that the elution flow rate in step (6) was 4 mL/min. Detecting the contents of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 99.4 percent, and the yield is 79 percent; pyruvic acid: the purity was 99.8% and the yield was 75.8%.
Example 5
The procedure was as in example 1, except that the elution flow rate in step (6) was 10 mL/min. Detecting the contents of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 98.8 percent, and the yield is 81.9 percent; pyruvic acid: the purity was 99.3% and the yield was 73.8%.
example 6
The implementation manner is the same as that of the embodiment 1, except that the silica gel mesh number of the column in the step (6) is 100-200 meshes. Detecting the contents of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 99.8 percent, and the yield is 85.2 percent; pyruvic acid: the purity was 99.9% and the yield was 67.2%.
Example 7
The implementation manner is the same as that of example 1, except that the silica gel mesh number in step (6) is 300-400 meshes. Detecting the contents of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 98.1 percent, and the yield is 87.1 percent; pyruvic acid: the purity was 99.4% and the yield was 76.4%.
example 8
The embodiment is different from the embodiment 1 in that the diameter-height ratio of the column in the step (6) is 1: 5.5. detecting the contents of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 98.3 percent, and the yield is 83.6 percent; pyruvic acid: the purity was 98.4% and the yield was 73.2%.
example 9
the embodiment is the same as example 1 except that the column in step (6) has a diameter-height ratio of 1: 7. Detecting the contents of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 98.7 percent, and the yield is 74.9 percent; pyruvic acid: the purity was 99.0% and the yield was 69.3%.
example 10
The embodiment is the same as example 1 except that the column in step (6) has a diameter-height ratio of 1: 10. Detecting the contents of the prepared alpha-ketoglutaric acid and pyruvic acid, wherein the content of the alpha-ketoglutaric acid: the purity is 98.7 percent, and the yield is 72.9 percent; pyruvic acid: the purity was 99.0% and the yield was 66.3%.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A method for extracting alpha-ketoglutaric acid and pyruvic acid from microbial fermentation liquor or enzyme conversion liquor simultaneously is characterized in that the microbial fermentation liquor or the enzyme conversion liquor containing the alpha-ketoglutaric acid and the pyruvic acid is concentrated, acidified, decolored, desalted, chromatographed, distilled and refined;
The method comprises the following steps:
1) pretreatment: separating the microbial fermentation liquor or enzyme conversion liquor containing alpha-ketoglutaric acid and pyruvic acid to remove thallus and other visible solid substances and macromolecular substances, and concentrating to make the concentrations of alpha-ketoglutaric acid and pyruvic acid be greater than 200 g/L; acidifying the fermentation liquor by concentrated hydrochloric acid, decolorizing by cation exchange resin, and removing high-valence metal ions to obtain a pretreatment solution containing alpha-ketoglutaric acid and pyruvic acid;
2) Separation of alpha-ketoglutarate and pyruvate: directly adding the pretreatment solution obtained in the step (1) into a silica gel filler glass chromatographic column filled with the column by a wet method, standing, and then eluting and separating alpha-ketoglutaric acid and pyruvic acid by using a mixed organic solvent, wherein the mixed organic solvent is ethyl acetate and petroleum ether, or ethyl acetate and n-hexane, or ethyl acetate and dichloromethane; pyruvic acid is eluted before alpha-ketoglutaric acid, and the pyruvic acid part is eluted by ethyl acetate with the volume ratio of 2:3 to 1: a mixed solvent of petroleum ether; or a mixed solvent of ethyl acetate and n-hexane in a volume ratio of 4:7 to 5: 8; or the mixed solvent of ethyl acetate and dichloromethane with the volume ratio of 2:9 to 2:7, when the elution solvent does not contain pyruvic acid, the polarity of the elution solvent is increased, alpha-ketoglutaric acid is eluted, the mixed solvent ratio adopted for eluting the alpha-ketoglutaric acid part is larger than the maximum ratio of the pyruvic acid elution solvent, or the ethyl acetate is singly adopted for eluting the alpha-ketoglutaric acid; the flow rate of the solvent during elution is 4-10 mL/min;
3) Obtaining a refined product of alpha-ketoglutaric acid: distilling the collected alpha-ketoglutaric acid solution under reduced pressure, concentrating, drying and crushing to obtain an alpha-ketoglutaric acid refined product;
4) Obtaining a fine pyruvic acid product: and (4) carrying out rotary evaporation on the collected pyruvic acid crude solution to obtain a pyruvic acid refined product.
2. the method of claim 1, wherein the separating and filtering of step (1) comprises one or more of centrifugation, ultrafiltration, microfiltration, or plate and frame filtration.
3. The method according to claim 1 or 2, wherein the cation exchange resin for decoloring in step (1) is 732 cation exchange resin.
4. The method of claim 1 or 2, wherein the acidification in step (1) is to adjust the pH of the solution to be less than or equal to 1.5.
5. The method as claimed in claim 1, wherein the wet packing in step (2) is performed by weighing 200-mesh 300-mesh silica gel 15 times, 20 times, 25 times or 30 times the total amount of α -ketoglutaric acid and pyruvic acid, and adding a single solvent with the least polarity in the mixed elution solvents.
6. the method according to claim 1, wherein the fine α -ketoglutarate product obtained in step (3) is obtained by concentrating the separated and collected α -ketoglutarate solution by rotary evaporation, drying and pulverizing; the drying condition is 50-80 ℃; and (4) rotationally evaporating the collected pyruvic acid solution under the conditions of: 120rpm,40 ℃.
7. The method according to claim 2, wherein the fine α -ketoglutarate product obtained in step (3) is obtained by concentrating the separated and collected α -ketoglutarate solution by rotary evaporation, drying and pulverizing; the drying condition is 50-80 ℃; and (4) rotationally evaporating the collected pyruvic acid solution under the conditions of: 120rpm,40 ℃.
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