CN117466760A - Valine separation and purification method - Google Patents
Valine separation and purification method Download PDFInfo
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- CN117466760A CN117466760A CN202311344319.1A CN202311344319A CN117466760A CN 117466760 A CN117466760 A CN 117466760A CN 202311344319 A CN202311344319 A CN 202311344319A CN 117466760 A CN117466760 A CN 117466760A
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- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 title claims abstract description 318
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 239000004474 valine Substances 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 48
- 238000000926 separation method Methods 0.000 title abstract description 23
- 238000000746 purification Methods 0.000 title abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 66
- 230000004151 fermentation Effects 0.000 claims abstract description 66
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 56
- 239000011347 resin Substances 0.000 claims abstract description 47
- 229920005989 resin Polymers 0.000 claims abstract description 47
- 238000001035 drying Methods 0.000 claims abstract description 33
- 239000008367 deionised water Substances 0.000 claims abstract description 32
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 32
- 239000012528 membrane Substances 0.000 claims abstract description 32
- 239000000919 ceramic Substances 0.000 claims abstract description 30
- 238000001914 filtration Methods 0.000 claims abstract description 26
- 238000002425 crystallisation Methods 0.000 claims abstract description 25
- 230000008025 crystallization Effects 0.000 claims abstract description 25
- 238000011068 loading method Methods 0.000 claims abstract description 16
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 13
- 239000002245 particle Substances 0.000 claims abstract description 8
- 229960004295 valine Drugs 0.000 claims description 187
- 239000000047 product Substances 0.000 claims description 67
- 239000007788 liquid Substances 0.000 claims description 42
- 239000012535 impurity Substances 0.000 claims description 36
- 239000000706 filtrate Substances 0.000 claims description 31
- 239000011148 porous material Substances 0.000 claims description 20
- 238000001816 cooling Methods 0.000 claims description 19
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 13
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 13
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 12
- 235000004279 alanine Nutrition 0.000 claims description 12
- 238000001704 evaporation Methods 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- 230000002209 hydrophobic effect Effects 0.000 claims description 6
- 125000000524 functional group Chemical group 0.000 claims description 4
- 239000000178 monomer Substances 0.000 claims description 4
- 230000000379 polymerizing effect Effects 0.000 claims description 4
- 150000003384 small molecules Chemical class 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 3
- -1 specifically Substances 0.000 claims 1
- 239000002351 wastewater Substances 0.000 abstract description 8
- 238000010828 elution Methods 0.000 abstract description 5
- 230000004913 activation Effects 0.000 abstract description 4
- 238000013375 chromatographic separation Methods 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 65
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- 238000005342 ion exchange Methods 0.000 description 11
- 229960003136 leucine Drugs 0.000 description 11
- 229940024606 amino acid Drugs 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 10
- 239000012467 final product Substances 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 239000012452 mother liquor Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 5
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 150000001450 anions Chemical class 0.000 description 4
- 150000001768 cations Chemical class 0.000 description 4
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 4
- 229960000310 isoleucine Drugs 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
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- 239000002537 cosmetic Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000001471 micro-filtration Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 150000005693 branched-chain amino acids Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 239000012539 chromatography resin Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000020939 nutritional additive Nutrition 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
- C07C227/42—Crystallisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a valine separation and purification method, which comprises the following operation steps: filtering fermentation liquor by a ceramic membrane, carrying out first vacuum concentration and crystallization, dissolving, loading to a column, carrying out second vacuum concentration and centrifugal drying; wherein, in the step of loading, the stationary phase of the chromatographic column is equal particle weak polarity chromatographic resin. According to the valine separation and purification method, continuous chromatographic separation is carried out by adopting a chromatographic column with a specific gel type resin as a stationary phase, and the gel type resin only needs deionized water for activation and elution, so that the operation is simple and convenient, the yield is improved, the discharge of acid-base wastewater is greatly reduced, and the environmental protection pressure is reduced.
Description
Technical Field
The invention relates to the technical field of biological fermentation engineering, in particular to a separation and purification method of valine.
Background
L-valine (called valine for short), the chemical name is 2-amino-3-methyl butyric acid, belongs to branched chain amino acid, and is one of eight amino acids necessary for human body; its molecular formula is HO 2 CCH(NH 2 )CH(CH3) 2 . L-valine is a main raw material for medicines and cosmetics, and is also an important component of animal feeds. Therefore, the commercial market is increasingly applying L-valine in medicines, seasonings, cosmetics, nutritional additives, feed additives and the like.
The fermentation process is an important process for producing L-valine. Since L-valine produces many impurities including alanine, leucine, isoleucine and the like at the same time during fermentation production, it is necessary to separate and remove these impurities from L-valine in the separation and purification of L-valine.
The separation method of L-valine used in the prior art mainly comprises a precipitation method, a whole membrane method and an ion exchange method. (1) The precipitation method is to use a specific precipitant to specifically combine with L-valine so as to form precipitate, and then separate and extract, and the method has high yield, simple operation steps and relatively large environmental pollution; (2) Although the whole membrane method has little wastewater, the membrane filtration method can only remove a large amount of inorganic salt ions and macromolecular hybrid proteins, amino acid with similar properties to L-valine in fermentation liquor can not be eradicated, and the obtained L-valine has higher impurity; (3) The ion exchange method is to adjust the pH of the fermentation broth from which the thalli are removed to be acidic, separate L-valine and impurity amino acid by using strong acid cation exchange resin, and finally separate L-valine by eluting with ammonia water. If the purity of L-valine needs to be improved, multistage ion exchange columns are needed for series extraction, which can certainly lead to large production wastewater in the production process, complicated operation steps and low yield.
For example, chinese patent application CN105274182a relates to a method for producing L-valine, which comprises the following steps: and (3) obtaining valine fermentation liquor by a fermentation method, filtering the fermentation liquor by a microfiltration membrane, evaporating, concentrating and crystallizing the microfiltration liquor to obtain a crude product, dissolving the crude product, adding water for dissolving, performing ion exchange by a strong acid ion exchange resin, filtering effluent by an ultrafiltration membrane, and concentrating and crystallizing the filtrate in vacuum to obtain the product. The method is subjected to a strong acid ion exchange column separation process, but a large amount of acid-base wastewater is discharged.
Disclosure of Invention
The invention aims to provide a valine separation and purification method, which adopts a chromatographic column with a specific gel type resin as a stationary phase for continuous chromatographic separation, wherein the gel type resin only needs deionized water for activation and elution, and the method is simple and convenient to operate, improves the yield, simultaneously greatly reduces the discharge of acid-base wastewater and lightens the environmental protection pressure.
In order to achieve the above object, the present invention provides the following technical solutions:
a separation and purification method of valine comprises the following operation steps:
filtering fermentation liquor by a ceramic membrane, carrying out first vacuum concentration and crystallization, dissolving, loading to a column, carrying out second vacuum concentration and centrifugal drying;
wherein, in the step of loading, the stationary phase of the chromatographic column is equal particle weak polarity chromatographic resin.
As a further description of the technical scheme of the invention, the separation and purification method of valine specifically comprises the following steps:
s1, filtering fermentation liquor by a ceramic membrane: filtering the fermentation liquor by adopting a ceramic membrane, and collecting micro-filtrate;
s2, vacuum concentration for the first time: evaporating and concentrating the micro filtrate obtained in the step S1, and collecting a first concentrated solution;
s3, crystallizing: crystallizing the concentrated solution obtained in the step S2 to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into deionized water, and redissolving to obtain L-valine solution;
s5, column loading: passing the L-valine solution of the S4 through a gel type resin chromatographic column, eluting with ionized water, and collecting effluent;
s6, second vacuum concentration: the effluent liquid in the step S5 is filtered by filter paper for one time, and then vacuum concentration is carried out, and a second concentrated solution is collected;
s7, centrifugal drying: and (3) cooling, crystallizing, centrifuging and drying the second concentrated solution obtained in the step (S6) to obtain an L-valine finished product.
As a further description of the technical scheme of the present invention, in S5, the stationary phase of the gel-type resin chromatographic column is a homogeneous weak-polarity chromatographic resin, the chromatographic resin is prepared by polymerizing a monomer with a small dipole moment, and the chromatographic resin does not have any functional group.
In one preferred embodiment, the pore surfaces of the chromatographic resin are hydrophobic and bind to hydrophobic moieties within small molecules to adsorb organics in solution.
The invention adopts specific chromatographic resin to carry out continuous chromatographic separation, the stationary phase of the gel type resin chromatographic column is average particle weak polar chromatographic resin, the chromatographic resin is prepared by polymerizing monomers with small dipole moment, does not have any functional group, has strong hydrophobicity on the pore surface, and can adsorb organic matters in solution by combining with a hydrophobic part in small molecules.
The chromatographic resin is used as a stationary phase and is suitable for adsorbing nonpolar substances in a polar solvent. Valine belongs to nonpolar amino acid, main impurities leucine and isoleucine in valine fermentation liquid belong to nonpolar amino acid, and alanine belongs to polar amino acid. When the aqueous valine solution with impurities passes through the weak polar chromatography resin, valine, leucine and isoleucine are adsorbed on the resin, and alanine is carried out by the aqueous solution. Then, water is used as a mobile phase for elution, and valine, leucine and isoleucine can be sequentially separated due to different polarities, so that the purpose of separating and purifying valine is achieved.
As a further description of the technical solution of the present invention, in S1, the pore diameter of the ceramic membrane is 0.05-0.2 μm.
As a further description of the technical scheme of the present invention, in S2, the content concentration of the first concentrated solution is 300-400g/L.
As a further description of the technical scheme of the invention, in the S4, the mass concentration of the L-valine solution is 4-5%.
As a further description of the technical scheme of the present invention, in S5, eluting with ionic water, specifically, eluting with deionized water, the post-impurity including leucine; and eluting the effluent liquid again by using deionized water to remove the pre-impurities, wherein the pre-impurities comprise alanine.
As a further description of the technical scheme of the present invention, in the step S6, the pore diameter of the filter paper is 0.2-0.45 μm.
As a further description of the technical scheme of the invention, the separation and purification method of valine specifically comprises the following steps:
s1, filtering fermentation liquor by a ceramic membrane: the valine is produced by adopting a fermentation method, after fermentation is finished, fermentation liquor is taken, ceramic membrane filtration is adopted, the pore diameter of the ceramic membrane is 0.05 mu m, water is used for washing, and when the L-valine content of filter residues is lower than 1.5g/L, micro filtrate is collected;
s2, vacuum concentration for the first time: evaporating and concentrating the micro filtrate obtained in the step S1, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.084Mpa until valine content is 350g/L, and collecting first concentrated solution;
s3, crystallizing: slowly cooling the first concentrated solution obtained in the step S2 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, and centrifuging to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into 14600L of deionized water, and redissolving at the water temperature of 80 ℃ to obtain an L-valine solution;
s5, column loading: passing the L-valine solution of S4 through a gel-type resin chromatographic column, eluting the rear impurities with deionized water, wherein the rear impurities comprise leucine; eluting the effluent liquid again by deionized water for the second time, removing the pre-impurities including alanine, and finally collecting the effluent liquid;
s6, second vacuum concentration: the effluent liquid of the step S5 is passed through filter paper with a pore diameter of 0.45 μm, and the filtrate is added into an evaporator for vacuum concentration under the conditions of: concentrating at 60deg.C under vacuum of 0.085Mpa until valine content is 250g/L, and collecting second concentrated solution;
s7, centrifugal drying: cooling the second concentrated solution in the step S6 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, centrifuging to obtain an L-valine wet product, and carrying out double-cone drying on the L-valine wet product under the double-cone drying condition: vacuum-0.09 Mpa, chamber temperature 70 deg.C, and steam pressure 0.2Mpa to obtain L-valine product.
Based on the technical scheme, compared with the prior art, the invention has the following technical effects:
(1) According to the separation and purification method of valine, the extraction yield of the pharmaceutical grade L-valine is about 64%, the purity of the pharmaceutical grade L-valine reaches 99% through HPLC detection, and the pharmaceutical grade L-valine reaches the pharmaceutical grade standard; the extraction yield of L-valine in the traditional process is about 60%, and the purity of L-valine is about 95% as detected by HPLC.
(2) According to the valine separation and purification method, continuous chromatographic separation is carried out by adopting the chromatographic column with the specific gel type resin as the stationary phase, and the gel type resin only needs deionized water for activation and elution, so that the operation is simple and convenient, the yield is improved, the discharge of acid-base wastewater is greatly reduced, and the environmental protection pressure is reduced.
Drawings
FIG. 1 is a purity detection chromatogram of L-valine of example 1.
FIG. 2 is a purity detection chromatogram of L-valine of example 2.
FIG. 3 is a purity detection chromatogram of L-valine of example 3.
FIG. 4 is a purity detection chromatogram of L-valine of example 4.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings. The drawings illustrate preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
A separation and purification method of valine comprises the following steps:
s1, filtering fermentation liquor by a ceramic membrane: filtering the fermentation liquor by adopting a ceramic membrane, and collecting micro-filtrate;
s2, vacuum concentration for the first time: evaporating and concentrating the micro filtrate obtained in the step S1, and collecting a first concentrated solution;
s3, crystallizing: crystallizing the first concentrated solution in the step S2 to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into deionized water, and redissolving to obtain L-valine solution;
s5, column loading: passing the L-valine solution of S4 through a gel type resin chromatographic column, eluting with ionized water, and collecting effluent;
s6, second vacuum concentration: the effluent liquid in the step S5 is filtered by filter paper for one time, and then vacuum concentration is carried out, and a second concentrated solution is collected;
s7, centrifugal drying: and (3) cooling and crystallizing the second concentrated solution in the step (S6), centrifuging and drying to obtain an L-valine finished product.
Wherein in S1, the pore diameter of the ceramic membrane is 0.05-0.2 μm;
in S2, the content concentration of the first concentrated solution is 300-400g/L;
in S4, the mass concentration of the L-valine solution is 4-5%;
in S5, the stationary phase of the gel-type resin chromatographic column is a chromatographic resin with uniform particle and weak polarity, the chromatographic resin is prepared by polymerizing monomers with small dipole moment, and the chromatographic resin does not have any functional group.
The pore surface of the chromatographic resin has hydrophobicity, and can adsorb organic matters in solution by combining with hydrophobic parts in small molecules.
The gel-type resin chromatographic column is preferably:
the manufacturing factory: western security blue is available from new materials, inc.;
stationary phase resin model:s3850, average particle weak polarity chromatographic resin;
eluting with ion water to obtain impurities including leucine; and eluting the effluent liquid again by using deionized water to remove the pre-impurities, wherein the pre-impurities comprise alanine.
In S6, the pore size of the filter paper is 0.2-0.45 μm.
Example 1
A separation and purification method of valine specifically comprises the following steps:
s1, filtering fermentation liquor by a ceramic membrane: valine was produced by fermentation, after completion of fermentation, samples were taken from 8400L of the fermentation broth, and the L-valine content in the fermentation broth was measured to be 105g/L by High Performance Liquid Chromatography (HPLC).
Filtering with ceramic membrane with pore diameter of 0.05 μm, washing with water until L-valine content of residue is lower than 1.5g/L, and collecting micro filtrate;
s2, vacuum concentration: evaporating and concentrating the micro filtrate in the step S1, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.084Mpa until valine content is 350g/L, and collecting first concentrated solution;
s3, crystallizing: slowly cooling the first concentrated solution obtained in the step S2 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, and centrifuging to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into 14600L of deionized water, and redissolving at the water temperature of 80 ℃ to obtain an L-valine solution;
s5, column loading: passing the L-valine solution of S4 through a gel type resin chromatographic column, eluting the impurities with deionized water, wherein the impurities comprise leucine; eluting the effluent liquid again by deionized water for the second time, removing the pre-impurities including alanine, and finally collecting the effluent liquid;
s6, second vacuum concentration: the effluent liquid in the step S5 is passed through filter paper with the aperture of 0.45 mu m, and the filtrate is added into an evaporator for vacuum concentration under the conditions of: concentrating at 60deg.C under vacuum of 0.085Mpa until valine content is 250g/L, and collecting second concentrated solution;
s7, centrifugal drying: cooling the second concentrated solution in the step S6 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, centrifuging to obtain an L-valine wet product, and carrying out double-cone drying on the L-valine wet product under the double-cone drying condition: vacuum-0.09 Mpa, chamber temperature 70 deg.C, and steam pressure 0.2Mpa to obtain L-valine product.
Detecting content by High Performance Liquid Chromatography (HPLC), calculating weight of L-valine in the fermentation liquid, and dividing weight of the final product by weight of L-valine in the fermentation liquid to obtain yield. The yield of the primary crystallization is 64 percent, and the product meets the pharmaceutical grade standard. The mother liquor is recovered to be used as a feed-grade product, and the total yield is 82 percent. The purity of the final product was 100% by detecting ninhydrin positive substances with an amino acid analyzer, and the purity detection chromatogram of L-valine in this example is shown in FIG. 1.
Example 2
A separation and purification method of valine specifically comprises the following steps:
s1, filtering fermentation liquor by a ceramic membrane: valine was produced by fermentation, after fermentation was completed, sampled in 8400L of the fermentation broth, and the L-valine content in the fermentation broth was measured to be 99.5g/L by High Performance Liquid Chromatography (HPLC).
Filtering with ceramic membrane with pore diameter of 0.05 μm, washing with water until L-valine content of residue is lower than 1.5g/L, and collecting micro filtrate;
s2, vacuum concentration: evaporating and concentrating the micro filtrate in the step S1, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.084Mpa until valine content is 350g/L, and collecting first concentrated solution;
s3, crystallizing: slowly cooling the first concentrated solution obtained in the step S2 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, and centrifuging to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into 13800L of deionized water, and redissolving the crude L-valine product at the water temperature of 80 ℃ to obtain an L-valine solution;
s5, column loading: passing the L-valine solution of S4 through a gel type resin chromatographic column, eluting the impurities with deionized water, wherein the impurities comprise leucine; eluting the effluent liquid again by deionized water for the second time, removing the pre-impurities including alanine, and finally collecting the effluent liquid;
s6, second vacuum concentration: the effluent liquid in the step S5 is passed through filter paper with the aperture of 0.45 mu m, and the filtrate is added into an evaporator for vacuum concentration under the conditions of: concentrating at 60deg.C under vacuum of 0.085Mpa until valine content is 250g/L, and collecting second concentrated solution;
s7, centrifugal drying: cooling the second concentrated solution in the step S6 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, centrifuging to obtain an L-valine wet product, and carrying out double-cone drying on the L-valine wet product under the double-cone drying condition: vacuum-0.09 Mpa, chamber temperature 70 deg.C, and steam pressure 0.2Mpa to obtain L-valine product.
Detecting content by High Performance Liquid Chromatography (HPLC), calculating weight of L-valine in the fermentation liquid, and dividing weight of the final product by weight of L-valine in the fermentation liquid to obtain yield. The yield of the primary crystallization is 61 percent, and the product meets the pharmaceutical grade standard. The mother liquor is recovered to be used as a feed-grade product, and the total yield is 80 percent. The purity of the final product was 100% by detecting ninhydrin positive substances with an amino acid analyzer, and the purity detection chromatogram of L-valine in this example is shown in FIG. 2.
Example 3
A separation and purification method of valine specifically comprises the following steps:
s1, filtering fermentation liquor by a ceramic membrane: valine was produced by fermentation, and after completion of fermentation, samples were taken from 8270L of the fermentation broth, and the L-valine content in the fermentation broth was measured to be 98.1g/L by High Performance Liquid Chromatography (HPLC).
Filtering with ceramic membrane with pore diameter of 0.05 μm, washing with water until L-valine content of residue is lower than 1.5g/L, and collecting micro filtrate;
s2, vacuum concentration: evaporating and concentrating the micro filtrate in the step S1, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.084Mpa until valine content is 350g/L, and collecting first concentrated solution;
s3, crystallizing: slowly cooling the first concentrated solution obtained in the step S2 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, and centrifuging to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into 13400L of deionized water, and redissolving at the water temperature of 80 ℃ to obtain an L-valine solution;
s5, column loading: passing the L-valine solution of S4 through a gel type resin chromatographic column, eluting the impurities with deionized water, wherein the impurities comprise leucine; eluting the effluent liquid again by deionized water for the second time, removing the pre-impurities including alanine, and finally collecting the effluent liquid;
s6, second vacuum concentration: the effluent liquid in the step S5 is passed through filter paper with the aperture of 0.45 mu m, and the filtrate is added into an evaporator for vacuum concentration under the conditions of: concentrating at 60deg.C under vacuum of 0.085Mpa until valine content is 250g/L, and collecting second concentrated solution;
s7, centrifugal drying: cooling the second concentrated solution in the step S6 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, centrifuging to obtain an L-valine wet product, and carrying out double-cone drying on the L-valine wet product under the double-cone drying condition: vacuum-0.09 Mpa, chamber temperature 70 deg.C, and steam pressure 0.2Mpa to obtain L-valine product.
Detecting content by High Performance Liquid Chromatography (HPLC), calculating weight of L-valine in the fermentation liquid, and dividing weight of the final product by weight of L-valine in the fermentation liquid to obtain yield. The yield of the primary crystallization is 64 percent, and the product meets the pharmaceutical grade standard. The mother liquor is recovered to be used as a feed-grade product, and the total yield is 82 percent. The purity of the final product was 100% by detecting ninhydrin positive substances with an amino acid analyzer, and the purity detection chromatogram of L-valine in this example is shown in FIG. 3.
Example 4
A separation and purification method of valine specifically comprises the following steps:
s1, filtering fermentation liquor by a ceramic membrane: valine was produced by fermentation, and after completion of fermentation, samples were taken from 8500L of the fermentation broth, and the L-valine content of the fermentation broth was measured to be 100g/L by liquid chromatography (HPLC).
Filtering with ceramic membrane with pore diameter of 0.05 μm, washing with water until L-valine content of residue is lower than 1.5g/L, and collecting micro filtrate;
s2, vacuum concentration: evaporating and concentrating the micro filtrate in the step S1, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.086Mpa until valine content is 350g/L, and collecting first concentrated solution;
s3, crystallizing: slowly cooling the first concentrated solution obtained in the step S2 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, and centrifuging to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into 12818L deionized water, and redissolving the crude L-valine product at the water temperature of 80 ℃ to obtain an L-valine solution;
s5, column loading: passing the L-valine solution of S4 through the uniform particle weak polarity chromatographic resin column, eluting the impurities with deionized water, wherein the impurities comprise leucine; eluting the effluent liquid again by deionized water for the second time, removing the pre-impurities including alanine, and finally collecting the effluent liquid;
s6, second vacuum concentration: the effluent liquid in the step S5 is passed through filter paper with the aperture of 0.45 mu m, and the filtrate is added into an evaporator for vacuum concentration under the conditions of: concentrating at 60deg.C under vacuum of 0.085Mpa until valine content is 250g/L, and collecting second concentrated solution;
s7, centrifugal drying: cooling the second concentrated solution in the step S6 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, centrifuging to obtain an L-valine wet product, and carrying out double-cone drying on the L-valine wet product under the double-cone drying condition: vacuum-0.09 Mpa, chamber temperature 70 deg.C, and steam pressure 0.2Mpa to obtain L-valine product.
Detecting content by High Performance Liquid Chromatography (HPLC), calculating weight of L-valine in the fermentation liquid, and dividing weight of the final product by weight of L-valine in the fermentation liquid to obtain yield. The yield of the primary crystallization is 60 percent, and the product meets the pharmaceutical grade standard. The mother liquor is recovered to be used as a feed-grade product, and the total yield is 83 percent. The purity of the final product was 100.00% by detecting ninhydrin positive substances with an amino acid analyzer, and the purity detection chromatogram of L-valine in this example is shown in FIG. 4.
Comparative example 1
S1, filtering fermentation liquor by a ceramic membrane: valine was produced by fermentation, after fermentation was completed, sampled in 8400L of the broth, and the L valine content in the broth was measured to be 106g/L by High Performance Liquid Chromatography (HPLC).
Filtering with ceramic membrane with pore diameter of 0.05 μm, washing with water until L-valine content of residue is lower than 0.5g/L, and collecting micro filtrate;
s2, vacuum concentration: evaporating and concentrating the micro filtrate in the step S1, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.086Mpa until valine content is 350g/L, and collecting first concentrated solution;
s3, crystallizing: slowly cooling the first concentrated solution obtained in the step S2 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, and centrifuging to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into 13000L deionized water, and redissolving at the water temperature of 80 ℃ to obtain an L-valine solution;
s5, decoloring: adding 48kg of active carbon into the solution obtained in the step S4, keeping the temperature at 80 ℃ and decoloring for 30min, and filtering;
s6, second vacuum concentration: adding the filtrate obtained in the step S5 into an evaporator for vacuum concentration, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.085Mpa until valine content is 250g/L, and collecting second concentrated solution;
s7, centrifugal drying: cooling the second concentrated solution in the step S6 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, centrifuging to obtain an L-valine wet product, and carrying out double-cone drying on the L-valine wet product under the double-cone drying condition: vacuum-0.09 Mpa, chamber temperature 70 deg.C, and steam pressure 0.2Mpa to obtain L-valine product.
Detecting content by High Performance Liquid Chromatography (HPLC), calculating weight of L-valine in the fermentation liquid, and dividing weight of the final product by weight of L-valine in the fermentation liquid to obtain yield. The yield of the primary crystallization is 63%, and the product meets the pharmaceutical grade standard. The mother liquor is recovered to be used as a feed-grade product, and the total yield is 85 percent. The purity of the finished product is detected by a High Performance Liquid Chromatograph (HPLC) and is 98.71 percent.
Comparative example 2
A separation and purification method of valine specifically comprises the following steps:
s1, filtering fermentation liquor by a ceramic membrane: valine was produced by fermentation, after completion of the fermentation, samples were taken from 8300L of the fermentation broth, and the L valine content in the fermentation broth was measured to be 99.5g/L by liquid chromatography (HPLC).
Filtering with ceramic membrane with pore diameter of 0.05 μm, washing with water until L-valine content of residue is lower than 1.5g/L, and collecting micro filtrate;
s2, vacuum concentration: evaporating and concentrating the micro filtrate in the step S1, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.086Mpa until valine content is 350g/L, and collecting first concentrated solution;
s3, crystallizing: slowly cooling the first concentrated solution obtained in the step S2 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, and centrifuging to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into 13500L deionized water, and redissolving at the water temperature of 80 ℃ to obtain an L-valine solution;
s5, column loading: and (3) regulating the pH value of the L-valine solution of S4 to 4.5, then enabling the solution to pass through 732 strong acid cation resin, eluting with dilute ammonia water, enabling the eluent to pass through 711 strong alkaline anion resin, and collecting the ion exchange liquid treated by the anion resin and the cation resin.
S6, second vacuum concentration: the ion exchange liquid in the step S5 is filtered by filter paper with the aperture of 0.45 mu m, and the filtrate is added into an evaporator for vacuum concentration under the conditions of: concentrating at 60deg.C under vacuum of 0.085Mpa until valine content is 250g/L, and collecting second concentrated solution;
s7, centrifugal drying: cooling the second concentrated solution in the step S6 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, centrifuging to obtain an L-valine wet product, and carrying out double-cone drying on the L-valine wet product under the double-cone drying condition: vacuum-0.09 Mpa, chamber temperature 70 deg.C, and steam pressure 0.2Mpa to obtain L-valine product.
Detecting content by High Performance Liquid Chromatography (HPLC), calculating weight of L-valine in the fermentation liquid, and dividing weight of the final product by weight of L-valine in the fermentation liquid to obtain yield. The yield of the primary crystallization is 52 percent, and the product meets the pharmaceutical grade standard. The mother liquor is recovered to be used as a feed-grade product, and the total yield is 75 percent. The purity of the finished product is detected by a High Performance Liquid Chromatograph (HPLC) and is 98.62 percent.
Comparing examples 1-4 with comparative examples 1-2, it can be seen that the valine separation and purification method provided by the invention can be improved in purity from food grade to pharmaceutical grade compared with the method of concentrating and crystallizing after directly decolorizing the crude dissolution liquid.
Compared with the method for extracting valine finished products after removing impurities by anion and cation ion exchange columns, the valine separation and purification method provided by the invention has the advantages that the process for extracting valine after removing impurities by anion and cation ion exchange columns is complex, the yield is low, and a large amount of acid-base wastewater is discharged in the ion exchange column separation process. The invention adopts the chromatographic column with the uniform particle weak polarity chromatographic resin as the stationary phase for continuous ion exchange, and the gel type resin only needs deionized water for activation and elution, thereby greatly reducing the discharge of acid-base wastewater and relieving the environmental protection pressure. The method is simple and convenient to operate, the yield is improved, and the grade is the grade of medicines.
The foregoing is merely illustrative and explanatory of the invention as it is described in more detail and is not thereby to be construed as limiting the scope of the invention. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the spirit of the invention, and that these obvious alternatives fall within the scope of the invention.
Claims (10)
1. A method for separating and purifying valine, which is characterized by comprising the following steps:
filtering fermentation liquor by a ceramic membrane, carrying out first vacuum concentration and crystallization, dissolving, loading to a column, carrying out second vacuum concentration and centrifugal drying; wherein, in the step of loading, the stationary phase of the chromatographic column is equal particle weak polarity chromatographic resin.
2. The method for separating and purifying valine according to claim 1, comprising the steps of:
s1, filtering fermentation liquor by a ceramic membrane: filtering the fermentation liquor by adopting a ceramic membrane, and collecting micro-filtrate;
s2, vacuum concentration for the first time: evaporating and concentrating the micro filtrate obtained in the step S1, and collecting a first concentrated solution;
s3, crystallizing: crystallizing the first concentrated solution obtained in the step S2 to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into deionized water, and redissolving to obtain L-valine solution;
s5, column loading: passing the L-valine solution of the S4 through a gel type resin chromatographic column, eluting with ionized water, and collecting effluent;
s6, second vacuum concentration: the effluent liquid in the step S5 is filtered by filter paper for one time, and then vacuum concentration is carried out, and a second concentrated solution is collected;
s7, centrifugal drying: and (3) cooling, crystallizing, centrifuging and drying the second concentrated solution obtained in the step (S6) to obtain an L-valine finished product.
3. The method according to claim 2, wherein in S5, the stationary phase of the gel-type resin column is a homogeneous weak polar resin prepared by polymerizing a monomer having a small dipole moment, and the resin has no functional group.
4. The method according to claim 3, wherein the chromatographic resin has a hydrophobic pore surface, and is bound to a hydrophobic moiety in a small molecule to adsorb an organic substance in a solution.
5. The method for separating and purifying valine according to claim 2, wherein in S1, the ceramic membrane has a pore size of 0.05 to 0.2. Mu.m.
6. The method for separating and purifying valine according to claim 2, wherein in S2, the concentration of the first concentrated solution is 300 to 400g/L.
7. The method for separating and purifying valine according to claim 2, wherein in S4, the concentration of the L-valine solution is 4 to 5% by mass.
8. The method for separating and purifying valine according to claim 2, wherein in S5, the post-impurity is eluted with ion water, specifically, deionized water, the post-impurity including leucine; and eluting the effluent liquid again by using deionized water to remove the pre-impurities, wherein the pre-impurities comprise alanine.
9. The method for separating and purifying valine according to claim 2, wherein in S6, the pore size of the filter paper is 0.2 to 0.45. Mu.m.
10. The method for separating and purifying valine according to claim 1, comprising the steps of:
s1, filtering fermentation liquor by a ceramic membrane: the valine is produced by adopting a fermentation method, after fermentation is finished, fermentation liquor is taken, ceramic membrane filtration is adopted, the pore diameter of the ceramic membrane is 0.05 mu m, water is used for washing, and when the L-valine content of filter residues is lower than 1.5g/L, micro filtrate is collected;
s2, vacuum concentration for the first time: evaporating and concentrating the micro filtrate obtained in the step S1, wherein the concentration conditions are as follows: concentrating at 60deg.C under vacuum of 0.084Mpa until valine content is 350g/L, and collecting first concentrated solution;
s3, crystallizing: slowly cooling the first concentrated solution obtained in the step S2 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, and centrifuging to obtain a crude L-valine product;
s4, dissolving: adding the crude L-valine product obtained in the step S3 into 14600L of deionized water, and redissolving at the water temperature of 80 ℃ to obtain an L-valine solution;
s5, column loading: passing the L-valine solution of S4 through a gel-type resin chromatographic column, eluting the rear impurities with deionized water, wherein the rear impurities comprise leucine; eluting the effluent liquid again by deionized water for the second time, removing the pre-impurities including alanine, and finally collecting the effluent liquid;
s6, second vacuum concentration: the effluent liquid of the step S5 is passed through filter paper with a pore diameter of 0.45 μm, and the filtrate is added into an evaporator for vacuum concentration under the conditions of: concentrating at 60deg.C under vacuum of 0.085Mpa until valine content is 250g/L, and collecting second concentrated solution;
s7, centrifugal drying: cooling the second concentrated solution in the step S6 to 15 ℃ for crystallization, preserving heat for 0.5h at the temperature, centrifuging to obtain an L-valine wet product, and carrying out double-cone drying on the L-valine wet product under the double-cone drying condition: vacuum-0.09 Mpa, chamber temperature 70 deg.C, and steam pressure 0.2Mpa to obtain L-valine product.
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