CN108285912B - Method for preparing and extracting pharmaceutical grade valine by fermentation - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- 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
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Abstract
The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for preparing and extracting pharmaceutical grade valine by fermentation, which comprises the following steps: fermenting, filtering and concentrating to obtain a crude product of valine; carrying out primary acid dissolution on the crude product of valine by using hydrochloric acid, and then carrying out cooling crystallization and centrifugation to obtain a valine solid and a mother solution; carrying out secondary acid dissolution on the valine solid by using hydrochloric acid, and then carrying out cooling crystallization and centrifugation to obtain the valine solid and a mother solution again; dissolving the obtained valine solid in water with distilled water, and performing ion exchange, decolorization, fine filtration and crystallization to obtain the pharmaceutical grade valine. The method has high fermentation efficiency, can effectively remove the heteropolyacid, greatly improves the product quality and reaches the pharmaceutical grade standard.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly provides a method for preparing and extracting pharmaceutical grade valine by fermentation.
Background
Valine has the chemical name of 2-amino-3-methylbutyric acid and the molecular formula of C5H11NO2The relative molecular mass was 117.15. Valine is one of eight essential amino acids in human body, has multiple physiological functions, and plays an important role in many aspects such as metabolic regulation and information transmission in human life bodies. In the field of medicine, valine is mainly used for preparing amino acid infusion, and can be used for treating blood brain barrier, chronic liver cirrhosis, hepatic coma, renal failure, congenital metabolic defect, diabetes and the like. Valine can work together with isoleucine and leucine to promote normal growth of human body, repair tissue and regulate blood sugar.
The valine can be produced by three methods, namely extraction, chemical synthesis and biological fermentation. The extraction method is mainly to hydrolyze soybean protein and the like, and then to add hydrochloric acid to generate and precipitate valine-hydrochloride crystals. The method has the advantages of good separation effect, simple extraction operation and short production period, but the yield of the valine is lower, so that the production cost is high. The chemical synthesis method has various synthesis modes, but has the disadvantages of multiple reaction steps, complex reaction process and more byproducts, so that the production cost is higher, and the grafting is difficult to realize in industrial scale production. The biological fermentation method is a very economic production method which utilizes valine producing bacteria to produce products by fermentation, has the advantages of low raw material cost, mild reaction conditions and the like, is easy to realize large-scale production, and is a method for producing the valine products by fermentation.
The valine fermentation liquor contains complex components, not only contains the target product valine, but also contains pigment, protein, polysaccharide, reducing sugar and other heteroacid such as leucine, isoleucine, alanine and the like. At present, the extraction method of valine mainly comprises a precipitation method, a whole membrane method and an ion exchange method. The invention patent 201210260549.5 discloses a method for extracting valine by using a combined technology of membrane separation and electrodialysis, which comprises removing inorganic salts from a fermentation broth from which thalli are removed by an electrodialysis device, and obtaining a finished product of valine through the steps of protein removal, pigment removal, reverse osmosis preconcentration, concentration crystallization and the like. The invention patent 200710190536.4 relates to a method for preparing high-purity valine by using a simulated moving bed technology and using water as an eluent to extract from valine fermentation liquor. Some of the above methods have difficulty in producing valine of pharmaceutical grade purity, and some have difficulty in achieving an industrial scale. Therefore, the exploration of an extraction method for preparing valine with various pharmaceutical-grade purities has great significance for solving the key problems in the field of amino acid extraction.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for preparing and extracting pharmaceutical grade valine by fermentation. According to the invention, the valine fermentation liquor is prepared by a biological fermentation method, then a crude product of valine is obtained by a whole membrane method, and then the crude product is treated by acid dissolution, water dissolution, ion exchange, decoloration, fine filtration and other processes, so that the mixed acid can be effectively removed, and the pharmaceutical grade valine is obtained.
The invention is realized by the following technical scheme:
a method for preparing and extracting pharmaceutical grade valine by fermentation comprises the following steps: step 1) fermentation process, step 2) ultrafiltration and concentration to prepare a crude product of valine, step 3) acid dissolution, crystallization and centrifugation, and step 4) water dissolution, ion exchange, decoloration, fine filtration and crystallization.
Specifically, the method comprises the following steps:
step 1) fermentation process:
(1) inoculating lactobacillus fermentation brevibacterium into a fermentation culture medium according to the inoculation amount of 6-8%, and continuously fermenting for 50-55 hours to obtain valine fermentation liquor; controlling the temperature at 30 deg.C, pH at 6.5, and glucose concentration at 10g/L or higher during fermentation;
(2) filtering valine fermentation liquor by using a ceramic membrane to obtain filtrate A and wet thalli; adding 1-3 wt% of tourmaline powder into wet thalli, stirring at 100rpm for 30min, stopping stirring, heating to 50-55 ℃, preserving heat for 60-90s, naturally cooling to room temperature, adding into a dialysis culture medium with three times of weight, culturing at 30 ℃ for 4-6h at 100rpm, and filtering by ceramic membranes to collect thalli and filtrate B;
step 2) ultrafiltration and concentration to prepare a crude product of valine: combining the filtrate A and the filtrate B obtained in the step 1), performing ultrafiltration through an ultrafiltration membrane, collecting ultrafiltrate, concentrating the ultrafiltrate in vacuum until the content of valine is 30 wt%, cooling, crystallizing, and centrifuging to obtain a crude product of valine;
step 3) acid dissolution, crystallization and centrifugation: carrying out primary acid dissolution on a crude product of valine by using 7mol/L hydrochloric acid with the volume of 1.0-1.5 times, and then carrying out cooling crystallization and centrifugation to obtain a valine solid A and a mother solution; carrying out secondary acid dissolution on the valine solid A by adopting 1mol/L hydrochloric acid with the volume of 0.5-0.8 times, and then obtaining the valine solid B and mother liquor again through cooling crystallization and centrifugation;
step 4) water dissolving, ion exchange, decoloring, fine filtering and crystallizing: and dissolving the valine solid B in water by using distilled water with the volume of 8-10 times, and then carrying out ion exchange, decoloration, fine filtration and crystallization to obtain the pharmaceutical grade valine.
Preferably, the first and second electrodes are formed of a metal,
the fermentation medium comprises the following components: 50g/L glucose, 30g/L corn steep liquor, 10g/L soybean meal, 10g/L lactose, 6g/L ammonium sulfate, 0.1g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate, 6mg/L manganese sulfate, 6mg/L ferrous sulfate and 6mg/L potassium chloride.
Preferably, the first and second electrodes are formed of a metal,
the dialysis medium comprises the following components: 0.8% of monopotassium phosphate, 0.8% of dipotassium phosphate, 0.5% of ammonium sulfate, 0.08% of polyethylene glycol, 0.01% of ferrous sulfate, 0.01% of magnesium sulfate and 0.01% of zinc sulfate, wherein the pH value is adjusted to 6.5, and the mass ratio is above.
Preferably, the first and second electrodes are formed of a metal,
the membrane aperture of the ceramic membrane is 40-50 nm.
Preferably, the first and second electrodes are formed of a metal,
the ultrafiltration parameters are as follows: the temperature is 40 ℃, the pressure is 7-8bar, and the pressure is 6-7 bar.
Preferably, the first and second electrodes are formed of a metal,
the step 4) specifically comprises the following steps:
(1) dissolving valine solid B in 8-10 times of distilled water, adjusting pH of the solution to 4.5, introducing into strong acid ion exchange resin, and resolving with 2% ammonia water to obtain eluate;
(2) concentrating and deaminating the eluate with single-effect evaporator, adding 1.8-3 ‰ of active carbon for decolorizing for 45min, adjusting pH of the decolorized feed liquid to 6.0, and removing active carbon;
(3) adjusting pH of the feed liquid to 6.0 after removing active carbon, performing fine filtration with 0.2 μm filter core, concentrating the fine filtrate, crystallizing, centrifuging, drying, and pulverizing to obtain the final product.
The beneficial effects achieved by the invention mainly comprise but are not limited to the following aspects:
the method is improved aiming at the fermentation process, avoids the accumulation of valine concentration to cause feedback inhibition, and carries out secondary acid production treatment on the fermented waste thalli, thereby increasing the permeability of cell membranes and improving the acid production capability of the strains;
the acid production capability and cell membrane permeability of the strain can be improved by heat treatment at proper time and temperature, and the yield of valine is greatly improved by matching with a dialysis culture medium;
the tourmaline can automatically release negative ions which have strong oxidability and continuously generate direct current static electricity to release mineral substances and trace elements, thereby promoting the propagation of strains; the method adopts the dialysis culture medium to culture the strains, can change the biological membrane structure of cells, promote the utilization and the transportation of substances, greatly reduce the feedback inhibition and regulation caused by the accumulation of valine, improve the acid production efficiency, reduce the subsequent residual sugar, avoid the adhesion and flocculation of the strains, and facilitate the subsequent membrane filtration and separation;
after fermentation is finished, firstly, a separation technology is adopted to remove substances generating inhibition, and as the inhibition is relieved, the cell enzyme activity is recovered, so that secondary dialysis fermentation is carried out, and compared with common fermentation, the acid production period in the whole fermentation process is greatly prolonged;
according to the invention, the valine fermentation liquor is prepared by a biological fermentation method, then a crude product of valine is obtained by a whole membrane method, and then the crude product is subjected to processes such as acid dissolution, water dissolution, ion exchange, decoloration, fine filtration and the like, so that the mixed acid can be effectively removed, the pharmaceutical grade valine is obtained, the purity can reach more than 99.9%, and the application prospect is wide.
Drawings
FIG. 1: influence of Heat treatment temperature on the valine content of filtrate B.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be described more clearly and completely below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for preparing and extracting pharmaceutical grade valine by fermentation comprises the following steps:
seed liquid (1 × 10) of Brevibacterium lactofermentum ATCC14708cfu/ml) is inoculated into a fermentation medium according to the inoculation amount of 7 percent, and the valine fermentation liquid is obtained after continuous fermentation for 55 hours; controlling the temperature at 30 deg.C, pH at 6.5, and glucose concentration at 20g/L or higher during fermentation;
the fermentation medium comprises the following components: 50g/L glucose, 30g/L corn steep liquor, 10g/L soybean meal, 10g/L lactose, 6g/L ammonium sulfate, 0.1g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate, 6mg/L manganese sulfate, 6mg/L ferrous sulfate and 6mg/L potassium chloride;
filtering valine fermentation broth by using a ceramic membrane to obtain filtrate A (the content of valine is 4.9 percent) and wet thalli; adding 3 wt% tourmaline powder into wet thallus, stirring at 100rpm for 30min, stopping stirring, heating to 51 deg.C, keeping the temperature for 60s, naturally cooling to room temperature, adding into dialysis culture medium with three times of weight, culturing at 30 deg.C and stirring at 100rpm for 5h, and filtering with ceramic membrane to collect thallus and filtrate B; the dialysis culture medium comprises the following components in percentage by mass: potassium dihydrogen phosphate 0.8%, dipotassium hydrogen phosphate 0.8%, ammonium sulfate 0.5%, polyethylene glycol 0.08%, ferrous sulfate 0.01%, magnesium sulfate 0.01%, zinc sulfate 0.01%, adjusting pH to 6.5; the membrane aperture of the ceramic membrane is 40 nm;
2) mixing the filtrate A and the filtrate B, ultrafiltering, and collecting ultrafiltrate, wherein the ultrafiltration temperature is 40 deg.C, the inlet pressure is 7-8bar, and the outlet pressure is 6-7 bar; concentrating the ultrafiltrate in vacuum until the content of valine is 30 wt%, cooling to room temperature, and centrifuging to obtain a crude product of valine;
3) carrying out primary acid dissolution on a crude product of valine by using 7mol/L hydrochloric acid with the volume of 1.2 times, and then cooling, crystallizing and centrifuging to obtain a valine solid A and a mother solution; carrying out secondary acid dissolution on the valine solid A by adopting 1mol/L hydrochloric acid with the volume of 0.8 time, and then obtaining the valine solid B and mother liquor again through cooling crystallization and centrifugation;
4) dissolving valine solid B in 10 times of distilled water, performing ion exchange, decolorizing, fine filtering, and crystallizing to obtain pharmaceutical grade valine;
(1) adjusting the pH value of the water-dissolved liquid to 4.5, introducing into strong acid ion exchange resin, and resolving with 2% ammonia water;
(2) concentrating and deaminating the eluate with single-effect evaporator, adding 3 ‰ active carbon for decolorizing for 45min, adjusting pH of the decolorized feed liquid to 6.0, and removing active carbon;
(3) adjusting pH of the decarbonized feed liquid to 6.0, performing fine filtration by using a 0.2 μm filter element, concentrating, crystallizing, centrifuging, drying and pulverizing the fine filtration to obtain a finished product, wherein the purity of valine is 99.98% by detection.
Example 2
A method for preparing and extracting pharmaceutical grade valine by fermentation comprises the following steps:
seed liquid (1 × 10) of Brevibacterium lactofermentum ATCC14708cfu/ml) is inoculated into a fermentation medium according to the inoculation amount of 8 percent, and the valine fermentation liquid is obtained after continuous fermentation for 50 hours; controlling the temperature at 30 deg.C, pH at 6.5, and glucose concentration at 15g/L or higher during fermentation;
the fermentation medium comprises the following components: 50g/L glucose, 30g/L corn steep liquor, 10g/L soybean meal, 10g/L lactose, 6g/L ammonium sulfate, 0.1g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate, 6mg/L manganese sulfate, 6mg/L ferrous sulfate and 6mg/L potassium chloride;
filtering valine fermentation broth by using a ceramic membrane to obtain filtrate A (the content of valine is 4.7 percent) and wet thalli; adding 3 wt% tourmaline powder into wet thallus, stirring at 100rpm for 30min, stopping stirring, heating to 50 deg.C, keeping the temperature for 80s, naturally cooling to room temperature, adding into dialysis culture medium with three times of weight, culturing at 30 deg.C and stirring at 100rpm for 6h, and filtering with ceramic membrane to collect thallus and filtrate B; the dialysis culture medium comprises the following components in percentage by mass: potassium dihydrogen phosphate 0.8%, dipotassium hydrogen phosphate 0.8%, ammonium sulfate 0.5%, polyethylene glycol 0.08%, ferrous sulfate 0.01%, magnesium sulfate 0.01%, zinc sulfate 0.01%, adjusting pH to 6.5; the membrane aperture of the ceramic membrane is 50 nm;
2) mixing the filtrate A and the filtrate B, ultrafiltering, and collecting ultrafiltrate, wherein the ultrafiltration temperature is 40 deg.C, the inlet pressure is 7-8bar, and the outlet pressure is 6-7 bar; concentrating the ultrafiltrate in vacuum until the content of valine is 30 wt%, cooling to room temperature, and centrifuging to obtain a crude product of valine;
3) carrying out primary acid dissolution on a crude product of valine by using 7mol/L hydrochloric acid with the volume of 1.0 time, and then cooling, crystallizing and centrifuging to obtain a valine solid A and a mother solution; carrying out secondary acid dissolution on the valine solid A by adopting 1mol/L hydrochloric acid with the volume of 0.5 time, and then obtaining the valine solid B and mother liquor again through cooling crystallization and centrifugation;
4) dissolving valine solid B in 8 times of distilled water, performing ion exchange, decolorizing, fine filtering, and crystallizing to obtain pharmaceutical grade valine;
(1) adjusting the pH value of the water-dissolved liquid to 4.5, introducing into strong acid ion exchange resin, and resolving with 2% ammonia water;
(2) concentrating and deaminating the eluate with single-effect evaporator, adding 1.8 ‰ of active carbon, decolorizing for 45min, adjusting pH of the decolorized solution to 6.0, and removing active carbon;
(3) adjusting pH of the decarbonized feed liquid to 6.0, performing fine filtration by using a 0.2 μm filter element, concentrating, crystallizing, centrifuging, drying and pulverizing the fine filtration to obtain a finished product, wherein the purity of valine is 99.97% by detection.
Example 3
1. Taking example 1 as an example, the content of valine is detected, and the content of valine in the filtrate B is respectively detected; meanwhile, a comparison group is also arranged, wherein the comparison group 1 comprises: the same as example 1 except that only the dialysis culture treatment was carried out without using tourmaline powder and heat treatment; control group 2: after the fermentation, tourmaline powder and dialysis culture treatment is performed without heat treatment, which is otherwise the same as in example 1; control group 3: after the fermentation, the heat treatment and the dialysis culture treatment are carried out without using tourmaline powder, and the rest is the same as the example 1; the content of valine in the filtrate B of each group is detected, and the specific result is shown in the table 1:
TABLE 1
And (4) conclusion: as shown in Table 1, the valine with higher concentration can be generated after the re-culture treatment of the waste strains, the valine can be generated by the groups of example 1 and the comparison groups 1-3, but the acid-generating effect of the treatment by tourmaline powder, heat treatment and dialysis culture is better than that of the treatment by a single mode or two modes, which is 2.51 times that of the comparison group 1, 1.22 times that of the comparison group 2 and 1.32 times that of the comparison group 3.
2. Influence of Heat treatment temperature on the content of valine in filtrate B:
the procedure of example 2 was followed except that a plurality of concentration gradients were set at 30-60 ℃ to examine the influence of different temperature conditions on the production of valine by the strain. As shown in fig. 1, the temperature between 30 ℃ and 40 ℃ has little influence on the acid yield, and is not obviously increased, the valine yield is gradually increased with the temperature being increased by more than 40 ℃, and the peak value can be reached at 50 ℃, but when the temperature is continuously increased, the valine yield is obviously reduced, probably because the enzyme activity of the strain is reduced due to overhigh temperature, and the strain is greatly damaged, therefore, the selection of the proper temperature is more critical, and the effect of improving the valine yield cannot be achieved due to overhigh or overlow temperature.
The above description is a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiments according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (3)
1. A method for preparing and extracting pharmaceutical grade valine by fermentation, which is characterized by comprising the following steps:
step 1) fermentation process:
(1) inoculating lactobacillus fermentation brevibacterium into a fermentation culture medium according to the inoculation amount of 6-8%, and continuously fermenting for 50-55 hours to obtain valine fermentation liquor; controlling the temperature at 30 deg.C, pH at 6.5, and glucose concentration at 10g/L or higher during fermentation; the lactobacillus brevis is ATCC 1470;
(2) filtering valine fermentation liquor by using a ceramic membrane to obtain filtrate A and wet thalli; adding 1-3 wt% tourmaline powder into wet thallus, stirring at 100rpm for 30min, stopping stirring, heating to 50-55 deg.C, maintaining the temperature for 60-90s, naturally cooling to room temperature, adding into dialysis culture medium with three times of weight, controlling the temperature at 30 deg.C, stirring at 100rpm, culturing for 4-6h, and filtering with ceramic membrane to collect thallus and filtrate B; the dialysis medium comprises the following components: 0.8% of monopotassium phosphate, 0.8% of dipotassium phosphate, 0.5% of ammonium sulfate, 0.08% of polyethylene glycol, 0.01% of ferrous sulfate, 0.01% of magnesium sulfate and 0.01% of zinc sulfate, wherein the pH value is adjusted to 6.5, and the mass ratio is above;
step 2) ultrafiltration and concentration to prepare a crude product of valine: combining the filtrate A and the filtrate B obtained in the step 1), performing ultrafiltration through an ultrafiltration membrane, collecting ultrafiltrate, concentrating the ultrafiltrate in vacuum until the content of valine is 30 wt%, cooling, crystallizing, and centrifuging to obtain a crude product of valine;
step 3) acid dissolution, crystallization and centrifugation: carrying out primary acid dissolution on a crude product of valine by using 7mol/L hydrochloric acid with the volume of 1.0-1.5 times, and then carrying out cooling crystallization and centrifugation to obtain a valine solid A and a mother solution; carrying out secondary acid dissolution on the valine solid A by adopting 1mol/L hydrochloric acid with the volume of 0.5-0.8 times, and then obtaining the valine solid B and mother liquor again through cooling crystallization and centrifugation;
step 4) water dissolving, ion exchange, decoloring, fine filtering and crystallizing:
(1) dissolving valine solid B in 8-10 times of distilled water, adjusting pH of the solution to 4.5, introducing into strong acid ion exchange resin, and resolving with 2% ammonia water to obtain eluate;
(2) concentrating and deaminating the eluate with single-effect evaporator, adding 1.8-3 ‰ of active carbon for decolorizing for 45min, adjusting pH of the decolorized feed liquid to 6.0, and removing active carbon;
(3) adjusting pH of the feed liquid to 6.0 after removing active carbon, performing fine filtration with 0.2 μm filter core, concentrating the fine filtrate, crystallizing, centrifuging, drying, and pulverizing to obtain the final product.
2. The method of claim 1, wherein the fermentation medium comprises: 50g/L glucose, 30g/L corn steep liquor, 10g/L soybean meal, 10g/L lactose, 6g/L ammonium sulfate, 0.1g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate, 6mg/L manganese sulfate, 6mg/L ferrous sulfate and 6mg/L potassium chloride.
3. The method according to claim 1, wherein the ceramic membrane has a membrane pore size of 40-50 nm.
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| CN110092728A (en) * | 2019-06-12 | 2019-08-06 | 无锡荣丰生物工程有限公司 | The method that valine two imitates condensing crystallizing system and carries out condensing crystallizing using the system |
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| CN113773215B (en) * | 2020-06-10 | 2024-02-02 | 安徽华恒生物科技股份有限公司 | L-valine with high bulk density and preparation method and application thereof |
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