CN111057727B - Method for producing, separating and extracting L-glutamine - Google Patents
Method for producing, separating and extracting L-glutamine Download PDFInfo
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Abstract
The invention belongs to the field of L-glutamine production, and discloses a method for producing, separating and extracting L-glutamine, which comprises the following steps: 1) fermentation, 2) ceramic membrane filtration, 3) concentration and redissolution, 4) simulated moving bed chromatography, 5) activated carbon decolorization, 6) refining concentration, crystallization and centrifugation, and 7) fluidized bed drying. The invention improves the yield, purity, extraction yield, product quality and production efficiency of the L-glutamine, reduces the dosage of acid and alkali, reduces the discharge amount of waste water and waste, and lightens the pressure of environmental protection.
Description
Technical Field
The invention belongs to the technical field of L-glutamine production, and particularly relates to a method for producing, separating and extracting L-glutamine.
Background
L-Glutamine (L-Glutamine), also known as alpha-aminopentanamic acid, glutamic acid-5-amide, is a white crystal or crystalline powder, is water-soluble, odorless, non-toxic, hardly soluble in methanol, ethanol, diethyl ether, benzene, acetone, ethyl acetate, chloroform, ethyl acetate and the like, is unstable in neutral solution, is easily decomposed into glutaminol or propylester in alcohol, alkali or hot water to become pyrrolecarboxyl alcohol, is odorless, and has slight sweet taste.
L-glutamine is the most abundant amino acid in the human body and is a non-essential amino acid. Under normal conditions, glutamine synthesized in human bodies can meet metabolic needs, but under emergency and high metabolic states such as strenuous exercise, trauma, infection and the like, the content of glutamine in the bodies is reduced, and the glutamine must be supplemented from the outside. It has great effects in maintaining intestinal function, improving immunity, improving acid-base balance disorder, removing toxic substance, etc., and is amino acid with wide application in clinic, and can be used for treating various diseases. Meanwhile, glutamine is used as an amino acid infusion, a medical intermediate, a food additive and an important biochemical reagent.
However, domestic medicinal L-glutamine mainly depends on import, the international market demand is vigorous, the finished product of glutamine produced by the existing extraction process is difficult to meet the requirements of certain customers, how to improve the fermentation efficiency and reduce the fermentation cost is achieved, and the method of simulated moving bed chromatography is adopted to extract L-glutamine so as to meet the requirements of the customers and improve the product yield and quality is a key problem to be solved by the prior art.
Disclosure of Invention
In order to solve the problems and overcome the defects of the prior art, the invention provides the method for producing, separating and extracting the L-glutamine, the process is simple and easy to operate, and the aims of improving the fermentation yield, reducing the generation of sewage, reducing the production cost, reducing the labor intensity, improving the quality and the purity of the product and the like are fulfilled.
The purpose of the invention is realized by the following technical scheme.
A method for producing, isolating and extracting L-glutamine, comprising the steps of: 1) fermentation, 2) ceramic membrane filtration, 3) concentration and redissolution, 4) simulated moving bed chromatography, 5) activated carbon decolorization, 6) refining concentration, crystallization and centrifugation, and 7) fluidized bed drying.
Specifically, the method comprises the following steps:
step 1) fermentation: inoculating the corynebacterium glutamicum seed liquid into a fermentation tank containing a fermentation medium in an inoculation amount of 5-15%, wherein the fermentation time is 48 h; the fermentation time is divided into two stages, the first stage is 0-24h, the fermentation temperature is 32 ℃, and the pH is controlled at 5.0 by feeding ammonia water; the second stage is 24-48h, the fermentation temperature is 32 ℃, the pH is controlled at 7.0 by feeding ammonia water, and after the second stage is finished, L-glutamine fermentation liquor is obtained; in the whole fermentation process, the stirring speed is controlled at 200rpm, the dissolved oxygen is controlled at 20%, and the glucose concentration in the fermentation tank is controlled to be not lower than 0.4% by feeding glucose solution;
step 2) ceramic membrane filtration: adjusting the pH value of the L-glutamine fermentation liquid obtained in the step 1) to 5.0, then filtering the L-glutamine fermentation liquid through a ceramic membrane with the aperture of 0.05-0.1um to remove mycoprotein and other particle impurities, and collecting a ceramic membrane clear liquid;
step 3) concentration and redissolution: concentrating the clear liquid of the ceramic membrane obtained in the step 2) by a four-effect evaporator, collecting concentrated solution, then carrying out primary crystallization on the concentrated solution, centrifugally collecting obtained mother liquor and crude product, adding pure water into the crude product for dissolving, and collecting obtained solution;
step 4) simulated moving bed chromatography: separating L-glutamine and other impurities from the solution obtained in the step 3) by simulated moving bed chromatography; eluting L-glutamine to obtain an L-glutamine extracting solution;
step 5) activated carbon decoloration: adding activated carbon into the L-glutamine extracting solution obtained in the step 4) for decoloring, controlling the temperature at 55 ℃ and the decoloring time at 30min, and filtering to obtain a decoloring solution;
step 6) refining concentration, crystallization and centrifugation: controlling the temperature of the decolorized solution obtained in the step 5) at 45 ℃, refining and concentrating the decolorized solution by an evaporator to concentrate the concentration of the L-glutamine solution to 25%, cooling and crystallizing the concentrated solution, growing crystals for 15 hours, and centrifuging to obtain L-glutamine crystals;
step 7), fluidized bed drying: putting the L-glutamine crystals obtained in the step 6) into a fluidized bed for drying, controlling the temperature in the fluidized bed to be 60-65 ℃, and crushing to obtain the finished product of L-glutamine.
Further, in the step 1), when the fermentation time is up to 24 hours, adding a promoting liquid into the fermentation medium until the fermentation is finished.
Further, the promoting liquid comprises more than two of potassium dihydrogen phosphate, succinic acid and chitosan
Further, the fermentor medium comprises soybean oil.
Preferably, the components of the fermenter medium are: 1.5g/L potassium dihydrogen sulfate, 100g/L glucose, 0.01g/L manganese sulfate monohydrate, 0.01g/L ferrous sulfate heptahydrate, 0.01g/L zinc sulfate heptahydrate, 0.4g/L magnesium sulfate heptahydrate, 37.2g/L ammonium sulfate, VB10.01g/L, 5g/L of corn steep liquor and 15-25ml/L of soybean oil.
Preferably, the enhancing fluid comprises monopotassium phosphate, succinic acid and chitosan.
Preferably, the composition of the promoting fluid is: 20g/L of monopotassium phosphate, 15g/L of succinic acid and 200mg/L of chitosan.
Preferably, the ceramic membrane is an inorganic microfiltration membrane, the microfiltration temperature is 40 ℃, and the working pressure is as follows: the inlet pressure was 3.3bar and the outlet pressure was 1 bar.
Compared with the prior art, the technical scheme of the invention has the following outstanding advantages and uniqueness:
in the process of producing L-glutamine by aerobic fermentation of corynebacterium glutamicum, oxygen transfer from gas phase to liquid phase is an important factor influencing strain growth and L-glutamine generation; the solubility of oxygen in the culture medium is very low, soybean oil is used as an auxiliary liquid oxygen carrier which is not miscible in the water phase, and the soybean oil has higher oxygen solubility than water, so that the fermentation yield of L-glutamine can be improved.
The invention improves the yield of L-glutamine by optimizing the components of the promoting liquid. In the middle stage of fermentation, the proliferation speed of the strain is slowed down, the anabolic product is taken as a main component, amino on chitosan is combined with teichoic acid or lipopolysaccharide with negative charges in the bacterial cell wall, and metal cations are chelated, so that the permeability of the cell wall is changed, and the glutamine secretion outside the cell is promoted. Succinic acid has certain promotion effect on the tricarboxylic acid cycle, and has inhibition effect on the glyoxylate cycle, so that the intermediate metabolite flows to the tricarboxylic acid cycle more, and the fermentation conversion efficiency of L-glutamine is improved. Phosphate is necessary in the fermentation process of L-glutamine, but the concentration cannot be too high, otherwise the fermentation is shifted to valine fermentation, and the phosphate is slowly fed through the middle stage of the fermentation, so that the concentration of the phosphate is not suitable to be too high, and the effect of improving the conversion rate is achieved.
The invention adopts the simulated moving bed chromatographic separation technology to replace an ion exchange system to separate and extract L-glutamine, and separates other impurities generated in glutamine fermentation liquor by the principle of physical adsorption, thereby improving the purity of the glutamine. The invention adopts the four-effect evaporator, and the evaporation process is under the vacuum action, thereby not only ensuring the sanitation requirement of materials, but also ensuring the environmental protection requirement, simultaneously greatly reducing the evaporation temperature and saving the raw steam. The invention can effectively remove impurities through physical adsorption, improve the product quality, reduce the dosage of acid and alkali and reduce the discharge amount of wastewater.
Drawings
FIG. 1: the effect of soybean oil concentration on L-glutamine production;
FIG. 2: the influence of soybean oil concentration on the thallus concentration;
FIG. 3: influence of different components of the promoting liquid on the L-glutamine production.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described 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 producing, isolating and extracting L-glutamine comprising the steps of:
step 1) fermentation: the strain is Corynebacterium glutamicum GM34, which can be referred to as the research on optimizing L-glutamine fermentation medium by a response surface method, and the biotechnology communication is 2013.
Mixing Corynebacterium glutamicum seed liquid (OD)600nm13.1) inoculating 10% of the inoculum size into a 100L fermentation tank containing 50L of fermentation medium, wherein the fermentation time is 48 h; the fermentation time is divided into two stages, the first stage is 0-24h, the fermentation temperature is 32 ℃, and the pH is controlled at 5.0 by feeding ammonia water; the second stage is 24-48h, the fermentation temperature is 32 ℃, the pH is controlled at 7.0 by feeding ammonia water, and after the second stage is finished, L-glutamine fermentation liquor is obtained;
when the fermentation time is 24 hours, adding a promoting liquid into the fermentation medium at the speed of 0.05ml/min.L (0.05 ml per minute in each liter of fermentation liquid) until the fermentation is finished;
in the whole fermentation process, the stirring speed is controlled at 200rpm, the dissolved oxygen is controlled at 20%, and the glucose concentration in the fermentation tank is controlled to be not lower than 0.4% by feeding 60% of glucose solution by mass percent;
the components of the fermentation tank culture medium are as follows: 1.5g/L potassium dihydrogen sulfate, 100g/L glucose, 0.01g/L manganese sulfate monohydrate, 0.01g/L ferrous sulfate heptahydrate, 0.01g/L zinc sulfate heptahydrate, 0.4g/L magnesium sulfate heptahydrate, 37.2g/L ammonium sulfate, VB10.01g/L, 5g/L of corn steep liquor and 20ml/L of soybean oil.
Promoting liquid: 20g/L of monopotassium phosphate, 15g/L of succinic acid and 200mg/L of chitosan.
Step 2) ceramic membrane filtration: adjusting the pH value of the L-glutamine fermentation liquid obtained in the step 1) to 5.0, then filtering the L-glutamine fermentation liquid by a ceramic membrane with the aperture of 0.05-0.1um to remove mycoprotein and other particle impurities, and collecting the clear liquid of the ceramic membrane.
Step 3) concentration and redissolution: concentrating the clear liquid of the ceramic membrane obtained in the step 2) by a four-effect evaporator, collecting concentrated solution, then carrying out primary crystallization on the concentrated solution, centrifugally collecting obtained mother liquor and crude product, adding pure water into the crude product for dissolving, and collecting obtained solution.
Step 4) simulated moving bed chromatography: separating L-glutamine and other impurities from the solution obtained in the step 3) by simulated moving bed chromatography; and (3) dissociating the L-glutamine to obtain a high-purity L-glutamine extracting solution.
Step 5) activated carbon decoloration: adding a proper amount of activated carbon into the L-glutamine extracting solution obtained in the step 4) for decoloring, adjusting the pH value, controlling the temperature at 55 ℃, decoloring for 30min, and filtering to obtain a decoloring solution.
Step 6) refining concentration, crystallization and centrifugation: controlling the temperature of the decolorized liquid obtained in the step 5) at 45 ℃, refining and concentrating the decolorized liquid by an evaporator to concentrate the concentration of the L-glutamine liquid to 25%, cooling and crystallizing the concentrated liquid, starting frozen water to cool at the speed of 8 ℃/h, stopping stirring after the temperature of the liquid is reduced to 5 ℃, growing crystals for 15 hours, and starting a centrifugal machine to centrifuge to obtain the L-glutamine crystals.
Step 7), fluidized bed drying: and (3) putting the crystals obtained in the step 6) into a fluidized bed for drying, controlling the temperature in the fluidized bed to be 65 ℃, and crushing to obtain the finished product of the L-glutamine. The measured L-glutamine index is as follows: the yield of L-glutamine was 87.3%; the purity is more than 99%.
The separation technology can improve the quality of the product, further improve the yield, increase the yield and provide a product with high cost performance for the glutamine market; compared with the existing separation and extraction process, the product yield of the invention is improved by 2-5%, the discharge of sewage is reduced, the pollution to the environment is reduced, the social benefit is increased, and the technology has great market popularization prospect.
Example 2
A method for producing, isolating and extracting L-glutamine comprising the steps of:
mixing Corynebacterium glutamicum seed liquid (OD)600nm13.4) inoculating the mixture into a 100L fermentation tank containing 50L fermentation medium at an inoculation amount of 9%, wherein the fermentation time is 48 h; the fermentation time is divided into two stages, the first stage is 0-24h, the fermentation temperature is 32 ℃, and the pH is controlled at 5.0 by feeding ammonia water; the second stage is 24-48h, the fermentation temperature is 32 ℃, the pH is controlled at 7.0 by feeding ammonia water, and the fermentation liquor is collected after the second stage is finished;
when the fermentation time is 24 hours, adding a promoting liquid into the fermentation medium at the speed of 0.07ml/min.L (0.07 ml per minute per liter of fermentation liquid) until the fermentation is finished;
in the whole fermentation process, the stirring speed is controlled at 200rpm, the dissolved oxygen content is controlled at 25%, the glucose concentration in the fermentation tank is controlled to be not less than 0.4% by feeding glucose solution with the mass fraction of 65%,
the components of the fermentation tank culture medium are as follows: 1.5g/L potassium dihydrogen sulfate, 100g/L glucose, 0.01g/L manganese sulfate monohydrate, 0.01g/L ferrous sulfate heptahydrate, 0.01g/L zinc sulfate heptahydrate, 0.4g/L magnesium sulfate heptahydrate, 37.2g/L ammonium sulfate, VB10.01g/L, 5g/L of corn steep liquor and 15ml/L of soybean oil.
Promoting liquid: 20g/L of monopotassium phosphate, 20g/L of succinic acid and 150mg/L of chitosan.
The separation and extraction method was the same as in example 1.
Comparative example 1
A method for producing, isolating and extracting L-glutamine comprising the steps of:
mixing Corynebacterium glutamicum seed liquid (OD)600nm13.1) inoculating 10% of the inoculum size into a 100L fermentation tank containing 50L of fermentation medium, wherein the fermentation time is 48 h; the fermentation time is divided into two stages, the first stage is 0-24h, the fermentation temperature is 32 ℃, and the pH is controlled at 5.0 by feeding ammonia water; the second stage is 24-48h, the fermentation temperature is 32 ℃, the pH is controlled at 7.0 by feeding ammonia water, and after the second stage is finished, L-glutamine fermentation liquor is obtained;
in the whole fermentation process, the stirring speed is controlled at 200rpm, the dissolved oxygen is controlled at 20%, and the glucose concentration in the fermentation tank is controlled to be not lower than 0.4% by feeding 60% of glucose solution by mass percent;
the components of the fermentation tank culture medium are as follows: 1.5g/L potassium dihydrogen sulfate, 100g/L glucose, 0.01g/L manganese sulfate monohydrate, 0.01g/L ferrous sulfate heptahydrate, 0.01g/L zinc sulfate heptahydrate, 0.4g/L magnesium sulfate heptahydrate, 37.2g/L ammonium sulfate, VB10.01g/L and 5g/L of corn steep liquor.
The separation and extraction method was the same as in example 1.
Example 3
1. On the basis of comparative example 1, the effect of the addition of soybean oil to the fermentation medium on the production of L-glutamine was investigated. Setting the concentration of the soybean oil to be 0,5,10,15,20,25 and 30, and the unit ml/L respectively, as shown in figure 1-2, along with the increase of the concentration of the soybean oil, the thallus concentration and the L-glutamine fermentation yield are also improved, when the thallus concentration and the L-glutamine fermentation yield are increased to 15ml/L, the L-glutamine amplification begins to slow down, the concentration of the soybean oil reaches 20ml/L, the L-glutamine yield approaches to a peak value, and compared with the case of not adding the soybean oil, the L-glutamine yield is improved by 13.7 percent, and the thallus concentration is increased by 6.5 percent; the soybean oil concentration is continuously increased, and the influence on L-glutamine is small; in the process of producing L-glutamine by aerobic fermentation of corynebacterium glutamicum, oxygen transfer from a gas phase to a liquid phase is an important factor influencing strain growth and L-glutamine generation, while the solubility of oxygen in a culture medium is very low, soybean oil is adopted as an auxiliary liquid oxygen carrier which is not miscible in a water phase, and the soybean oil has higher oxygen solubility than water, so that the fermentation yield of L-glutamine can be improved.
2. Soybean oil is selectively added into the fermentation medium in an amount of 20ml/L, and the influence of the components of the accelerating solution on the yield of L-glutamine is researched. Considering that the increase of the fermentation volume can cause dilution effect on the product, the total amount of the accelerating liquid is properly selected to be between 5 and 10 percent, and when the test flow rate is set to be 0.05ml/min. Setting accelerating liquids of different raw materials; wherein group 1: potassium dihydrogen phosphate + succinic acid; group 2: potassium dihydrogen phosphate + chitosan; group 3: succinic acid + chitosan; group 4: inventive example 1; the concentrations of the starting materials of the respective sets of accelerating liquids were the same as in example 1. As shown in fig. 3, by comparing groups 1 to 4, group 4 had the greatest effect on L-glutamine production, and L-glutamine production could be significantly increased by 19.8%, 7.8% for group 2, 13.4% for group 3, and 11.4% for group 4, compared to the case where no booster liquid phase was used. Therefore, the combination of the three components of the monopotassium phosphate, the succinic acid and the chitosan has a certain positive promotion effect on the yield of the L-glutamine, and the synergistic performance is good.
While the invention has been described with reference to specific embodiments, it will be apparent to those skilled in the art that the invention is not limited thereto, and that various changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (4)
1. A method for producing, isolating and extracting L-glutamine, comprising the steps of: 1) fermentation, 2) ceramic membrane filtration, 3) concentration and redissolution, 4) simulated moving bed chromatography, 5) activated carbon decolorization, 6) refining concentration, crystallization and centrifugation, and 7) fluidized bed drying;
the method comprises the following steps:
step 1) fermentation: inoculating the corynebacterium glutamicum seed liquid into a fermentation tank containing a fermentation medium in an inoculation amount of 5-15%, wherein the fermentation time is 48 h; the fermentation time is divided into two stages, the first stage is 0-24h, the fermentation temperature is 32 ℃, and the pH is controlled at 5.0 by feeding ammonia water; the second stage is 24-48h, the fermentation temperature is 32 ℃, the pH is controlled at 7.0 by feeding ammonia water, and after the second stage is finished, L-glutamine fermentation liquor is obtained; in the whole fermentation process, the stirring speed is controlled at 200rpm, the dissolved oxygen is controlled at 20%, and the glucose concentration in the fermentation tank is controlled to be not lower than 0.4% by feeding glucose solution;
step 2) ceramic membrane filtration: adjusting the pH value of the L-glutamine fermentation liquid obtained in the step 1) to 5.0, then filtering the L-glutamine fermentation liquid through a ceramic membrane with the aperture of 0.05-0.1um to remove mycoprotein and other particle impurities, and collecting a ceramic membrane clear liquid;
step 3) concentration and redissolution: concentrating the clear liquid of the ceramic membrane obtained in the step 2) by a four-effect evaporator, collecting concentrated solution, then carrying out primary crystallization on the concentrated solution, centrifugally collecting obtained mother liquor and crude product, adding pure water into the crude product for dissolving, and collecting obtained solution;
step 4) simulated moving bed chromatography: separating L-glutamine and other impurities from the solution obtained in the step 3) by simulated moving bed chromatography; eluting L-glutamine to obtain an L-glutamine extracting solution;
step 5) activated carbon decoloration: adding activated carbon into the L-glutamine extracting solution obtained in the step 4) for decoloring, controlling the temperature at 55 ℃ and the decoloring time at 30min, and filtering to obtain a decoloring solution;
step 6) refining concentration, crystallization and centrifugation: controlling the temperature of the decolorized solution obtained in the step 5) at 45 ℃, refining and concentrating the decolorized solution by an evaporator to concentrate the concentration of the L-glutamine solution to 25%, cooling and crystallizing the concentrated solution, growing crystals for 15 hours, and centrifuging to obtain L-glutamine crystals;
step 7), fluidized bed drying: putting the L-glutamine crystals obtained in the step 6) into a fluidized bed for drying, controlling the temperature in the fluidized bed to be 60-65 ℃, and crushing to obtain a finished product of L-glutamine;
in the step 1), when the fermentation is carried out for 24 hours, adding a promoting liquid into a fermentation culture medium; the promoting liquid comprises more than two of potassium dihydrogen phosphate, succinic acid and chitosan;
adding a promoting liquid into a fermentation medium at a speed of 0.05ml/min.L, namely 0.05ml per minute of flow in each liter of fermentation liquid until the fermentation is finished;
the components of the promoting liquid are as follows: 20g/L of monopotassium phosphate, 15g/L of succinic acid and 200mg/L of chitosan.
2. The method of claim 1, wherein the fermentor medium comprises soybean oil.
3. The method of claim 2, wherein the components of the fermentor medium are: 1.5g/L potassium dihydrogen sulfate, 100g/L glucose, 0.01g/L manganese sulfate monohydrate, 0.01g/L ferrous sulfate heptahydrate, 0.01g/L zinc sulfate heptahydrate, 0.4g/L magnesium sulfate heptahydrate, 37.2g/L ammonium sulfate, VB10.01g/L, 5g/L of corn steep liquor and 15-25ml/L of soybean oil.
4. The method according to any one of claims 1 to 3, wherein the ceramic membrane is an inorganic microfiltration membrane, the microfiltration temperature is 40 ℃, the working pressure is: the inlet pressure was 3.3bar and the outlet pressure was 1 bar.
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