CN1205178C - Process for extracting glutamine from fermentation broth - Google Patents

Abstract

The invention relates to a process for extracting glutamine from fermentation liquid, belonging to the technical field of amino acid preparation for medicine and health care. This method is to pretreat the glutamine fermentation liquid first, remove bacteria and some proteins, pigments and polysaccharides, control the pH value of the fermented liquid after pretreatment, remove the inorganic salts in it through electrodialysis equipment, and re-adjust the pH of the feed liquid Value, and pass into the treated OH type anion exchange column, the impurities are exchanged with the resin and adsorbed, glutamine flows through the ion exchange column to be separated, the effluent is first decolorized, vacuum concentrated, and then isoelectric crystallization, the crude crystal is passed through the organic Solvent wash and dry. Utilizing the process of the present invention to extract glutamine in the fermentation broth can significantly reduce the amount of exchange resin, effectively avoid the conversion problem of glutamine in strong alkali and strong acid environment, greatly reduce the production cost, and the total extraction yield of glutamine reaches About 70%, suitable for industrial production.

Description

Process for extracting glutamine from fermentation broth

technical field

The invention relates to a process for extracting glutamine from fermentation liquid, belonging to the technical field of amino acid preparation for medicine and health care.

Background technique

L-glutamine (Gln) is the γ-carboxyl amidate of L-glutamic acid (Glu), and is one of the 20 basic amino acids that constitute proteins. Its chemical molecular formula is H ₂ NCO·CH ₂ ·CH ₂ ·CH(NH ₂ )·COOH, its molecular weight is 146.15, its decomposition temperature is 184-185°C, and its isoelectric point is 5.65. It is a neutral amino acid. In recent years, medical discoveries have shown that glutamine is a conditionally essential amino acid, which plays a pivotal role in biological metabolism, and a variety of diseases will be caused when it is lacking.

The main producers of glutamine by fermentation are Japan and South Korea. It is estimated that the annual output of glutamine in the world has reached about 4,000 tons, and it is showing an increasing trend. The research on the production of glutamine by fermentation has a history of nearly 20 years in my country, but at present, there is no mature downstream separation technology, and there is no enterprise that uses fermentation to produce glutamine on a large scale. The separation and extraction of glutamine at home and abroad basically adopts the anion-cation double column ion exchange method, but there are differences in the choice of resin, the combination of anion and cation columns and the specific operation method. It was reported in China in 1998 (Ion Exchange and Adsorption, 1998, 14(2), 97-103), and the double-column method was used for step-by-step elution, and the laboratory yield could reach 56.6%. In the early 1990s, foreign patents (Miyazawa et al. Method for purifying L-glutamine, Int. Cl. C07C 227/00, United States Patent 4,966,994 Filed: July.11, 1989 Date of Patent: Oct.30, 1990.) passed fermentation The process of combining liquid crude crystallization and ion exchange has achieved a yield of more than 70% in the laboratory. Due to the large amount of exchange resin used in the separation process of yin and yang, the elution and regeneration of acid and alkali consume too much, resulting in serious environmental pollution. In addition, glutamine is easily converted into glutamic acid in a strong alkali and strong acid environment, resulting in a very low yield; The coarse crystallization process consumes more energy.

Contents of the invention

In order to solve the problems existing in the existing ion exchange method, the object of the present invention is to provide a process for extracting glutamine from fermentation broth with high yield and purity, low product cost, low energy consumption and environmental protection.

The present invention proposes a process for extracting glutamine from fermented liquid, which is characterized in that: the process is to pretreat the glutamine fermented liquid, remove bacteria and some proteins, pigments and polysaccharides, control After the pretreatment, the pH value of the fermentation broth is removed by electrodialysis equipment, the pH value of the feed liquid is readjusted, and it is passed through the treated OH-type anion exchange column, the impurities are exchanged with the resin and adsorbed, and the glutamine flow It is separated by passing through an ion exchange column, the effluent is first decolorized, concentrated in vacuum, and then isoelectric crystallized, and the crude crystal is washed with an organic solvent and dried. The process steps are:

(1) Flocculation and ultrafiltration coupling pretreatment fermentation broth: adjust the glutamine fermentation broth to pH 3.0-7.0, add polymer flocculant to the fermentation broth under slow stirring, the final mass concentration is 500-1500ppm, and then stir rapidly , remove the solid phase by filtration, wash the solid phase with deionized water, collect the supernatant and washing water, and perform ultrafiltration after merging. The molecular weight cut-off of the ultrafiltration membrane is 5000-50000, and the permeate is standby;

(2) Electrodialysis desalination: adjust the pH of the permeate to 4-7, enter the electrodialysis device for desalination, control the operating voltage and current, when the current density drops to 10-30mA/ ^cm2 , stop the electrodialysis, and collect the feed liquid for later use;

(3) The processing of ion exchange resin: the basic ion exchange resin is processed by conventional methods and packed into columns, then converted into OH type for subsequent use;

(4) Ion exchange to remove impurities: adjust the pH of the feed liquid obtained in step (2) to 4.0-6.0, and pass it into the ion exchange column processed in step (3), monitor the pH value of the effluent, and determine the start of the feed liquid and the moment of termination of collection, to ensure that glutamic acid and other impurities do not penetrate the resin;

(5) Feed liquid decolorization: add gac to above-mentioned effluent, stir, filter;

(6) Vacuum concentration of feed liquid: vacuum concentration of feed liquid after decolorization;

(7) crystallization of concentrated solution: adjust the pH value of the concentrated solution after decolorization, cool down and crystallize, filter, and collect crystals;

(8) The primary crystallization mother liquor is first concentrated according to step (6), then crystallized according to step (7), and the crystals are combined with the crystals collected in step (7);

(9) Washing of glutamine crystals: add an organic solvent to the crude crystals in a ratio of 1-3 times, stir evenly, filter, and collect the crystals;

(10) Drying of glutamine crystals: drying the crystals obtained in step (9) and cooling to obtain the finished product of glutamine.

In the above process method, the fermented liquid is a glutamine fermented liquid, except for bacteria, miscellaneous protein and residual sugar, the content of glutamine must be higher than 30g/l, and the content of glutamic acid must be lower than 10g/l. 1, ammonium sulfate and ammonium chloride total amount 60g/l, also have other salts of a small amount of phosphate; The pH value of the fermented liquid described in step (1) is with mineral acid such as hydrochloric acid, sulfuric acid and phosphoric acid, organic acid such as acetic acid , any one of acetic acid and lactic acid is regulated; The macromolecule flocculant described in step (1) is any one in anionic, cationic, nonionic polyacrylamide and chitosan; Step (2) The acid used to regulate feed liquid pH is inorganic acid such as hydrochloric acid, sulfuric acid and phosphoric acid, organic acid such as any one in acetic acid, acetic acid and lactic acid, and used alkali is any one in sodium hydroxide, ammoniacal liquor, sodium carbonate; (3) used anion exchange resin is any one in D296, D261, D290, 201 * 4, 201 * 7, D301, D380, D314, D392; The organic solvent described in step (9) is dehydrated alcohol, Any one of 95% ethanol and methanol, acetone and butanone.

Because the present invention adopts the technology that electrodialysis desalination and impurity such as glutamic acid of single root anion exchange column are coupled to extract glutamine in the fermented liquid, can obviously reduce the consumption of exchange resin, has avoided glutamine effectively in strong alkali and The conversion problem in the strong acid environment greatly reduces the production cost, the total extraction yield of glutamine reaches about 70%, and the product quality conforms to the Japanese Pharmacopoeia. It also makes it possible to produce glutamine industrially by fermentation.

Detailed ways

In the invention, the glutamine fermented liquid is firstly pretreated to remove almost all bacteria and some proteins, pigments and polysaccharides. The pH value of the fermented liquid after pretreatment is controlled within a certain range, and the inorganic salts, mainly ammonium sulfate, ammonium chloride and phosphate substances are removed by electrodialysis equipment. Then the feed liquid readjusts the pH value, and passes through the treated OH-type anion exchange column at an appropriate flow rate. Impurities such as glutamic acid are adsorbed due to exchange with the resin, and most glutamine flows through the ion exchange column. , thus being separated. The effluent was first decolorized, concentrated in vacuo, and then isoelectric crystallized. The crude crystals are washed with an organic solvent and dried to obtain the finished product.

The first key point of the process method is the pretreatment of the fermentation broth. Through appropriate pretreatment methods, including flocculation, ultrafiltration or traditional centrifugation, all the bacteria and some foreign proteins and polysaccharides are removed to reduce the fermentation broth. The high viscosity not only fundamentally avoids the clogging and pollution of the membrane during the electrodialysis operation, but also facilitates the recrystallization of the mother liquor once, and is easy to improve the yield of the whole process.

The second key point of the process is the control of the pH of the feed solution during electrodialysis. By adding appropriate acid and alkali and controlling the operation time of electrodialysis, most of the inorganic salts can be removed in this process, and the loss of product glutamine is small at the same time.

The third key point of the process is the selection of the ion exchange resin and the adjustment of the pH of the feed solution during ion exchange. By selecting an appropriate resin and ensuring that the pH of the feed solution is within a certain range, all impurities such as glutamic acid can be removed, and most of the glutamine can be separated through the ion exchange column. Its process steps are:

1. Coagulation and ultrafiltration coupling pretreatment of fermentation broth: adjust the glutamine fermentation broth to pH 3.0-6.0, temperature 25°C, add polymer flocculant to the fermentation broth under slow stirring, the final mass concentration is 500-1500ppm, Then stir rapidly for 10-30 minutes, stand still for 30-60 minutes, filter to remove the solid phase, wash the solid phase with 2-4 times deionized water, collect the supernatant and washing water, combine and ultrafilter. The molecular weight cut-off of the ultrafiltration membrane is 5000-50000. The permeate is ready for use.

2. Electrodialysis desalination: the permeate is adjusted to pH 4-7, and enters the electrodialysis device for desalination. The electrodialysis equipment consists of multiple pairs of positive membranes, negative membranes and/or bipolar membranes connected in parallel or in series. The plates are made of stainless steel or titanium. The operating voltage and current are controlled to maintain the temperature of the feed solution at 10-40°C. When the current density drops to 10-30mA/cm ² , stop the electrodialysis and collect the feed solution for later use.

3. Treatment of ion-exchange resin: the basic ion-exchange resin is treated according to the conventional method and packed into a column, and then converted into OH type for later use.

4. Ion exchange to remove glutamic acid and other impurities: adjust the pH of the feed liquid obtained in step (2) to 4.0-6.0, and pass it into the ion exchange column treated in step (3) with a flow rate of 1.0-4.0BV/hour. Monitor the pH of the effluent to determine when feed begins and ends to collect to ensure that glutamic acid and other impurities do not penetrate the resin.

5. Feed liquid decolorization: Gradually add granular or powdered pharmaceutical grade activated carbon in the proportion of 0.5-2% to the above effluent, stir at 30°C for 20-60 minutes, and filter.

6. Vacuum concentration of the feed liquid: vacuum concentrate the decolorized feed liquid at 40-70°C until the glutamine content is 60-120g/l.

7. Crystallization of the concentrated solution: adjust the decolorized concentrated solution to pH 4-6, and then lower the temperature at a rate of 2-4°C/hour. Every time the temperature drops by about 10°C, keep warm for 1-4 hours to grow crystals, and then continue to cool down at the same rate until the temperature of the feed liquid drops to 3-5°C, maintain at this temperature for 8-12 hours to grow crystals, filter, and collect crystals.

8. The primary crystallization mother liquor is first concentrated according to step (6), then crystallized according to step (7), and the crystals are combined with the crystals collected in step (7).

9. Glutamine crystal washing: add an organic solvent to the crude crystal at a ratio of 1-3 times, stir at 30-50°C for 1-2 hours, and collect the crystal by filtration.

10. Drying of glutamine crystals: drying the crystals obtained in step (9) at 40-50°C for 3-6 hours, then at 80°C for 1-2 hours, and cooling to obtain the finished product of glutamine.

The fermented liquid is a glutamine fermented liquid, except bacteria, miscellaneous protein and residual sugar, the content of glutamine must be higher than 30g/l, the content of glutamic acid must be lower than 10g/l, ammonium sulfate and chlorine The total amount of ammonium chloride is about 60g/l, and there are also a small amount of other salts such as phosphate.

The pH value of the fermentation broth described in step 1 is adjusted with one of inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and organic acids such as acetic acid, acetic acid and lactic acid.

The polymer flocculant described in step 1 is one of anionic, cationic, nonionic polyacrylamide and chitosan.

The ultrafiltration device described in step 1 is one of tubular membrane, flat membrane, roll membrane and hollow fiber membrane.

The cathodic and cathodic membranes used in the electrodialysis equipment described in step 2 can be connected in parallel or in series.

The acid used in step 2 to adjust the pH of the feed solution is one of inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and one of organic acids such as acetic acid, acetic acid and lactic acid, and the used alkali is one of sodium hydroxide, ammonia water and sodium carbonate.

The anion exchange resin used in step 3 is one of D296, D261, D290, 201×4, 201×7, D301, D380, D314, D392.

The organic solvent described in step 9 is one of alcohols such as absolute ethanol, 95% ethanol and methanol or ketones such as acetone and butanone.

Below in conjunction with example the present invention will be further elaborated.

Example 1:

The main components of glutamine fermentation broth are: glutamine 35g/l, glutamic acid 8g/l, ammonium sulfate 50g/l, ammonium chloride 10g/l, a small amount of other inorganic salts, pH 6.7, fermentation broth volume 5L .

5g of cationic polyacrylamide was used as a flocculant, added into the fermentation broth under slow stirring, then stirred rapidly for 10 minutes, left to stand for 30 minutes, and filtered. Wash the solid phase with 500 ml deionized water. The supernatant and the washing water are combined and then ultrafiltered through a tubular membrane module. The molecular weight cut off of the ultrafiltration membrane is 6000. Adjust the pH of the fermented liquid to 6 with 2M sulfuric acid, and perform electrodialysis. The electrodialyzer consists of two positive membranes and one negative membrane. During this process, use 2M sulfuric acid and 2M sodium hydroxide to control the pH of the fermentation broth to stabilize at about 6, and control the temperature to about 30°C. When the current decreased to 20mA/cm ² , the electrodialysis was stopped. Then adjust the pH to 5.0 with the above-mentioned acid and alkali, and pass it into the treated D380 anion exchange column with a flow rate of 3BV/hour, and the column bed volume is 1 liter. Monitor the pH value of the effluent not lower than 5 to ensure that impurities such as glutamic acid do not penetrate the resin. Collect the effluent, add powdered activated carbon 30g, and filter twice after stirring slowly for 20 minutes. The decolorized solution was concentrated in vacuo to 1/5 of the original volume. Use hydrochloric acid to adjust the pH value of the concentrated solution to 5.6, lower the temperature at a rate of 2°C/hour, keep warm for 3 hours every time the temperature drops by 10°C, and keep warm for 10 hours to crystallize after dropping to 5°C. Filter to collect crystals. The mother liquor was re-concentrated and crystallized under the above conditions, and the secondary crystals were combined with the primary crystals. Add absolute ethanol to the crystals in an amount of 1ml/g, stir at 35°C for 1 hour, then filter, dry the crystals at 50°C for 5 hours, dry at 80°C for 2 hours, and cool to obtain the finished product of glutamine. The yield of glutamine in this process is 68.5%, and the purity is 98.8%.

Example 2:

The main components of glutamine fermentation broth are: glutamine 32g/l, glutamic acid 7g/l, ammonium sulfate 50g/l, ammonium chloride 6g/l, a small amount of other inorganic salts, pH 6.6, volume of fermentation broth 5L .

6g of chitosan was used as flocculant, added into the fermentation broth under slow stirring, then stirred rapidly for 15 minutes, left to stand for 30 minutes, and filtered. Wash the solid phase with 500 ml deionized water. The supernatant and the washing water are combined and ultrafiltered through a flat-panel membrane ultrafiltration device, and the molecular weight cut-off of the ultrafiltration membrane is 10,000. Use 2M hydrochloric acid to adjust the pH of the fermentation broth to 5, and perform electrodialysis. The electrodialyzer consists of two positive membranes and one negative membrane. During this process, use 2M hydrochloric acid and 20% ammonia water to control the pH of the fermentation broth to be stable at about 5, and control the temperature to about 35°C. When the current decreased to 25 mA/cm ² , the electrodialysis was stopped. Then adjust the pH to 4.5 with the above-mentioned acid and alkali, pass it into the treated 201×7 anion exchange column with a flow rate of 1.5BV/hour, and the column bed volume is 1 liter. Monitor the pH value of the effluent not lower than 5 to ensure that impurities such as glutamic acid do not penetrate the resin. Collect the effluent, add granular activated carbon 25g, and filter twice after stirring slowly for 30 minutes. The decolorized solution was concentrated in vacuo to 1/4 of the original volume. Use hydrochloric acid to adjust the pH value of the concentrated solution to 5.6, lower the temperature at a rate of 3°C/hour, keep the temperature for 4 hours every time the temperature is lowered by 10°C, and keep it for 10 hours to crystallize after dropping to 5°C. Filter to collect crystals. The mother liquor was re-concentrated and crystallized under the above conditions, and the secondary crystals were combined with the primary crystals. Then add methanol in an amount of 1ml/g to the combined crystals, stir at 30°C for 1.5 hours, filter, dry the crystals at 45°C for 3 hours, dry at 80°C for 2 hours, and obtain the glutamine product after cooling. The yield of glutamine in this process is 67.6%, and the purity is 99.3%.

Example 3:

The main components of glutamine fermentation broth are: glutamine 38g/l, glutamic acid 10g/l, ammonium sulfate 40g/l, ammonium chloride 20g/l, a small amount of other inorganic salts, pH6.7, fermentation broth volume 5L .

7g of non-ionic polyacrylamide was used as a flocculant, added to the fermentation broth under slow stirring, then stirred rapidly for 15 minutes, left to stand for 30 minutes, and filtered. Wash the solid phase with 500 ml deionized water. The supernatant and the washing water are combined and ultrafiltered through a hollow fiber membrane device, and the molecular weight cut-off of the ultrafiltration membrane is 30,000. Use 2M nitric acid to adjust the pH of the fermentation broth to 6.5, and perform electrodialysis. The electrodialyzer consists of two positive membranes and one negative membrane. In this process, 2M sulfuric acid and 2M sodium hydroxide are used to control the pH of the fermentation broth to be stable at about 6.5, and the temperature is controlled to be about 25°C. When the current decreased to 15mA/cm ² , the electrodialysis was stopped. Then adjust the pH to 5.5 with the above-mentioned acid and alkali, and pass it into the treated D330 anion exchange column with a flow rate of 2BV/hour, and the column bed volume is 1 liter. Monitor the pH value of the effluent to not be lower than 5.5 to ensure that impurities such as glutamic acid do not penetrate the resin. Collect the effluent, add powdery activated carbon 28g, and filter twice after stirring slowly for 20 minutes. The decolorized solution was concentrated in vacuo to 1/4 of the original volume. Use hydrochloric acid to adjust the pH value of the concentrated solution to 5.6, lower the temperature at a rate of 3°C/hour, keep warm for 3 hours every time the temperature drops by 10°C, and keep warm for 12 hours to crystallize after dropping to 4°C. Filter to collect crystals. The mother liquor was re-concentrated and crystallized under the above conditions, and the secondary crystals were combined with the primary crystals. Add 95% ethanol to the crude crystals in an amount of 1ml/g, stir at 30°C for 1 hour, then filter, dry the crystals at 55°C for 4 hours, and at 80°C for 2 hours, and get the glutamine product after cooling. The yield of glutamine in this process is 70.8%, and the purity is 99.0%.

Claims (7)

1, a kind of processing method of from fermented liquid, extracting glutamine, it is characterized in that: described processing method is earlier glutamine ferment liquid to be carried out pre-treatment, remove thalline and partial protein, pigment and polysaccharide, the pH value of control pretreatment secondary fermentation liquid, by electrodialysis appts removal inorganic salt wherein, readjust material liquid pH value, and feed the OH type anion-exchange column handle well, exchange takes place and is adsorbed in impurity and resin, and glutamine flows through ion exchange column and obtains separating, and effluent liquid is decolouring earlier, vacuum concentration, isoelectric point crystallization then, coarse crystal is by organic solvent washing, and drying gets final product, and its processing step is:

(1) flocculation and ultrafiltration coupling pre-treatment fermented liquid: glutamine ferment liquid is adjusted to pH3.0-7.0, slowly stir down and add polymeric flocculant to fermented liquid, final quality concentration is 500-1500ppm, stir fast then, remove by filter solid phase, use the deionized water wash solid phase, collect supernatant liquor and washing water, merge the back ultrafiltration, the molecular weight cut-off of ultra-filtration membrane is 5000-50000, and it is standby to see through liquid;

(2) electrodialytic desalting: see through liquid and regulate pH4-7, enter the electrodialysis unit desalination, the red-tape operati voltage and current is when current density drops to 10-30mA/cm ²The time, stopping electrodialysis, the collection feed liquid is standby;

(3) TREATMENT OF ION EXCHANGE RESINS: deacidite is handled back dress post according to a conventional method, changes into behind the OH type standby then;

(4) impurity is removed in ion-exchange: with the feed adjustment pH4.0-6.0 of step (2) gained, and feed the ion exchange column that step (3) is handled well, the pH value of monitoring stream fluid is determined the moment that feed liquid begins and stops collecting, and guarantees that L-glutamic acid and other impurity do not penetrate resin;

(5) feed liquid decolouring: in above-mentioned effluent liquid, add gac, stir, filter;

(6) vacuum concentration of feed liquid: the feed liquid vacuum concentration after will decolouring;

(7) concentrated solution crystallization: the concentrated solution pH value after the adjusting decolouring is to 4-6, and decrease temperature crystalline filters, and collects crystal;

(8) the primary crystallization mother liquor concentrates according to step (6) earlier, carries out crystallization according to step (7) then, and the crystal of crystal and step (7) collection is merged;

(9) glutamine crystal washing: according to quality is that 1-3 ratio doubly adds organic solvent in coarse crystal, and the after-filtration that stirs is collected crystal;

(10) glutamine crystal drying:, promptly get the glutamine finished product after the cooling with step (9) gained crystal drying.

2, according to the described a kind of processing method of from fermented liquid, extracting glutamine of claim 1, it is characterized in that: remove outside thalline, foreign protein and the residual sugar, the content of glutamine must be higher than 30g/l, L-glutamic acid content must be lower than 10g/l, ammonium sulfate and ammonium chloride total amount 60g/l also have the carbamate additives for low phosphorus hydrochlorate.

3, according to the described a kind of processing method of extracting glutamine from fermented liquid of claim 1, it is characterized in that: the pH value of the described fermented liquid of step (1) is with hydrochloric acid, sulfuric acid and phosphoric acid, any adjusting in acetate, acetic acid and the lactic acid.

4, according to the described a kind of processing method of extracting glutamine from fermented liquid of claim 1, it is characterized in that: the described polymeric flocculant of step (1) is any in anionic, cationic, non-ionic polyacrylamide and the chitosan.

5, according to the described a kind of processing method of from fermented liquid, extracting glutamine of claim 1, it is characterized in that: it is hydrochloric acid, sulfuric acid and phosphoric acid that step (2) is regulated the used acid of material liquid pH, any in acetate, acetic acid and the lactic acid, used alkali are any in sodium hydroxide, ammoniacal liquor, the yellow soda ash.

6, according to the described a kind of processing method of extracting glutamine from fermented liquid of claim 1, it is characterized in that: the used anionite-exchange resin of step (3) is any among D296, D261, D290,201 * 4,201 * 7, D301, D380, D314, the D392.

7, according to the described a kind of processing method of extracting glutamine from fermented liquid of claim 1, it is characterized in that: the described organic solvent of step (9) is a dehydrated alcohol, 95% ethanol, methyl alcohol, acetone, any in the butanone.