CN117736122A - Separation and purification method of arginine fermentation liquor - Google Patents

Abstract

The invention provides a separation and purification method of arginine fermentation broth, which comprises the following steps: adding calcium oxide into the arginine fermentation broth for reaction, and filtering to obtain arginine clear liquid; and (3) adsorbing and desalting the arginine clear liquid by using ion exchange resin to finish purification. The separation and purification method is simple to operate, reduces the use amount of the ion exchange resin, further reduces the generation of resin wastewater, and has high purity and high yield of the obtained product; and ammonia in the purification process can be absorbed by sulfuric acid and converted into ammonium sulfate, and the recovered ammonium sulfate can be used as a culture medium component for fermentation culture.

Description

Separation and purification method of arginine fermentation liquor

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a separation and purification method of arginine fermentation liquor.

Background

Arginine (arginine) chemical name: 2-amino-5-guanidino-pentanoic acid, which is an aliphatic basic polar alpha amino acid containing a guanidino group, is positively charged under physiological conditions. The natural products are largely present in protamine, and are also the basic components of various proteins.

Arginine plays an important role in cell division, ammonia excretion in the body, regulation of the immune system by affecting hormone synthesis and release, cardiovascular and sexual function by NO, and the like. Arginine is a direct precursor of nitric oxide, urea, ornithine and albumine, is an important prime for the synthesis of inosinic acid, and is used for the synthesis of polyamine, citrulline and glutamine. Arginine, as a precursor of nitric oxide, can directly relax blood vessels, preventing ventricular and vascular hypertrophy. Arginine also antagonizes the vasoconstrictor effects of endogenous hormones, such as Asymmetric Dimethylarginine (ADMA), and metabolites. Thus, arginine is widely used in foods, medicines, cosmetics, and feeds.

The existing arginine production method mainly comprises a chemical synthesis method, an enzyme method, a protein hydrolysis method and a fermentation method, the former three production methods have the problems of complex process, high cost, diversity of substrate selection, low yield, environmental pollution and the like, and the fermentation method for producing L-arginine has the advantages of simple process, high yield, environmental protection and the like, so the research on the production of L-arginine by the fermentation method is mainly focused at home and abroad.

The L-arginine is produced by a fermentation method, ammonium sulfate is added in the fermentation process to adjust the pH of the fermentation and provide a nitrogen source, and the concentration of the added ammonium sulfate can reach 25-30g/L; therefore, in addition to the high concentration of L-arginine, the fermentation broth also contains more complex impurity components such as microbial metabolites, inorganic salts such as ammonium sulfate and the like, organic pigments and the like; therefore, it is necessary to purify the fermentation broth to obtain a purified L-arginine solution having a high concentration and a high purity.

In the prior art, the L-arginine fermentation broth is generally acidified, then is adsorbed by using strong acid cation exchange resin, and is eluted by ammonia water for purification, as disclosed in Chinese patent CN114436899A, a large amount of ammonia water and inorganic acid are needed in the purification process, the resin usage amount is large, a large amount of wastewater is generated, and the purification cost of L-arginine is high.

Therefore, the development of the extraction process of the L-arginine fermentation liquor with more environmental protection, low cost and reliable product quality has a certain significance for the wide application of fermentation production of L-arginine.

Disclosure of Invention

The invention mainly aims to provide a separation and purification method of arginine fermentation liquor, which aims to solve the problems of large use amount of ion exchange resin and large resin wastewater amount in the process of purifying the arginine fermentation liquor in the prior art.

In order to achieve the above object, according to a first aspect of the present invention, there is provided a separation and purification method of arginine fermentation broth, comprising: adding calcium oxide into the arginine fermentation broth for reaction, and filtering to obtain arginine clear liquid; and (3) adsorbing and desalting the arginine clear liquid by using ion exchange resin to finish purification.

Further, the concentration of arginine in the arginine fermentation broth is 60-100g/L; preferably, the pH of the arginine fermentation broth is between 6.8 and 7.2.

Further, the separation and purification method further comprises, before adding the calcium oxide: filtering the arginine fermentation broth; preferably, the filtering comprises: filtering arginine fermentation liquor sequentially through a ceramic membrane and an ultrafiltration membrane; preferably, the ultrafiltration membrane has a molecular weight cut-off of 500 to 2000D, more preferably 800 to 1000D.

Further, after calcium oxide is added, the pH value of the system is controlled to be 10.5-11.0; preferably, the temperature of the reaction is 65-75 ℃; preferably, the reaction time is 1-2 hours.

Further, performing adsorption desalination includes: sequentially treating arginine clear liquid with calcium cation exchange resin and anion exchange resin to obtain arginine purified liquid.

Further, the calcium type cation exchange resin comprises LSC-100, LSC-500 or LSC-501; preferably, the anion exchange resin comprises D151, D152, D113 or 110, preferably D113.

Further, after the arginine purified solution is obtained, the separation and purification method further comprises the steps of: and (3) sequentially decoloring, concentrating, cooling, crystallizing and centrifuging the arginine purified solution to obtain an arginine product.

Further, decolorizing the arginine purified solution by using activated carbon; preferably, the temperature of decolorization is 30-65deg.C, preferably 50-60deg.C; preferably, the decolorizing time is 30-60min.

Further, the concentration of arginine in the concentrated arginine purified solution is 300-600g/L.

Further, the temperature of the cooling crystallization is 5-15 ℃; preferably, stirring is performed after cooling for 2-4 hours.

By applying the technical scheme of the invention, calcium oxide is combined with a large amount of sulfate radicals in the fermentation liquor to form calcium sulfate precipitate, ammonia gas in inorganic salt ammonium sulfate in the arginine fermentation liquor is released, and inorganic salt impurities are further removed through ion exchange resin adsorption, so that the arginine purified liquor is obtained. The separation and purification method is simple to operate, reduces the use amount of the ion exchange resin, further reduces the generation of resin wastewater, and has high purity and high yield of the obtained product; and ammonia in the purification process can be absorbed by sulfuric acid and converted into ammonium sulfate, and the recovered ammonium sulfate can be used as a culture medium component for fermentation culture.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.

As mentioned in the background art, the purification of arginine in arginine fermentation liquor by using ion exchange resin in the prior art requires a large amount of ammonia water and hydrochloric acid, and the large amount of resin is used, so that a large amount of resin wastewater harmful to the environment is generated, and the purification and emission of the resin wastewater are required, and the method has certain defects in terms of production cost and environmental protection. Therefore, the application aims to protect a separation and purification method capable of obtaining an arginine product with higher purity and more environment-friendly.

In a first exemplary embodiment of the present application, there is provided a method for separating and purifying an arginine fermentation broth, the method comprising: adding calcium oxide into the arginine fermentation broth for reaction, and filtering to obtain arginine clear liquid; and (3) adsorbing and desalting the arginine clear liquid by using ion exchange resin to finish purification.

The calcium oxide is combined with a large amount of sulfate radical in the arginine fermentation broth to form calcium sulfate precipitate, calcium hydroxide is generated by the reaction of the calcium oxide and water, ammonia is released by combining with ammonium ions, and the excessive ammonia can be absorbed by sulfuric acid to finish the preliminary purification of the arginine fermentation broth, so that impurities are further removed by resin absorption, and the purified arginine product is obtained. The separation and purification method is simple in operation, high in product purity, and on the basis of meeting the quality requirement, the cost for treating the production waste is saved, and the separation and purification method has economic value and is environment-friendly.

Any arginine fermentation broth that can be purified by the methods of the present application is suitable for use in the present application, and in a preferred embodiment, the arginine concentration in the arginine fermentation broth is from 60 to 100g/L, depending on the actual application requirements; preferably, the pH of the arginine fermentation broth is between 6.8 and 7.2.

In order to improve the purification effect, the arginine fermentation broth may be filtered to remove particles or impurities having a relatively large molecular weight, such as microbial cell debris, microbial metabolites, and the like, before the calcium oxide is added. In a preferred embodiment, the separation and purification method comprises, prior to the addition of calcium oxide: filtering the arginine fermentation broth; preferably, the filtering comprises: filtering arginine fermentation liquor sequentially through a ceramic membrane and an ultrafiltration membrane; preferably, the ultrafiltration membrane has a molecular weight cut-off of 500 to 2000D, more preferably 800 to 1000D.

To further remove the inorganic salt ammonium sulfate impurity relatively more thoroughly from the arginine broth, in a preferred embodiment, the pH after the addition of calcium oxide is 10.5-11.0; preferably, the temperature of the reaction is 65-75 ℃; preferably, the reaction time is 1-2 hours.

To further remove inorganic ionic impurities from the solution of ammonium sulfate in arginine fermentation broth, in a preferred embodiment, the adsorption desalting comprises: sequentially treating arginine clear liquid with calcium cation exchange resin and anion exchange resin to obtain arginine purified liquid. Wherein the calcium cation exchange resin is a resin with selective adsorption of cations, and is more suitable for adsorption and removal of calcium ions, and the anion exchange resin is a resin with selective adsorption of anions. As the ammonium sulfate in the fermentation liquor is primarily removed by precipitation, the inorganic salt components which are required to be removed by the ion exchange resin are greatly reduced, so that the impurity removal effect can be achieved by reducing the using amount of the subsequent ion exchange resin.

To further remove calcium impurities from the arginine serum, in a preferred embodiment, the calcium cation exchange resin comprises LSC-100, LSC-500, or LSC-501; preferably, the arginine serum feed flow rate is 5-15bv/h.

To further remove anions from the arginine serum, in a preferred embodiment, the anion exchange resin comprises D151, D152, D113 or 110, preferably D113.

After obtaining the purified arginine solution, to further meet the application requirements, the purified arginine solution may be prepared into arginine solid, and in a preferred embodiment, the separation and purification method comprises: and (3) sequentially decoloring, concentrating, cooling, crystallizing and centrifuging the arginine purified solution to obtain an arginine product.

To further remove organic pigments from the arginine purification solution, in a preferred embodiment, the arginine purification solution is decolorized with activated carbon; preferably, the temperature of decolorization is 30-65deg.C, preferably 50-60deg.C; preferably, the decolorizing time is 30-60min.

The arginine purified solution may be concentrated to a suitable concentration as desired, and in a preferred embodiment, the concentration of arginine in the concentrated arginine purified solution is 300-600g/L. To obtain higher yields of arginine, in a preferred embodiment, the temperature of the reduced crystallization is 5-15 ℃; preferably, stirring is performed after cooling for 2-4 hours.

The present application is described in further detail below in conjunction with specific embodiments, which should not be construed as limiting the scope of the claims.

Example 1:

taking L-arginine fermentation liquor, wherein the concentration of arginine in the initial fermentation liquor is 100g/L, the pH value is 6.8, and the fermentation liquor is subjected to ceramic membrane to remove thalli and other insoluble impurities with larger particles; the clear solution of the ceramic membrane is filtered by an ultrafiltration membrane with 1000D of molecular weight cut-off, the membrane filtration pressure is 1.5Mpa, and the membrane filtration temperature is controlled below 50 ℃.

Collecting 20L of obtained ultrafiltration membrane clear liquid, adding calcium oxide into the obtained ultrafiltration membrane clear liquid, stirring, maintaining the pH of the system at 10.5 and the temperature at 70 ℃ for 1h, and filtering the obtained feed liquid to remove calcium sulfate precipitate;

the obtained arginine clear solution is adsorbed and filtered by 0.5L of LSC-100 cationic resin (Cean blue dawn technology New material Co., ltd.) at a flow rate of 5BV/h, and the obtained feed solution is further passed by 0.5L of D113 anionic resin (clam port three-resin technology Co., ltd.) at a flow rate of 5BV/h to obtain arginine purified solution; adding active carbon accounting for 1% of the volume of the arginine purified solution into the arginine purified solution, decoloring for 30min at 50 ℃, and filtering to remove the active carbon; further concentrating the arginine purified solution under reduced pressure to obtain arginine concentrated solution with arginine concentration of 500 g/L.

In order to further obtain solid arginine product, the arginine concentrate is cooled to 10 ℃, the temperature is kept, stirring and crystallization are carried out for 2 hours, the crystallized product is centrifuged by a three-leg centrifuge, the L-arginine wet product is obtained, and the L-arginine wet product is dried in a vacuum drying oven (temperature 60 ℃).

The quality of the obtained L-arginine is 1300.3g, the total yield is 65.0%, the L-arginine content is 99.51%, and the water content is less than 0.5%; the crystallization mother liquor can be recrystallized, and the overall yield is more than 90%.

Example 2:

taking L-arginine fermentation liquor, wherein the concentration of arginine in the initial fermentation liquor is 100g/L, the pH value is 6.8, and the L-arginine fermentation liquor is subjected to ceramic membrane to remove a large amount of thalli and other insoluble impurities with larger particles; passing the ceramic membrane clear liquid through an ultrafiltration membrane with molecular weight of 800D, wherein the membrane passing pressure is 1.8Mpa, and the membrane passing temperature is controlled below 50 ℃.

Collecting 20L of obtained ultrafiltration membrane clear liquid, adding calcium oxide into the obtained ultrafiltration membrane clear liquid, stirring, adjusting the pH to 11.0, maintaining the temperature at 65 ℃ for 1.5h, and filtering the obtained feed liquid to remove calcium sulfate precipitate;

the obtained arginine clear solution is adsorbed and filtered by 0.5L of LSC-100 cationic resin (Cean blue dawn scientific and technological new materials Co., ltd.) at the flow rate of 10BV/h, and the obtained feed solution is further passed through 0.5L of D113 anionic resin at the flow rate of 10 BV/h; adding active carbon accounting for 1% of the volume of the arginine purification liquid into the arginine purification liquid, decoloring for 60min at 30 ℃, and filtering to remove the active carbon; further concentrating the arginine purified solution under reduced pressure to obtain arginine concentrated solution with arginine concentration of 600g/L.

In order to further obtain solid arginine product, the arginine concentrate is cooled to 5 ℃, the temperature is kept, stirring and crystallization are carried out for 3 hours, the crystallized product is centrifuged by a three-leg centrifuge, the L-arginine wet product is obtained, and the L-arginine wet product is dried in a vacuum drying oven (temperature 65 ℃).

The quality of the obtained L-arginine is 1359.8g, the total yield is 68.0%, the L-arginine content is 99.41%, and the water content is less than 0.5%; the crystallization mother liquor can be recrystallized, and the overall yield is more than 90%.

Example 3:

taking L-arginine fermentation liquor, wherein the concentration of arginine in the initial fermentation liquor is 88g/L, the pH value is 7.2, and the L-arginine fermentation liquor is subjected to ceramic membrane to remove a large amount of thalli and other insoluble impurities with larger particles; passing the ceramic membrane clear liquid through an ultrafiltration membrane with a molecular weight of 2000D, wherein the membrane passing pressure is 2.0Mpa, and the membrane passing temperature is controlled below 50 ℃.

Collecting 20L of obtained ultrafiltration membrane clear liquid, adding calcium oxide into the obtained ultrafiltration membrane clear liquid, stirring, adjusting the pH to 10.5, maintaining the temperature at 75 ℃ for 2 hours, and filtering the obtained feed liquid to remove calcium sulfate precipitate;

the obtained arginine clear solution is adsorbed and filtered by 0.5L of LSC-501 cationic resin (Xiyan Xiao technology New Material Co., ltd.) at a flow rate of 5BV/h, and the obtained feed solution is further passed through 0.5L of D113 anionic resin at a flow rate of 5 BV/h; adding active carbon accounting for 2% of the volume of the arginine purification liquid into the arginine purification liquid, decoloring for 40min at 60 ℃, and filtering to remove the active carbon; further concentrating the arginine purified solution under reduced pressure to obtain arginine concentrated solution with arginine concentration of 400 g/L.

In order to further obtain solid arginine product, the arginine concentrate is cooled to 5 ℃, the temperature is kept, stirring and crystallization are carried out for 4 hours, the crystallized product is centrifuged by a three-leg centrifuge, the L-arginine wet product is obtained, and the L-arginine wet product is dried in a vacuum drying oven (temperature 65 ℃).

The quality of the obtained L-arginine is 1179.2g, the total yield is 67.0%, the L-arginine content is 99.38%, and the water content is less than 0.50%; the crystallization mother liquor can be recrystallized, and the overall yield is more than 90%.

Example 4:

taking L-arginine fermentation liquor, wherein the concentration of arginine in the initial fermentation liquor is 70g/L, the pH value is 7.0, and the L-arginine fermentation liquor is subjected to ceramic membrane to remove a large amount of thalli and other insoluble impurities with larger particles; passing the ceramic membrane clear liquid through an ultrafiltration membrane with molecular weight of 500D, wherein the membrane passing pressure is 1.5Mpa, and the membrane passing temperature is controlled below 50 ℃.

Collecting 20L of obtained ultrafiltration membrane clear liquid, adding calcium oxide into the obtained ultrafiltration membrane clear liquid, stirring, adjusting the pH to 10.5, maintaining the temperature at 70 ℃ for 2 hours, and filtering the obtained feed liquid to remove calcium sulfate precipitate;

the obtained arginine clear solution is adsorbed and filtered by 0.5L of LSC-500 cationic resin (Cean blue dawn scientific and technological new materials Co., ltd.) at a flow rate of 5BV/h, and the obtained feed solution is further passed through 0.5L of D113 anionic resin at a flow rate of 5 BV/h; adding active carbon with the volume of 1.0% of that of the arginine purification liquid into the arginine purification liquid, decoloring for 40min at 55 ℃, and filtering to remove the active carbon; further concentrating the arginine purified solution under reduced pressure to obtain arginine concentrated solution with arginine concentration of 500 g/L.

In order to further obtain solid arginine product, the arginine concentrate is cooled to 5 ℃, the temperature is kept, stirring and crystallization are carried out for 3 hours, the crystallized product is centrifuged by a three-leg centrifuge, the L-arginine wet product is obtained, and the L-arginine wet product is dried in a vacuum drying oven (temperature 65 ℃).

The quality of the obtained L-arginine is 966.5g, the total yield is 69.0%, the L-arginine content is 99.43%, and the water content is less than 0.50%; the crystallization mother liquor can be recrystallized, and the overall yield is more than 90%.

Comparative example 1:

taking L-arginine fermentation liquor, wherein the concentration of arginine in the initial fermentation liquor is 100g/L, the pH value is 6.8, and the L-arginine fermentation liquor is subjected to ceramic membrane to remove a large amount of thalli and other insoluble impurities with larger particles; and (3) passing the ceramic membrane clear liquid through an ultrafiltration membrane with the molecular weight of 1000D, wherein the membrane passing pressure is 1.5Mpa, and the membrane passing temperature is controlled below 50 ℃.

Collecting 20L of obtained ultrafiltration membrane clear liquid, adsorbing the ultrafiltration membrane clear liquid by using LSC-010 cation (hydrogen type) exchange resin (Siemens Lan Xiao), detecting the outflow concentration of L-arginine in the resin volume of 10L, and when the outflow concentration of L-arginine is equal to or close to the concentration of the fed L-arginine, stopping adsorption, and adding water with the volume of 2 times of that of the column body for cleaning when the single ultrafiltration membrane clear liquid treatment capacity of 10L is achieved; preparing ammonia water with the concentration of 7 percent as analysis liquid, adding the ammonia water with the concentration of 7 percent into the cation resin which is completely washed, and collecting the analysis liquid, wherein the flow rate is 2.0 column volume/hour; preparing hydrochloric acid with concentration of 4% from the completely resolved cation resin (replacing the cation resin with hydrogen form from ammonium form), and regenerating the resin for later use for adsorbing L-arginine next time; adding active carbon accounting for 1% of the volume of the analysis liquid into the collected analysis liquid, decoloring at 50 ℃ for 30min, filtering to remove the active carbon, concentrating under reduced pressure to remove water and redundant ammonia, concentrating until the concentration of L-arginine is 600g/L, cooling to 10 ℃ for 3h, centrifuging the crystallized product by using a tripodia centrifuge to obtain an L-arginine wet product, and drying the L-arginine wet product in a vacuum drying oven (the temperature is 65 ℃).

The quality of the obtained L-arginine is 662.5g, the total yield is 66.25%, the L-arginine content is 99.48%, and the water content is 0.5%; the crystallization mother liquor can be recrystallized for reuse, and the overall yield can be maintained to be more than 90%.

Comparing the comparative example with the example, it can be seen that arginine purification by using the technical scheme of the application can obtain arginine products with better yield, content and moisture; from the aspect of environmental protection, a large amount of ammonia water and hydrochloric acid are needed in the use process of the comparative example 1, the amount of the used resin is large, and meanwhile, more waste water is generated by resin regeneration; the purification operation is simpler, the ammonium sulfate generated in the purification process can be used for fermentation again, and the calcium sulfate can be reused by the construction industry, so that the use amount of the ion exchange resin is greatly reduced, the economic cost of wastewater treatment is greatly reduced, and the technical scheme is more green and environment-friendly and the purification cost is lower.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the calcium oxide is combined with a large amount of sulfate radical in the arginine fermentation broth to form calcium sulfate precipitate, calcium hydroxide is generated by the reaction of the calcium oxide and water, ammonia is released by combining with ammonium ions, and the excessive ammonia is volatilized and then absorbed by sulfuric acid, so that the arginine fermentation broth is subjected to preliminary purification, and impurities are further removed by resin adsorption, so that the purified arginine product is obtained. The separation and purification method is simple in operation, high in product purity, and on the basis of meeting the quality requirement, the use amount of the ion exchange resin is greatly reduced, meanwhile, the discharge amount and the treatment cost of resin wastewater are reduced, no production waste is generated, the cost of treating the production waste is saved, and the method has economic value and is environment-friendly.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for separating and purifying arginine fermentation broth, which is characterized by comprising the following steps:

adding calcium oxide into the arginine fermentation broth for reaction, and filtering to obtain arginine clear liquid;

and (3) adsorbing and desalting the arginine clear liquid by using ion exchange resin to finish the purification.

2. The method according to claim 1, wherein the arginine concentration in the arginine fermentation broth is 60-100g/L;

preferably, the pH value of the arginine fermentation broth is 6.8-7.2.

3. The separation and purification method according to claim 1, wherein before the addition of calcium oxide, the separation and purification method further comprises:

filtering the arginine fermentation broth;

preferably, the filtering comprises: sequentially filtering the arginine fermentation broth through a ceramic membrane and an ultrafiltration membrane;

preferably, the ultrafiltration membrane has a molecular weight cut-off of 500-2000D, more preferably 800-1000D.

4. The method according to claim 1, wherein the pH of the system is controlled to be 10.5-11.0 after the calcium oxide is added;

preferably, the temperature of the reaction is 65-75 ℃;

preferably, the reaction time is 1-2 hours.

5. The separation and purification method according to claim 1, wherein the performing adsorption desalting comprises:

and sequentially treating the arginine clear solution by using a calcium type cation exchange resin and an anion exchange resin to obtain the arginine purified solution.

6. The separation and purification method according to claim 5, wherein the calcium cation exchange resin comprises LSC-100, LSC-500 or LSC-501;

preferably, the anion exchange resin comprises D151, D152, D113 or 110, preferably D113.

7. The method according to claim 5, wherein after obtaining the arginine purified solution, the method further comprises:

and (3) sequentially decoloring, concentrating, cooling, crystallizing and centrifuging the arginine purified solution to obtain an arginine product.

8. The method according to claim 7, wherein the arginine purified solution is decolorized with activated carbon;

preferably, the decolorization temperature is 30-65deg.C, preferably 50-60deg.C;

preferably, the decolorizing time is 30-60min.

9. The method according to claim 7, wherein the concentration of arginine in the arginine purified solution after concentration is 300 to 600g/L.

10. The separation and purification method according to claim 7, wherein the temperature of the reduced crystallization is 5-15 ℃;

preferably, stirring is performed after cooling, wherein the stirring time is 2-4h.