Extraction process of l-aspartic acid

Abstract

The application belongs to the technical field of organic chemistry, and particularly relates to an extraction process of L-aspartic acid. The extraction process of L-aspartic acid comprises five steps of hydrolysis extraction, decolorization purification, acidification separation, purification extraction and impurity removal. The application improves the resource utilization efficiency, increases the added value, reduces the production cost, improves the purity and yield of the product L-aspartic acid by improving the ion exchange extraction separation method, and improves the safety and market value of the product.

Description

Technical Field

[0001] This invention belongs to the field of organic chemistry technology, specifically relating to an extraction process for L-aspartic acid. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] L-Aspartic acid is an amino acid, one of the 20 standard amino acids. It plays an important physiological role in organisms, especially in protein synthesis and metabolism. The main functions of aspartic acid include: (1) Protein synthesis: Aspartic acid is a component of proteins and participates in the protein synthesis process; (2) Metabolic function: In the body, it participates in amino acid metabolism as an important metabolic intermediate, especially in the urea cycle; (3) Neurotransmitter: Aspartic acid can act as a neurotransmitter in the nervous system, affecting the activity and signal transmission of nerve cells; (4) Synthesis of other molecules: It also participates in the synthesis of other important amino acids and biomolecules, such as adenosine deacidification (ATP) and adenosine diphosphate (ADP).

[0004] Aspartic acid has many uses, including: (1) as an electrolyte supplement for amino acid infusions, potassium, calcium and other inorganic ion supplements, fatigue recovery agents, etc. Aspartic acid potassium magnesium injection or oral solution is used for arrhythmias and premature beats, tachycardia, hypokalemia, hypomagnesemia, heart failure, myocardial infarction, angina pectoris, hepatitis, cirrhosis and other diseases caused by cardiac glycoside poisoning. It is low in toxicity. This product should not be injected without dilution. It should be used with caution in patients with renal insufficiency and atrioventricular block. (2) It can be used as an ammonia detoxifier, liver function promoter, fatigue recovery agent and other pharmaceutical products. It can be used to make sodium aspartate food additives and additives for various soft drinks. It can also be used as a biochemical reagent, culture medium and organic synthesis intermediate. (3) It is used to synthesize sweeteners. In medicine, it is used to treat heart disease. It is used as a liver function promoter, ammonia detoxifier, fatigue reliever and amino acid infusion component, etc. (4) Nutritional supplement and flavor enhancer. It is added to various soft drinks. In medicine, it is used as an ammonia detoxifier, a liver function promoter, and a fatigue recovery agent.

[0005] Generally, aspartic acid is produced through two methods: synthesis and fermentation. The synthesis method primarily uses maleic acid or fumaric acid, or their esters, as raw materials, treating them with ammonia under pressure, followed by hydrolysis. The fermentation method, under enzymatic action, involves the addition of ammonia to fumaric acid, yielding the product in high yield. Many legumes, grains, and vegetables are also rich in aspartic acid, including asparagus, ginger, *Xanthoceras sorbifolium*, green asparagus, spinach, beans, bean sprouts, pears, peaches, and meats. Currently, there are also reports of methods for directly isolating and extracting aspartic acid.

[0006] The Wuhan Botanical Garden, Chinese Academy of Sciences, reported a method for separating L-aspartic acid from cottonseed meal hydrolysate. Cottonseed meal was hydrolyzed with hydrochloric acid. The resulting mixture of amino acids and hydrochloric acid was used to adsorb the amino acids onto a cation exchange column using a treated cation exchange resin. Chloride ions and other impurities on the column were washed away with distilled water. Ammonia was then used as the eluent to separate acidic, neutral, and some basic amino acids in groups. The eluent containing acidic amino acids was further separated using an anion exchange resin to obtain L-aspartic acid. (Separation of L-arginine, L-glutamic acid, and L-aspartic acid from cottonseed meal hydrolysate [J]. Pharmaceutical Industry, 1979, (10): 43-47.) Chinese Patent Publication No. CN105294470A discloses a method for extracting rose aspartic acid from the waste liquid after extracting essential oil from rose flowers. The method includes the following steps: first step, pretreatment; second step, acidification; third step, separation and purification with cation exchange resin; fourth step, decolorization and precipitation with activated carbon; and fifth step, recrystallization.

[0007] Chinese Patent Publication No. CN116354839A discloses a method for extracting aspartic acid from the plant Robinia pseudoacacia. The extraction method is as follows: using fresh Robinia pseudoacacia roots as raw material, the root bark is peeled off, crushed, and extracted by reflux with 50% ethanol. The extract is concentrated under reduced pressure, cooled to room temperature, and allowed to stand, at which point crude aspartic acid crystals precipitate out. The crude aspartic acid crystals are dissolved in boiling water, filtered, cooled to room temperature, and allowed to stand to obtain the finished aspartic acid product.

[0008] Many studies have shown that spinach is rich in aspartic acid, but there are currently no reported methods for extracting aspartic acid from spinach. Summary of the Invention

[0009] To overcome the shortcomings of existing technologies, this invention provides a process for extracting L-aspartic acid from spinach, which improves resource utilization efficiency, increases added value, and reduces production costs. At the same time, by improving the extraction method, the purity and yield of the product L-aspartic acid are increased.

[0010] Specifically, the method for extracting L-aspartic acid from spinach is as follows: (1) Hydrolysis extraction: Wash and cut 1000 parts by weight of spinach, add 2 to 4 times the amount of boiling water and boil for 2 to 5 minutes, filter and take the filtrate; (2) Decolorization and purification: Concentrate the filtrate to 1 / 5 to 1 / 2 of its original volume, cool, add 0.5 to 1 times the amount of 85% to 95% ethanol by mass, shake well, filter, remove the solvent by rotary evaporation under reduced pressure, and control the water bath temperature at 30 to 35℃ for 5 to 15 min; 35 to 45℃ for 10 to 20 min; 45 to 55℃ for 10 to 20 min; 25 to 30℃ for 10 to 20 min; 40 to 50℃ for 10 to 20 min; 55 to 70℃ for 5 to 15 min; 70 to 80℃ for 5 to 15 min to obtain solution A; add 0.005 to 0.012 parts by weight of calcium chloride to solution A, stir well, filter, and obtain solution B; (3) Acidification separation: Add acid to solution B, adjust the pH to 2.0~3.0, cool, adsorb using H-type 732 cation exchange resin, rinse with deionized water 2~4 times to remove impurities, use ammonia-glycerol-nonadienol as eluent, elute 3~5 times, and collect the eluent; (4) Purification and extraction: Add 0.01 to 0.03 parts by weight of activated carbon to the eluent for decolorization, filter, concentrate the filtrate to half of the original volume, add 0.2 to 0.8 times the volume of diethyl ether, extract, and take the lower layer.

[0011] (5) Refining and removing impurities: The lower layer of liquid is placed in a water bath and evaporated under reduced pressure. The crystals are dried, ground, washed with ethanol 2-3 times, and dried to obtain the final product.

[0012] Furthermore, the acid in step (3) is either hydrochloric acid or sulfuric acid.

[0013] Furthermore, the mass fractions of ammonia, glycerol, and nonadienol in the eluent are 4.5%~6.5%, 0.5%~1.5%, and 0.1%~0.5%, respectively. In a preferred embodiment, the eluent is 5.5% ammonia - 1.0% glycerol - 0.4% nonadienol.

[0014] The roles of glycerol include: (1) polarity adjustment: Glycerol has strong polarity and can interact with the polar groups of aspartic acid, thereby affecting the elution behavior of aspartic acid with cation exchange resin; (2) fluidity adjustment: Glycerol has a certain viscosity, which can change the fluidity of the eluent and affect the contact time with the resin and the elution efficiency. The roles of ammonia include: (1) pH adjustment: Ammonia can adjust the pH value of the solution, change the charge state of the ion exchange resin, and affect the interaction between aspartic acid and the resin; (2) ionic strength: Ammonia ions in ammonia can compete with ions on the resin, thereby helping to elute aspartic acid. A suitable mixture of ammonia and glycerol as an eluent can play a synergistic role and achieve more efficient elution. Glycerol can adjust fluidity and polarity, while ammonia can change pH and ionic strength, thereby better eluting aspartic acid. It can also improve selectivity: By adjusting the ratio of glycerol and ammonia, the properties of the eluent can be finely adjusted, thereby achieving more precise separation of the target compound and improving specific selectivity and purity. Furthermore, it can reduce non-specific interactions, minimize background interference, and avoid competition between other compounds and the resin, thereby increasing the yield and purity of aspartic acid. Finally, it can also improve stability: the mixture of glycerol and ammonia makes the eluent more stable, reducing potential fluctuations during elution and improving the consistency and reproducibility of the separation process. However, nonadienol has good solubility, especially for some nonpolar and weakly polar compounds. In addition, nonadienol can reduce the surface tension of the liquid, thus helping to improve elution efficiency. Through continuous experimentation, the inventors unexpectedly discovered that nonadienol can effectively synergize with ammonia to improve elution capacity, thereby enhancing separation efficiency. Ammonia provides an alkaline environment, which can enhance the selective elution of certain compounds; its combination with nonadienol allows for optimization targeting specific molecules.

[0015] Furthermore, to ensure sufficient elution, the elution flow rate is 110~160 ml / min, and in a preferred embodiment, the elution flow rate is 130 ml / min.

[0016] In the preferred extraction scheme, the filtrate after cooling in step (1) is concentrated to 1 / 4 to 1 / 3 of its original volume.

[0017] Compared with the prior art, the technical advantages of the present invention are as follows: It is known that spinach contains L-aspartic acid, which is soluble in boiling water. This invention utilizes the wastewater from blanching spinach to extract L-aspartic acid, which can reduce the waste of raw materials, improve the efficiency of resource utilization, and be used to develop new products or applications, thereby increasing added value. Furthermore, by recycling the effective components in the wastewater, the demand for new raw materials can be reduced, thereby lowering production costs.

[0018] This invention combines existing methods for extracting L-aspartic acid from natural products and explores an improved ion exchange extraction method suitable for spinach wastewater, which improves the extraction efficiency and purity of the product, enhances product safety, and increases production capacity and market value. Attached Figure Description

[0019] Figure 1 Infrared characterization of the product in Example 1.

[0020] Figure 2 Example 1: Mass spectrometry characterization of the product.

[0021] Figure 3 The yield and content of L-aspartic acid obtained from groups A to H.

[0022] Figure 4 Purity of products from Examples 1-3 and Comparative Examples 1-6. Detailed Implementation

[0023] To make the objectives and technical solutions of this invention clearer, the following embodiments are provided for further explanation. However, the scope of protection of this invention is not limited to these embodiments; the embodiments are merely for illustrative purposes. Those skilled in the art should understand that any changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0024] Single-factor experimental investigation of eluent To improve the purity and yield of L-aspartic acid, the eluent in the ion exchange method was optimized and screened. Other methods were the same as in Example 1. The variable factor was the eluent, and all were expressed as mass fractions.

[0025] A: 4.5% ammonia solution - 0.5% glycerol - 0.0% nonadienol solution B: 5.0% ammonia solution - 0.0% glycerol - 0.4% nonadienol solution C: 5.5% ammonia solution - 0.2% glycerol - 0.8% nonadienol solution D: 6.0% ammonia solution - 0.5% glycerol - 0.1% nonadienol solution E: 6.5% ammonia solution - 1.0% glycerol - 0.3% nonadienol solution F: 5.0% ammonia solution - 1.5% glycerol - 0.5% nonadienol solution G: 5.0% ammonia solution - 2.5% glycerol - 1.0% nonadienol solution H: 5.0% ammonia water - 3.5% glycerol - 1.5% nonadienol solution Figure 3The yield and purity of L-aspartic acid obtained from groups A to H are shown. L-aspartic acid extracted using 4.5% ammonia as the eluent had a purity of 98.2% and a yield of 0.201 g. To improve the purity and yield of L-aspartic acid, the eluent was modified by adding appropriate amounts of glycerol and nonadienol. The suitable concentrations of glycerol and nonadienol were determined to be 0.5%–1.5% by mass, and 0.1%–0.5% by mass. On the one hand, nonadienol can reduce the surface tension of the liquid, thus helping to improve elution efficiency. On the other hand, the polarity of glycerol can interact with the polarity of aspartic acid, affecting its elution behavior on the resin and helping to reduce non-specific interactions between the target molecule and the ion exchange resin, thereby making elution more effective. Furthermore, the ammonia-glycerol-nonadienol combination has a synergistic effect in enhancing elution. However, high concentrations of glycerol can also affect the purity of L-aspartic acid and the yield of the product. Experimental screening revealed that the most suitable eluents are ammonia, glycerol, and nonadienol with mass fractions of 4.5%~6.5%, 0.5%~1.5%, and 0.1%~0.5%, respectively.

[0026] Example 1: L-Aspartic Acid Extraction Process (1) Hydrolysis extraction: Weigh 1000g of spinach, wash and cut it into sections, add 3 times the amount of boiling water and boil for 3 minutes, filter, and keep the filtrate for later use; (2) Decolorization and purification: The filtrate was heated and concentrated to 1 / 4 of its original volume, cooled, and 0.8 times the amount of 90% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The water bath heating program for reduced pressure distillation was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 8g of calcium chloride was added to solution A, stirred and mixed, and the precipitate was filtered out to obtain solution B; (3) Acidification separation: Add hydrochloric acid to solution B to adjust the pH to 2.5. After cooling, use H-type 732 cation exchange resin for adsorption. Wash with deionized water 3 times to remove impurities. Use a mixture of 5.5% ammonia water, 1.0% glycerol and 0.4% nonadienol as the eluent. The elution flow rate is 130 ml / min. Perform 4 elutions and collect the eluent. (4) Purification and extraction: Add 20g of activated carbon to the eluent for decolorization, filter, heat and concentrate the filtrate to half of its original volume, add 0.6 times the volume of diethyl ether, extract, let stand, and take the lower layer.

[0027] (5) Refining and impurity removal: The lower layer liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The temperature can be increased stepwise until crystals appear and no more distillate is produced. The crystals are dried, ground, washed with ethanol 2~3 times, and dried again to obtain 0.235g. The crystals are characterized by infrared spectroscopy and mass spectrometry as follows: Figure 1 and Figure 2 .

[0028] Example 2: L-Aspartic Acid Extraction Process (1) Hydrolysis extraction: Weigh 1000g of spinach, wash and cut it into sections, add twice the amount of boiling water and boil for 2 minutes, filter, and keep the filtrate for later use; (2) Decolorization and purification: The filtrate was heated and concentrated to 1 / 2 of its original volume, cooled, and 0.5 times the amount of 85% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The water bath heating program for the reduced pressure distillation was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 5g of calcium chloride was added to solution A, stirred and mixed, and the precipitate was filtered out to obtain solution B; (3) Acidification separation: Add hydrochloric acid to solution B to adjust the pH to 2.0. After cooling, use H-type 732 cation exchange resin for adsorption. Rinse twice with deionized water to remove impurities. Use a mixture of 4.5% ammonia, 0.5% glycerol and 0.1% nonadienol as the eluent. The elution flow rate is 110 ml / min. Perform elution twice and collect the eluent. (4) Purification and extraction: Add 10g of activated carbon to the eluent for decolorization, filter, heat and concentrate the filtrate to half of its original volume, add 0.2 times the volume of diethyl ether, extract, let stand, and take the lower layer.

[0029] (5) Refining and removing impurities: The lower layer liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The temperature can be increased stepwise until crystals appear and no more distillate is produced. The crystals are dried, ground, washed with ethanol 2~3 times, and dried to obtain 0.221g.

[0030] Example 3: L-Aspartic Acid Extraction Process (1) Hydrolysis extraction: Weigh 1000g of spinach, wash and cut it into sections, add 4 times the amount of boiling water and boil for 5 minutes, filter, and keep the filtrate for later use; (2) Decolorization and purification: The filtrate was heated and concentrated to 1 / 5 of its original volume, cooled, and 1 times the amount of 95% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The water bath heating program for reduced pressure distillation was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 12g of calcium chloride was added to solution A, stirred and mixed, and the precipitate was filtered out to obtain solution B. (3) Acidification separation: Add sulfuric acid to solution B to adjust the pH to 3.0. After cooling, use H-type 732 cation exchange resin for adsorption. Wash with deionized water 4 times to remove impurities. Use a mixture of 6.5% ammonia water, 1.5% glycerol and 0.5% nonadienol as the eluent. The elution flow rate is 160 ml / min. Perform 5 elutions and collect the eluent. (4) Purification and extraction: Add 30g of activated carbon to the eluent for decolorization, filter, heat and concentrate the filtrate to half of its original volume, add 0.8 times the volume of diethyl ether, extract, let stand, and take the lower layer.

[0031] (5) Refining and removing impurities: The lower layer liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The temperature can be increased stepwise until crystals appear and no more distillate is produced. The crystals are dried, ground, washed with ethanol 2~3 times, and dried to obtain 0.240g.

[0032] Comparative Example 1: L-Aspartic Acid Extraction Process (1) Hydrolysis extraction: Weigh 1000g of spinach, wash and cut it into sections, add 3 times the amount of boiling water and boil for 3 minutes, filter, and keep the filtrate for later use; (2) Decolorization and purification: The filtrate was heated and concentrated to 1 / 4 of its original volume, cooled, and 0.8 times the amount of 90% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The water bath heating program for the reduced pressure distillation was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 8g of calcium chloride was added to solution A, stirred and mixed, and the precipitate was filtered out to obtain solution B; (3) Acidification separation: Add hydrochloric acid to solution B to adjust the pH to 2.5. After cooling, use H-type 732 cation exchange resin for adsorption. Wash with deionized water 3 times to remove impurities. Use a mixture of 5.5% ammonia water, 1.0% glycerol and 0.4% nonadienol as the eluent. The elution flow rate is 130 ml / min. Perform 4 elutions and collect the eluent. (4) Purification and extraction: Add 0.02 times the mass of activated carbon to the eluent for decolorization, filter, heat and concentrate the filtrate to half the original volume, add 0.6 times the volume of diethyl ether, extract, let stand, and take the lower layer.

[0033] (5) Refining and removing impurities: The lower layer liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The temperature can be increased stepwise until crystals appear and no more distillate is produced. The crystals are dried, ground, washed with ethanol 2~3 times, and dried to obtain 0.173g.

[0034] Comparative Example 2: L-Aspartic Acid Extraction Process (1) Hydrolysis extraction: Weigh 1000g of spinach, wash and cut it into sections, add 3 times the amount of boiling water and boil for 3 minutes, filter, and keep the filtrate for later use; (2) Decolorization and purification: The filtrate was heated and concentrated to 1 / 4 of its original volume, cooled, and 0.8 times the amount of 90% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The water bath heating program for reduced pressure distillation was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 8g of calcium chloride was added to solution A, stirred and mixed, and the precipitate was filtered out to obtain solution B; (3) Acidification separation: Add hydrochloric acid to solution B to adjust the pH to 2.5. After cooling, use H-type 732 cation exchange resin for adsorption. Wash with deionized water 3 times to remove impurities. Use 4.0% ammonia water as eluent and the elution flow rate is 130 ml / min. Perform 4 elutions and collect the eluent. (4) Purification and extraction: Add 20g of activated carbon to the eluent for decolorization, filter, heat and concentrate the filtrate to half of its original volume, add 0.6 times the volume of diethyl ether, extract, let stand, and take the lower layer.

[0035] (5) Refining and removing impurities: The lower layer liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The temperature can be increased stepwise until crystals appear and no more distillate is produced. The crystals are dried, ground, washed with ethanol 2~3 times, and dried to obtain 0.181g.

[0036] Comparative Example 3: L-Aspartic Acid Extraction Process (1) Hydrolysis extraction: Weigh 1000g of spinach, wash and cut it into sections, add 3 times the amount of boiling water and boil for 3 minutes, filter, and keep the filtrate for later use; (2) Decolorization and purification: The filtrate was heated and concentrated to 1 / 4 of its original volume, cooled, and 0.8 times the amount of 90% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The water bath heating program for reduced pressure distillation was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 8g of calcium chloride was added to solution A, stirred and mixed, and the precipitate was filtered out to obtain solution B; (3) Acidification separation: Add hydrochloric acid to solution B to adjust the pH to 2.5. After cooling, use H-type 732 cation exchange resin for adsorption. Rinse with deionized water 3 times to remove impurities. Use a mixture of 5.5% ammonia and 1.5% potassium chloride as the eluent. The elution flow rate is 130 ml / min. Perform 4 elutions and collect the eluent. (4) Purification and extraction: Add 20g of activated carbon to the eluent for decolorization, filter, heat and concentrate the filtrate to half of its original volume, add 0.6 times the volume of diethyl ether, extract, let stand, and take the lower layer.

[0037] (5) Refining and removing impurities: The lower layer liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The temperature can be increased stepwise until crystals appear and no more distillate is produced. The crystals are dried, ground, washed with ethanol 2~3 times, and dried to obtain 0.177g.

[0038] Comparative Example 4: L-Aspartic Acid Extraction Process (1) Hydrolysis extraction: Weigh 1000g of spinach, wash and cut it into sections, add 3 times the amount of boiling water and boil for 3 minutes, filter, and keep the filtrate for later use; (2) Decolorization and purification: The filtrate was heated and concentrated to 1 / 4 of its original volume, cooled, and 0.8 times the amount of 90% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The water bath heating program for reduced pressure distillation was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 8g of calcium chloride was added to solution A, stirred and mixed, and the precipitate was filtered out to obtain solution B; (3) Acidification separation: Hydrochloric acid was added to solution B to adjust the pH to 2.5. After cooling, adsorption was performed using H-type 732 cation exchange resin. The solution was rinsed three times with deionized water to remove impurities. A pH 2.2, 0.1 mol / L citrate buffer was used as the eluent. The elution flow rate was 130 ml / min, and elution was performed four times. The eluent was collected. (4) Purification and extraction: Add 20g of activated carbon to the eluent for decolorization, filter, heat and concentrate the filtrate to half of its original volume, add 0.6 times the volume of diethyl ether, extract, let stand, and take the lower layer.

[0039] (5) Refining and removing impurities: The lower layer liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The temperature can be increased stepwise until crystals appear and no more distillate is produced. The crystals are dried, ground, washed with ethanol 2~3 times, and dried to obtain 0.152g.

[0040] Comparative Example 5: L-Aspartic Acid Extraction Process (1) Hydrolysis extraction: Weigh 1000g of spinach, wash and cut it into sections, add 3 times the amount of boiling water and boil for 3 minutes, filter, and keep the filtrate for later use; (2) Decolorization and purification: The filtrate was heated and concentrated to 1 / 4 of its original volume, cooled, and 0.8 times the amount of 90% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The water bath heating program for reduced pressure distillation was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 8g of calcium chloride was added to solution A, stirred and mixed, and the precipitate was filtered out to obtain solution B; (3) Acidification separation: Add hydrochloric acid to solution B to adjust the pH to 2.5. After cooling, use H-type 732 cation exchange resin for adsorption. Rinse with deionized water 3 times to remove impurities. Use a mixture of 5.5% ammonia water, 1.0% sodium hyaluronate and 0.4% nonadienol as the eluent. The elution flow rate is 130 ml / min. Perform 4 elutions and collect the eluent. (4) Purification and extraction: Add 20g of activated carbon to the eluent for decolorization, filter, heat and concentrate the filtrate to half of its original volume, add 0.6 times the volume of diethyl ether, extract, let stand, and take the lower layer.

[0041] (5) Refining and removing impurities: The lower layer liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The temperature can be increased stepwise until crystals appear and no more distillate is produced. The crystals are dried, ground, washed with ethanol 2~3 times, and dried to obtain 0.114g.

[0042] Comparative Example 6: L-Aspartic Acid Extraction Process (1) Pretreatment: Weigh 1000g of spinach, wash and cut it into sections, add 3 times the amount of boiling water and boil for 3 minutes, filter and obtain a first mixture; (2) Acidification: Add concentrated hydrochloric acid to the primary mixture and adjust the pH to 1.5-2.5 to obtain the secondary mixture; (3) Separation and purification: Place the H-type 732 cation exchange resin in a 200-400 mesh filter cloth, place the filter cloth in the secondary mixture, stir the secondary mixture until the exchange adsorption is saturated, wash the 732 cation exchange resin with deionized water until the ninhydrin reaction is negative; pack the 732 cation exchange resin column by wet method, elute the 732 cation exchange resin column with 4% ammonia solution at an elution rate of 1.0 l / h, collect the tertiary mixture when the ninhydrin reaction is positive; concentrate the tertiary mixture under reduced pressure to obtain the quaternary mixture; (4) Decolorization and precipitation: Add 1% activated carbon to the four mixtures for decolorization treatment for 20 min, filter to obtain the fifth mixture; add 1 / 4 volume of ethanol to the fifth mixture, stir slowly until cooled, place at 4-7℃ for 36-48 h, filter, wash the filter cake with anhydrous ethanol, dry to obtain a light yellow crude amino acid product. (5) Recrystallization: Add crude amino acid and 50 times the mass of deionized water to a round-bottom flask equipped with a reflux condenser, heat to reflux, and wait for the crude amino acid to dissolve. Add anhydrous ethanol dropwise to the round-bottom flask at 70-80℃ until the solution is saturated. Stop adding anhydrous ethanol and stop heating. Seal and refrigerate at 4-8℃. Add aspartic acid seed crystals to the flask and continue to refrigerate at 0-4℃ for 12-24 hours. Filter and wash the crystals with anhydrous ethanol at 0-4℃. Dry at 60℃ for 2 hours to obtain pale yellow crystals. Repeat this process once more to obtain the final product.

[0043] Product Detection in Examples 1-3 According to GB 29938 / AJI92, the products obtained from the L-aspartic acid extraction process in Examples 1-3 were tested and evaluated in various aspects.

[0044] Table 1. Aspartic acid detection report obtained in Examples 1-3 Table 1 shows that the L-aspartic acid prepared by the methods in Examples 1-3 of this invention has high purity and meets the GB 9938 / AJI92 standard, indicating that the method is reliable and highly practical.

[0045] Purity of the extracts from Examples 1-3 and Comparative Examples 1-4 The purity of the extracts from Examples 1-3, Comparative Examples 1-5, and the product obtained by the method disclosed in Chinese Patent CN105294470 B (Comparative Example 6) was determined according to GB 9938 / AJI92 standard. The results are as follows: Figure 4 As shown, each embodiment, comparative example, and prior art patent group underwent five parallel experiments to verify the stability and accuracy of the method.

Claims

1. An extraction process for L-aspartic acid, characterized in that, The extraction process includes the following steps: (1) Hydrolysis extraction: Wash and cut 1000 parts by weight of spinach, add 2 to 4 times the amount of boiling water and boil for 2 to 5 minutes, filter and take the filtrate; (2) Decolorization and purification: The filtrate is concentrated to 1 / 5 to 1 / 2 of its original volume, cooled, and 0.5 to 1 times the amount of 85% to 95% ethanol is added. The mixture is shaken and mixed, filtered, and the solvent is removed by rotary evaporation under reduced pressure and heating to obtain solution A. 0.005 to 0.012 parts by weight of calcium chloride is added to solution A, stirred and filtered to obtain solution B. (3) Acidification separation: Add acid to solution B, adjust the pH to 2.0~3.0, cool, adsorb using cation exchange column, rinse with deionized water 2~4 times to remove impurities, use ammonia water-glycerol-nonadienol with mass fractions of 4.5%~6.5%, 0.5%~1.5%, 0.1%~0.5% as eluent, elute 3~5 times, and collect the eluent; (4) Purification and extraction: Add 0.01 to 0.03 parts by weight of activated carbon to the eluent for decolorization, filter, concentrate the filtrate to half of the original volume, add 0.2 to 0.8 times the volume of diethyl ether, extract, and take the lower layer; (5) Refining and removing impurities: The lower layer of liquid is placed in a water bath and evaporated under reduced pressure. The crystals are dried, ground, washed with ethanol 2-3 times, and dried to obtain the final product.

2. The extraction process for L-aspartic acid according to claim 1, characterized in that, The vacuum rotary evaporation heating program in step (2) is as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min.

3. The extraction process for L-aspartic acid according to claim 1, characterized in that, The acid in step (3) is either hydrochloric acid or sulfuric acid.

4. The extraction process for L-aspartic acid according to claim 1, characterized in that, The eluent is 5.5% ammonia, 1.0% glycerol, and 0.4% nonadienol.

5. The extraction process for L-aspartic acid according to claim 1, characterized in that, The cation exchange column is an H-type 732 cation exchange resin.

6. The extraction process for L-aspartic acid according to claim 1, characterized in that, The elution flow rate is 110~160 ml / min.

7. The extraction process for L-aspartic acid according to claim 1 or 6, characterized in that, The elution flow rate was 130 ml / min.

8. The extraction process for L-aspartic acid according to claim 1, characterized in that, In step (1), the cooled filtrate is concentrated to 1 / 4 to 1 / 3 of its original volume.

9. The extraction process for L-aspartic acid according to claim 1, characterized in that, The extraction process includes the following steps: (1) Hydrolysis extraction: 1000 parts by weight of spinach were washed, cut into sections, boiled in 3 times the amount of boiling water for 3 minutes, filtered, and the filtrate was collected. (2) Decolorization and purification: The filtrate was concentrated to 1 / 4 of its original volume, cooled, and 0.8 times the amount of 90% ethanol was added. The mixture was shaken and mixed, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The heating program was as follows: 30~35℃, 5~15min; 35~45℃, 10~20min; 45~55℃, 10~20min; 25~30℃, 10~20min; 40~50℃, 10~20min; 55~70℃, 5~15min; 70~80℃, 5~15min, to obtain solution A; 0.008 parts by weight of calcium chloride was added to solution A, stirred and filtered to obtain solution B. (3) Acidification separation: Add hydrochloric acid to solution B, adjust the pH to 2.5, cool, adsorb using H-type 732 cation exchange resin, rinse with deionized water 3 times to remove impurities, use 5.5% ammonia water-1.0% glycerol-0.4% nonadienol as eluent, elute at a flow rate of 130 ml / min, perform 4 elutions, and collect the eluent; (4) Purification and extraction: Add 0.02 parts by weight of activated carbon to the eluent for decolorization, filter, concentrate the filtrate to half of the original volume, add 0.6 times the volume of diethyl ether, extract, and take the lower layer; (5) Refining and removing impurities: The lower layer of liquid is placed in a water bath and evaporated under reduced pressure at 30℃~60℃. The crystals are dried, ground, washed with ethanol 2~3 times, and dried to obtain the final product.

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