A method for separating and purifying 5-amino levulinic acid

Abstract

The present application relates to the technical field of separation and purification, and particularly relates to a separation and purification method of 5-amino levulinic acid. The present application optimizes the separation and purification process of 5-amino levulinic acid, realizes efficient purification of 5-ALA through steps such as resin ion exchange, ammonia elution, precise temperature control and cooling crystallization, and is suitable for large-scale production of 5-ALA separated and purified from fermentation broth.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, and in particular to a method for separating and purifying 5-aminolevulinic acid. Background Technology

[0002] 5-Aminolevulinic acid (5-ALA) is a key bioactive substance with irreplaceable application value in the fields of medicine, agriculture, and biotechnology. Its production mainly relies on microbial fermentation. However, 5-ALA itself has chemically sensitive properties to temperature and pH. In addition, impurities such as proteins, polysaccharides, and pigments in the fermentation broth system have similar physicochemical properties to the target product. As a result, the crystallization stage has become the core bottleneck restricting the purity, yield, and environmental friendliness of the product during the separation and purification process.

[0003] Existing crystallization techniques have many insurmountable drawbacks: 1. Disadvantages of organic reagent crystallization: It relies on a large amount of organic solvents (such as ethanol, acetone, etc.) as crystallization medium, which not only greatly increases production costs, but also easily leads to excessive solvent residues, posing safety and environmental risks; at the same time, organic solvents can easily destroy the stable structure of 5-ALA, causing degradation, and can easily cause uneven crystal morphology, affecting the stability of product quality. 2. Inherent defects of evaporation crystallization: Evaporation crystallization requires high-temperature heating to remove the solvent, but 5-ALA is prone to hydrolysis and oxidative degradation under high-temperature conditions (the degradation rate accelerates significantly when the temperature exceeds 80℃), resulting in a significant decrease in yield; moreover, the high-temperature environment will exacerbate the co-crystallization phenomenon between impurities and the target product, reducing product purity; in addition, salts are prone to thermal decomposition during high-temperature evaporation, generating corrosive gases, damaging equipment and introducing secondary pollution; 3. Poor synergy between crystallization and pretreatment processes: Existing crystallization technology lacks effective synergy with pretreatment processes such as desalination and decolorization. Trace impurities remaining during pretreatment are easily enriched during the crystallization stage, further reducing product purity. At the same time, traditional crystallization processes cannot adapt to the sensitive characteristics of 5-ALA, making it difficult to balance the contradiction between "impurity removal" and "product stability". The technical defects in the existing 5-ALA crystallization process directly result in product purity generally being below 90%, yield being less than 70%, and there are also problems of high environmental pressure and high production costs.

[0004] Therefore, it is necessary to optimize and improve the 5-ALA separation process. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a highly efficient separation and purification process for 5-aminolevulinic acid.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for the separation and purification of 5-aminolevulinic acid, comprising the following steps: (1) Inactivation: Adjust the pH of the 5-ALA fermentation broth to 3.5-4.0 with acid, and then heat to inactivate and sterilize; (2) Microfiltration: The 5-ALA fermentation broth after step (1) is filtered through a ceramic membrane, and the clear filtrate that has passed through is the microfiltration light liquid. (3) Ion exchange: The microfiltration liquid obtained in step (2) is passed through a resin column for adsorption. The resin is washed forward and backwashed with purified water until the permeate is clear. Then, ammonia is used for elution and the eluent is collected. The resin column is a mixture of a strong acid cation exchange resin and an iminodiacetic acid chelating resin. (4) Decolorization and concentration: The eluent obtained in step (3) was decolorized and concentrated using a mixture of LX-B40 resin and modified diatomaceous earth to obtain a concentrated solution. (5) Crystallization: The concentrated solution obtained in step (4) is cooled to 4-8℃ at a rate of 0.1-0.5℃ / min to crystallize, centrifuge and dry to obtain 5-ALA crystals.

[0007] This invention optimizes the separation and purification process of 5-aminolevulinic acid (ALA). First, the pH of the fermentation broth is adjusted before inactivation, effectively inhibiting the hydrolytic degradation of 5-ALA. If the pH is below 3.5, the molecular structure of 5-ALA will be destroyed, leading to a significant increase in the degradation rate; if the pH is above 4.0, contaminating bacteria in the fermentation broth cannot be completely inactivated, and their metabolism will contaminate the target product. The ion exchange step uses a combination of 95% strongly acidic cation exchange resin and 5% iminodiacetic acid-type chelating resin, achieving both efficient adsorption of 5-ALA and removal of metal impurities. The decolorization and concentration step uses a combination of LX-B40 resin and modified diatomaceous earth, effectively removing pigments without residue. The crystallization step, with a cooling rate of 0.1-0.5℃ / min and a final temperature of 4-8℃, achieves the directional and slow precipitation of 5-ALA, forming high-purity, uniform crystals. Too rapid a cooling rate leads to crystal agglomeration and impurity encapsulation, resulting in decreased purity; too slow a rate prolongs the process cycle. A final temperature below 4℃ increases energy consumption and is prone to freezing, while a temperature above 8℃ results in incomplete crystallization and a significant decrease in yield. Through resin ion exchange, ammonia elution, and precise temperature-controlled crystallization, this invention achieves highly efficient purification of 5-ALA, suitable for large-scale production scenarios of separating and purifying 5-ALA from fermentation broth. Temperature-controlled crystallization, as a novel crystallization technology, achieves the directional precipitation of the target product through precise temperature control, offering significant advantages over traditional crystallization methods.

[0008] Preferably, in step (2), the pore size of the ceramic membrane is 40-60 nm. In the microfiltration process, if the pore size of the ceramic membrane is too small, it will lead to membrane clogging, decreased filtration efficiency, and increased production costs; if the pore size is too large, it will be unable to remove impurities such as large molecular proteins and polysaccharides.

[0009] Preferably, in step (2), the feed pressure is controlled at 4-6 kg, and the filtrate is filtered until the OD of the filtrate is... 600nm A feed pressure of 0.5-1.0 yields a microfiltration light liquid. Deviations in feed pressure can lead to unstable filtration flow rate and OD. 600nm If the impurities are removed beyond this range, the subsequent ion exchange effect will be affected.

[0010] Preferably, in step (3), the strong acid cation exchange resin includes a styrene-divinylbenzene backbone strong acid cation exchange resin; and / or, the iminodiacetic acid type chelating resin includes an IDA chelating resin.

[0011] Preferably, in step (3), the resin column is composed of 95% strong acid cation exchange resin and 5% iminodiacetic acid chelating resin.

[0012] Preferably, in step (3), the ammonia concentration is 0.8-1.2 M. Using this concentration of ammonia ensures sufficient elution and reduces product residue.

[0013] Preferably, in step (3), the ammonia elution step includes: eluting at a flow rate of 0.8-1.2 BV / h, and collecting the first low flow liquid, the high flow liquid and the second low flow liquid respectively.

[0014] Preferably, in step (4), the mass ratio of LX-B40 resin to modified diatomaceous earth is 1:1.

[0015] Preferably, in step (4), the concentration of modified diatomaceous earth is 3%.

[0016] Preferably, the separation and purification method further includes a reconstitution and purification step: dissolving the obtained 5-ALA crystals in hydrochloric acid, adding citric acid, and then adding ethanol for recrystallization.

[0017] Preferably, the separation and purification method specifically comprises: (1) Inactivation: After fermentation, use 85% phosphoric acid to precisely adjust the pH of 10L fermentation liquid to 3.5-4.0. Then, pass the adjusted fermentation liquid into the inactivation tank, heat it to 75-85℃ and keep it at a constant temperature for 10-20 minutes, stirring continuously during the process (80-120r / min). (2) Microfiltration: The 5-ALA fermentation broth after step (1) is filtered through a 50nm ceramic membrane and fed under a specific pressure of 4-6kg (pressure fluctuation ±0.2kg). The OD600nm of the light liquid is controlled at 0.5-1.0. The microfiltered light liquid is collected for later use, and the microfiltered heavy liquid is processed separately.

[0018] (3) Ion exchange: Ion exchange is performed using a resin column composed of 95% strong acid cation exchange resin (styrene-divinylbenzene skeleton strong acid cation exchange resin) and 5% iminodiacetic acid type chelating resin (IDA chelating resin) to adsorb 5-ALA and remove metal ion impurities. The resin is pre-treated with 5% hydrochloric acid and 5% sodium hydroxide alternately until it is neutral for later use.

[0019] Taking a 18 g / L (pure AA) ceramic membrane clear solution as an example, the actual working exchange capacity reaches 90 g AA / L resin, and the column loading volume is 5 L. The microfiltration light solution is loaded onto the column for adsorption at a flow rate of 1.5-2.5 BV / h, and the pH of the permeate is monitored in real time. Column loading is stopped when the pH of the permeate drops to approximately 1.8-2.2. The resin is then forward-washed (flow rate 2.5-3.5 BV / h) and back-washed (flow rate 1.5-2.5 BV / h) with purified water until the permeate is clear and the pH reaches 4.5-6.5. Elute with 0.8-1.2M ammonia at a flow rate of 0.8-1.2 BV / h. Collect the first low-flow eluent when the pH of the eluent is approximately 5-6; collect the high-flow eluent when the pH reaches 6; and collect the last low-flow eluent when the pH is approximately 7-8. After mixing the first and high-flow eluents, adjust the pH to 2.0-3.0 with phosphoric acid. Adjust the pH of the last low-flow eluent to 3.5-4.5 (to keep 5-ALA in its most stable pH range, inhibiting degradation and ensuring yield; simultaneously adapting to the optimal conditions for resin adsorption, elution, decolorization, and crystallization, improving separation efficiency and product purity). The eluent can be re-adsorbed or mixed with the microfiltration heavy liquid and spray-dried. Finally, regenerate the resin by forward and backwashing with purified water until the pH of the permeate is 4.5-6.5.

[0020] (4) Decolorization and concentration: A decolorization system (1:1) of LX-B40 resin and 3% modified diatomaceous earth was used. The eluent collected in step (3) was passed through the decolorization column at a flow rate of 1.5-2.5 BV / h, and then eluted with 1 BV of purified water. The decolorized liquid was collected. The decolorized liquid was then passed into a vacuum concentration device, with the temperature set at 65-75℃ and the pressure at 0.08-0.10 MPa. The concentration was increased by 7-11 times until the 5-ALA content in the concentrate reached 750-850 g / L. The concentrate was then transferred to a crystallization tank.

[0021] (5) Cooling and crystallization: Start the temperature control system of the crystallizer and set the cooling rate to 0.1-0.5℃ / min to cool from room temperature (20-28℃) to 4-8℃. After reaching the target temperature, maintain the temperature for 1-3 hours. During this period, the stirring speed is 40-60 r / min to form crystals and avoid crystal agglomeration. If the yield of the first crystallization is less than 80%, the mother liquor is de-alcoholized and then subjected to secondary crystallization. The secondary mother liquor is then sent to the spray drying process.

[0022] (6) Centrifugation and drying: Pass the crystallization liquid into a three-legged centrifuge, use a 200-mesh filter cloth, set the speed to 2800-3200 r / min, and centrifuge for 12-18 minutes to obtain wet crude product. Put the wet crude product into a double cone vacuum dryer, set the temperature to 55-65℃, and dry for 10-26 hours until constant weight to obtain crude 5-ALA product; (7) Refining: Take 1 kg of crude 5-ALA, add an equimolar amount of concentrated hydrochloric acid and stir to redissolve, then add 0.05% citric acid (based on the mass of crude product) and stir to dissolve. Then slowly add 2 L of anhydrous ethanol, stir at room temperature for 25-35 minutes, let stand at 4-8℃ for 1.5-2.5 h, centrifuge to obtain crystals, and dry at 55-65℃ for 6-10 hours to obtain refined product with a content ≥98% and moisture ≤0.2%. The refining process uses an enamel crystallization tank to avoid chloride ion corrosion.

[0023] Secondly, the present invention provides 5-aminolevulinic acid obtained by the separation and purification method described above.

[0024] The beneficial effects of this invention are as follows: This invention enhances the stability of 5-ALA by reducing the degradation rate of 5-ALA from 15-20% in traditional processes to below 5% through precise inactivation conditions, compound resin adsorption, crystallization rate control, and compounding with citric acid stabilizer.

[0025] In the separation and purification process of this invention, ion exchange decolorization uses a compound decolorization system of LX-B40 resin and modified diatomaceous earth. Compared with traditional activated carbon decolorization, the reagents are more environmentally friendly, the pigment removal rate is increased by 30%, and there is no activated carbon residue pollution. The product purity reaches over 98%, and can reach up to 98.8%. In the elution process, ammonia water is used to replace traditional hydrochloric acid decolorization, which significantly improves the 5-ALA yield (from 65% to 95%), and the cost of ammonia water is only 1 / 3 of that of hydrochloric acid, greatly reducing production costs.

[0026] This invention combines a compound resin system with specific microfiltration parameters to achieve a removal rate of over 99% for impurities such as proteins and polysaccharides, thus solving the core problem of incomplete impurity separation in traditional processes.

[0027] The separation and purification process of this invention mainly consists of ceramic membrane microfiltration, compound ion exchange, and precise cooling crystallization. The process is simple, requires no complex equipment, achieves high-efficiency purification, and the crystallization process does not involve a large amount of organic reagent pollution, making it suitable for large-scale industrial production. Detailed Implementation

[0028] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0029] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products that can be obtained through commercial channels.

[0030] IDA-chelating resin, model: Suqing D402, Jiangsu Suqing Water Treatment Engineering Group Co., Ltd.; model: LX-B40, batch number: 20250308GS001, Xi'an Lanxiao Technology New Materials Co., Ltd.; model: LX-D001, batch number: 20250107P6006, Xi'an Lanxiao Technology New Materials Co., Ltd.

[0031] The method of this invention can be applied to the separation and purification of fermentation broth containing 5-aminolevulinic acid obtained from fermentation by any strain. Specifically, the "fermentation broth containing 5-aminolevulinic acid" used in the embodiments and comparative examples of this invention refers to fermentation broth produced by engineered Escherichia coli (E. coli). Escherichia coli The engineered bacteria are fermentation bacteria, and fermentation production is carried out in the same fermentation tank. The fermentation liquid after being discharged from the tank.

[0032] Example 1: An embodiment of the method for separating and purifying 5-aminolevulinic acid according to the present invention includes the following steps: (1) Inactivation: After fermentation, adjust the pH of 10L fermentation broth to 3.80 with 85% phosphoric acid, and then inactivate it at 80℃ for 15 minutes, stirring continuously during the process (80-120r / min). (2) Microfiltration: The 5-ALA fermentation broth treated in step (1) is filtered through a 50nm ceramic membrane and fed under a specific pressure of 5kg (pressure fluctuation ±0.2kg) to control the OD of the light liquid. 600nm At 0.5, collect the light microfiltrate for later use, while the heavy microfiltrate (solids retained by the membrane) is processed separately.

[0033] (3) Ion exchange: Ion exchange of resin columns consisting of 95% strong acid cation exchange resin (styrene-divinylbenzene skeleton strong acid cation exchange resin) and 5% iminodiacetic acid type chelating resin (IDA chelating resin) is carried out in advance. The resin is treated with 5% hydrochloric acid and 5% sodium hydroxide alternately until it is neutral for later use.

[0034] Taking a 18 g / L (pure AA) ceramic membrane clear solution as an example, the actual working exchange capacity reaches 90 g AA / L resin, and the column loading volume is 5 L. The microfiltration light solution is loaded onto the column for adsorption at a flow rate of 2 BV / h, and the pH of the permeate is monitored in real time. When the pH of the permeate reaches about 2.0, the column loading is stopped. The resin is forward-washed and back-washed with purified water until the permeate is clear and the pH reaches 5.0-6.0. Elute with 1M ammonia water at a flow rate of 1 BV / h. Collect the first low-flow eluent when the pH of the eluent is approximately 5-6; collect the high-flow eluent when the pH reaches 6; and collect the last low-flow eluent when the pH is approximately 7-8. After mixing the first low-flow eluent and the high-flow eluent, adjust the pH to 2.0-3.0 with phosphoric acid. Adjust the pH of the last low-flow eluent to 3.5-4.5. It can then be re-adsorbed or mixed with the microfiltration heavy liquid and spray-dried. Finally, regenerate the resin by two forward and backwashing cycles with purified water until the pH of the permeate is 4.5-6.5.

[0035] (4) Decolorization and concentration: A decolorization system of LX-B40 resin and 3% modified diatomaceous earth was used. The eluent collected in step (3) was passed through the decolorization column at a flow rate of 1.5-2.5 BV / h, and then eluted with 1 BV of purified water. The decolorized liquid was collected. The decolorized liquid was then passed into a vacuum concentration device, with the temperature set at 65-75℃ and the pressure at 0.08-0.10 MPa. The concentration was increased by 7-11 times until the 5-ALA content in the concentrate reached 750-850 g / L. The concentrate was then transferred to a crystallization tank.

[0036] (5) Cooling and crystallization: Start the temperature control system of the crystallizer and set the cooling rate to 0.1-0.5℃ / min to cool from room temperature (20-28℃) to 4-8℃. After reaching the target temperature, maintain the temperature for 1-3 hours. During this period, the stirring speed is 40-60 r / min to form crystals and avoid crystal agglomeration. If the yield of the first crystallization is less than 80%, the mother liquor is de-alcoholized and then subjected to secondary crystallization. The secondary mother liquor is then sent to the spray drying process.

[0037] (6) Centrifugation and drying: Pass the crystallization liquid into a three-legged centrifuge, use a 200-mesh filter cloth, set the speed to 2800-3200 r / min, and centrifuge for 12-18 minutes to obtain wet crude product. Put the wet crude product into a double cone vacuum dryer, set the temperature to 55-65℃, and dry for 10-26 hours until constant weight to obtain crude 5-ALA product; (7) Refining: Take 1 kg of crude 5-ALA, add an equimolar amount of concentrated hydrochloric acid and stir to redissolve, then add 0.05% citric acid (based on the mass of crude product) and stir to dissolve. Then slowly add 2 L of anhydrous ethanol, stir at room temperature for 25-35 minutes, let stand at 4-8℃ for 1.5-2.5 h, centrifuge to obtain crystals, and dry at 55-65℃ for 6-10 hours to obtain refined product with a content ≥98% and moisture ≤0.2%. The refining process uses an enamel crystallization tank to avoid chloride ion corrosion.

[0038] Example 2: An embodiment of the method for separating and purifying 5-aminolevulinic acid according to the present invention includes the following steps: (1) Inactivation: After fermentation, adjust the pH of 10L fermentation broth to 3.50 with 85% phosphoric acid, and then inactivate it at 80℃ for 15 minutes, stirring continuously during the process (80-120r / min). (2) Microfiltration: The 5-ALA fermentation broth treated in step (1) was filtered through a 40nm ceramic membrane and fed under a specific pressure of 4kg (pressure fluctuation ±0.2kg) to control the OD of the light liquid. 600nm At 0.5, collect the light microfiltrate for later use, while the heavy microfiltrate (solids retained by the membrane) is processed separately.

[0039] The subsequent steps are the same as in Example 1.

[0040] Example 3: An embodiment of the method for separating and purifying 5-aminolevulinic acid according to the present invention includes the following steps: (1) Inactivation: After fermentation, adjust the pH of 10L fermentation broth to 4.0 with 85% phosphoric acid, and then inactivate it at 80℃ for 15 minutes, stirring continuously during the process (80-120r / min). (2) Microfiltration: The 5-ALA fermentation broth treated in step (1) was filtered through a 60nm ceramic membrane and fed under a specific pressure of 6kg (pressure fluctuation ±0.2kg) to control the OD of the light liquid. 600nm In version 1.0, the light microfiltrate is collected for later use, while the heavy microfiltrate (solids retained by the membrane) is processed separately.

[0041] The subsequent steps are the same as in Example 1.

[0042] Comparative Example 1: This invention provides a comparative example of the method for separating and purifying 5-aminolevulinic acid. The only difference between this comparative example and Example 1 is that the ceramic membrane pore size is 65 nm in step (2), and the other steps are the same as in Example 1.

[0043] Comparative Example 2: This invention provides a comparative example of the method for separating and purifying 5-aminolevulinic acid. The only difference between this comparative example and Example 1 is that the ceramic membrane microfiltration step (2) is not performed, and the fermentation broth is directly subjected to ion exchange. The remaining steps are the same as in Example 1.

[0044] Comparative Example 3: This invention provides a comparative example of the method for separating and purifying 5-aminolevulinic acid. The only difference between this comparative example and Example 1 is that in step (3), an equal amount of hydrochloric acid is used instead of ammonia. The remaining steps are the same as in Example 1.

[0045] Test example: Preliminary research in this invention revealed that Examples 1-3 all yielded 5-ALA with high yield and purity, with Example 1 showing the best results. Therefore, it was used as a representative example for subsequent experiments.

[0046] 1. Yield Test method: High performance liquid chromatography (HPLC) was used to determine the initial total mass of 5-ALA in the fermentation broth and the pure mass of the final purified 5-ALA crystals (both based on anhydrous pure product). The fermentation broth needed to be pretreated before the test, and the crystals needed to be dissolved and diluted to a fixed volume. The chromatographic conditions were adapted to the separation and detection of 5-ALA.

[0047] Yield (%) = (mass of the final purified 5-ALA product ÷ initial total mass of 5-ALA in the fermentation broth) × 100%.

[0048] 2. Product purity Test method: High performance liquid chromatography (HPLC) external standard method was used for detection. Standard curves were plotted by preparing 5-ALA standard solutions of different concentrations. The purified 5-ALA crystals were dissolved and diluted to a fixed volume before being injected into the sample. The pure 5-ALA content in the sample was calculated by substituting the peak area into the standard curve. At the same time, the content of impurities such as moisture and ash in the sample was detected and calibrated comprehensively.

[0049] Product purity (%) = (mass of pure 5-ALA in the sample ÷ total mass of the sample to be tested) × 100% (anhydrous basis).

[0050] 3. Pigment removal rate Test method: The ultraviolet-visible spectrophotometry method was used to detect the absorbance A0 of the eluent before decolorization at 420nm (the characteristic absorption peak of the pigment) and the absorbance A1 of the clarified solution after decolorization at the same wavelength. Before the test, the two samples should be adjusted to the same pH and the same concentration and made up to a fixed volume to ensure that the test conditions are consistent.

[0051] Pigment removal rate (%) = [(A0- A1) ÷ A0] × 100%.

[0052] 4.5-ALA degradation rate Test method: High performance liquid chromatography (HPLC) was used to detect the mass M0 of 5-ALA in the feed at each stage of the process (such as ion exchange feed, crystallization feed) and the mass M1 of 5-ALA remaining after the corresponding stage discharge. The entire process was tracked and detected, and the degradation data of the entire process was finally obtained. At the same time, process separation loss was excluded, and only the product degradation caused by hydrolysis and oxidation was counted.

[0053] Calculation formula: 5-ALA degradation rate (%) = [(M0 - M1 - process separation loss) ÷ M0] × 100%.

[0054] Note: In this invention, the process separation loss refers to controllable and measurable losses such as solid-liquid separation and resin adsorption residue, which are statistically separated from the degradation amount.

[0055] 5. Solvent Residue Test method: Gas chromatography (GC) headspace sampling method was used. For the organic solvents used in this process and the traditional process, standard solutions of the corresponding solvents were prepared and standard curves were plotted. The 5-ALA crystal samples were pretreated by headspace sampling and then injected. The residual amount of solvent in the sample (unit: mg / kg) was calculated by the chromatographic peak area.

[0056] Solvent residue (mg / kg) = Solvent mass obtained from the standard curve ÷ Mass of the sample to be tested; Result determination: If the residual amount is <0.01%, that is, <100mg / kg (the index of this invention).

[0057] 6. Process cycle Test method: Timing starts from the inactivation process of fermentation broth and ends when the 5-ALA product is finally refined. The total time spent on the actual process operation (including equipment operation and material handling time, but excluding idle time other than equipment standby and material settling) is recorded as the process cycle (unit: h).

[0058] Table 1 The results are shown in Table 1. The technical solution of the present invention can obtain 5-ALA with high yield and purity. The ceramic membrane of Comparative Example 1 has a pore size range of 40-60 nm, Comparative Example 2 lacks a ceramic membrane microfiltration step, and Comparative Example 3 uses hydrochloric acid desorption instead of ammonia desorption and purification. The yield and purity of the 5-ALA product obtained by these methods are significantly reduced, and there is more degradation of 5-ALA.

[0059] Comparative Example 1 (Ceramic membrane pore size out of range): It cannot effectively intercept impurities such as large molecular proteins and polysaccharides in the fermentation broth. These impurities enter the decolorization stage with subsequent processes, combine with pigment molecules to form a complex system, and hinder the adsorption of pigments by LX-B40 resin and modified diatomaceous earth, resulting in a significant decrease in pigment removal rate. At the same time, the residual large molecular impurities destroy the stable structure of 5-ALA, catalyze its hydrolysis and oxidation reactions, and the excessively large pore size leads to incomplete filtration, an increase in the amount of residual bacteria, and the metabolism of bacteria further accelerates the degradation of 5-ALA, ultimately resulting in a significant increase in the degradation rate.

[0060] Comparative Example 2 (lacking ceramic membrane microfiltration step): Without microfiltration treatment, a large number of bacteria, proteins, polysaccharides and other impurities in the fermentation broth directly enter the ion exchange and decolorization process. On the one hand, this will clog the resin pores and reduce the resin's adsorption capacity for pigments. At the same time, the pigments carried by the impurities themselves cannot be effectively removed, resulting in a decrease in pigment removal rate. On the other hand, harmful substances produced by the metabolism of bacteria will destroy the molecular stability of 5-ALA, and the large molecular impurities that are not removed will aggravate the degradation of 5-ALA during the subsequent concentration and crystallization process, resulting in a significant increase in the degradation rate.

[0061] Comparative Example 3 (hydrochloric acid elution instead of ammonia elution): The hydrochloric acid used is a strong acid, which causes a sharp drop in the pH of the eluent during the elution process, exceeding the stable pH range of 3.5-4.0 for 5-ALA. This directly causes damage to the molecular structure of 5-ALA, accelerating its hydrolysis and oxidative degradation, resulting in an increased degradation rate. At the same time, hydrochloric acid elution alters the adsorption properties of the resin, reducing its ability to adsorb pigments. Furthermore, hydrochloric acid reacts with impurities in the eluent, potentially generating new colored substances, further reducing the pigment removal rate. Ultimately, this leads to a significant decrease in both pigment removal efficiency and product stability.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for separating and purifying 5-aminolevulinic acid, characterized in that, Includes the following steps: (1) Inactivation: Adjust the pH of the 5-ALA fermentation broth to 3.5-4.0 with acid, and then heat to inactivate and sterilize; (2) Microfiltration: The 5-ALA fermentation broth after step (1) is filtered through a ceramic membrane, and the clear filtrate that has passed through is the microfiltration light liquid. (3) Ion exchange: The microfiltration liquid obtained in step (2) is passed through a resin column for adsorption. The resin is washed forward and backwashed with purified water until the permeate is clear. Then, ammonia is used for elution and the eluent is collected. The resin column is a mixture of a strong acid cation exchange resin and an iminodiacetic acid chelating resin. (4) Decolorization and concentration: The eluent obtained in step (3) was decolorized and concentrated using a mixture of LX-B40 resin and modified diatomaceous earth to obtain a concentrated solution. (5) Crystallization: The concentrated solution obtained in step (4) is cooled to 4-8℃ at a rate of 0.1-0.5℃ / min to crystallize, centrifuge and dry to obtain 5-ALA crystals.

2. The separation and purification method according to claim 1, characterized in that, In step (2), the pore size of the ceramic membrane is 40-60 nm.

3. The separation and purification method according to claim 1, characterized in that, In step (2), the feed pressure is controlled at 4-6 kg, and the filtrate is filtered until the OD of the filtrate is... 600nm A concentration of 0.5-1.0 yields a microfiltrate.

4. The separation and purification method according to claim 1, characterized in that, In step (3), the strong acid cation exchange resin includes a styrene-divinylbenzene backbone strong acid cation exchange resin; and / or, the iminodiacetic acid type chelating resin includes an IDA chelating resin.

5. The separation and purification method according to claim 1, characterized in that, In step (3), the resin column is composed of 95% strong acid cation exchange resin and 5% iminodiacetic acid chelating resin.

6. The separation and purification method according to claim 1, characterized in that, In step (3), the concentration of ammonia water is 0.8-1.2 M.

7. The separation and purification method according to claim 1, characterized in that, In step (3), the ammonia elution step includes: eluting at a flow rate of 0.8-1.2 BV / h, and collecting the first low flow liquid, the high flow liquid and the second low flow liquid respectively.

8. The separation and purification method according to claim 1, characterized in that, In step (4), the mass ratio of LX-B40 resin to modified diatomaceous earth is 1:

1.

9. The separation and purification method according to claim 1, characterized in that, The separation and purification method further includes a reconstitution and purification step: after dissolving the obtained 5-ALA crystals in hydrochloric acid, citric acid is added, and then ethanol is added for recrystallization.

10. 5-Aminolevulinic acid obtained by the separation and purification method according to any one of claims 1-9.

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