A method for refining l-lysine acetate

Abstract

The application discloses a refining method of L-lysine acetate. The fermentation liquor is pretreated by membrane separation in the application, and the cell protein, pigment and inorganic salt in the fermentation liquor are removed, so that the subsequent crystallization load is reduced by 40%. When crystallization is carried out, a pulse gradient cooling method is adopted, and ultrasonic assistance is combined, so that the crystallization time is greatly shortened, the crystal type is good, and the crystal morphology is hexagonal flaky. Further refining is carried out, so that the product purity is increased to more than 99.8%, and the single impurity content is less than 0.1%. The application realizes efficient purification and industrial production of L-lysine acetate.

Description

Technical Field

[0001] This invention relates to a method for refining L-lysine acetate, belonging to the field of biochemical and pharmaceutical raw material preparation technology. Background Technology

[0002] L-Lysine acetate is a salt compound formed by the combination of the essential amino acid L-lysine and acetic acid. Its chemical formula is C6H2O. 14 N2O2·C2H4O2 has a molecular weight of 206.24. As an important amino acid derivative, L-lysine acetate not only retains the basic nutritional functions of L-lysine, but also has been widely used in the fields of medicine, food, and feed additives due to its unique physicochemical properties.

[0003] The pharmaceutical field is the primary application area for L-lysine acetate. Compared to traditional lysine hydrochloride, compound amino acid infusions formulated with lysine acetate have a lower chloride ion content, significantly reducing adverse reactions caused by hyperchloremia in patients, making them particularly suitable for the treatment of burns, liver disease, kidney disease, and encephalopathy. Studies have shown that L-lysine acetate not only provides essential nutritional support but also possesses anti-inflammatory and analgesic properties, protects hepatocytes, enhances immunity, and has potential anti-tumor effects. In clinical applications, lysine acetate is often used as a main component of compound amino acid injections in the formulation of various therapeutic infusion preparations. In the food industry, L-lysine acetate is used as a food fortifier and flavoring agent. Lysine is a common limiting amino acid in cereal proteins; adding L-lysine acetate can effectively improve the protein value of food and enhance nutritional balance. Compared to lysine hydrochloride, lysine acetate has a milder taste and does not introduce excessive chloride ions, making it more suitable for formulations of functional foods and foods for special medical purposes. Feed additives are another important application area for L-lysine acetate. As an essential amino acid for animal growth, L-lysine acetate promotes protein synthesis and improves feed conversion rate, playing a vital role in animal husbandry and aquaculture. Compared to its hydrochloride form, lysine acetate exhibits better stability and solubility, making it superior in feed processing and storage. Therefore, with the development of the health industry, the demand for high-purity L-lysine acetate continues to grow.

[0004] Currently, the main industrial method for producing L-lysine is microbial fermentation, followed by neutralization with acetic acid to obtain L-lysine acetate. However, the crude product typically contains the following impurities: fermentation residues (such as microbial proteins and polysaccharides), inorganic salts (such as ammonium sulfate and sodium chloride), other amino acid byproducts, organic pigments, and unknown impurities. Traditional purification techniques mainly include: recrystallization, which usually requires 3-5 repeated crystallizations to reach pharmaceutical standards, resulting in high solvent consumption and low yield (approximately 60-70%); ion exchange, which, while yielding high purity, presents challenges due to difficult resin regeneration and high wastewater treatment costs; and activated carbon adsorption, which is effective for pigment removal but poor for separating inorganic salts and similar amino acids, highlighting the need for technological improvements. Multi-step purification leads to significant product loss, high organic solvent consumption, increased costs and environmental burden, and difficulty in simultaneously meeting the requirements of high purity and high yield. Furthermore, the process exhibits poor stability during industrial scale-up. Therefore, developing an efficient, economical purification process suitable for large-scale production has significant industrial value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for purifying L-lysine acetate.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a method for purifying L-lysine acetate, comprising the following steps:

[0008] S1. Take the fermentation broth containing L-lysine acetate, add diatomaceous earth for adsorption treatment, and centrifuge to obtain a clear liquid;

[0009] S2. Pre-treat the clarified liquid sequentially using microfiltration and ultrafiltration membranes, and collect the permeate.

[0010] S3. Preheat the permeate to 40-60℃. Add 0.08-0.15% L-malic acid to the permeate by mass ratio. Cool the solution at a rate of 4-6℃ / 5min while stirring at 60-100 rpm. After 5min, maintain the temperature at a constant temperature for 8-12min. During the temperature maintenance phase, use ultrasound to promote crystal nucleation. Continue the pulsed gradient cooling process by repeating the above cooling and temperature maintenance cycle for a total of 5-8 cycles until the temperature drops to 15-25℃. Then stop stirring and maintain the temperature at a constant temperature to grow crystals until no new crystals are formed. Separate the crystals from the mother liquor to obtain crude lysine acetate.

[0011] S4. The crude lysine acetate was dissolved in water. The dissolved solution was then passed through an activated carbon column, a resin bed filled with D101 macroporous resin and AB-8 weakly polar resin, and a MIPs column prepared with L-histidine as the template molecule at a flow rate of 1-2 BV / h to obtain a purified lysine acetate solution.

[0012] S5. The purified lysine acetate solution is recrystallized in the same way as in step (3) to prepare lysine acetate.

[0013] In one embodiment of the present invention, the pulsed gradient cooling step is further preferably as follows: the permeate is preheated to 45-55°C, and 0.08-0.12% L-malic acid is added to the permeate by mass ratio. The permeate is cooled at a rate of 4-6°C / 5min while stirring at 80-100 rpm. After 5min, the temperature is held constant for 8-12min. During the holding phase, ultrasonic assistance is used to promote crystal nucleation. The pulsed gradient cooling process is continued in the manner described above, with the cooling and holding cycles repeated 5-7 times until the temperature drops to 18-22°C. Then, stirring is stopped, and crystal growth is carried out at a constant temperature.

[0014] In one embodiment of the present invention, in step S1, the amount of diatomaceous earth added is 0.1 to 1 g / 100 mL of fermentation broth.

[0015] In one embodiment of the present invention, in step S1, centrifugation is performed at 6000-10000 rpm for 10-20 min.

[0016] In one embodiment of the present invention, in step S2, the microfiltration membrane is a 0.22 μm microfiltration membrane.

[0017] In one embodiment of the present invention, in step S2, the cutoff value of the ultrafiltration membrane is 5 to 15 kDa.

[0018] In one embodiment of the present invention, in step S3, the frequency of the ultrasound assistance is 30-50 kHz and the power is 150-250 W.

[0019] In one embodiment of the present invention, in step S4, D101 macroporous resin and AB-8 weakly polar resin are filled at a volume ratio of 2 to 4:1.

[0020] A second objective of this invention is to provide the application of the purification method in the pharmaceutical field.

[0021] A third objective of this invention is to provide the application of the refining method in the field of animal feed.

[0022] The beneficial effects of this invention are:

[0023] This invention employs membrane separation to pretreat the fermentation broth, removing bacterial proteins, pigments, and inorganic salts, thus reducing the subsequent crystallization load by 40%. Furthermore, during crystallization, a pulsed gradient cooling method combined with ultrasonic assistance is used to significantly shorten the crystallization time and produce better crystal forms with a hexagonal plate-like morphology. Further purification increases the product purity to over 99.8%, with a single impurity content of <0.1%. This invention achieves efficient purification and industrial-scale production of L-lysine acetate. Detailed Implementation

[0024] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] Source of raw materials

[0026] Raw materials: Following the method described in patent CN118374426A for the construction and application of an L-lysine-producing Escherichia coli, L-lysine was first prepared. The L-lysine yield could reach 220 g / L in a 5L fermenter. Acetic acid was then added to the L-lysine fermentation broth to react and obtain a fermentation broth containing 149.4 g / L L-lysine acetate.

[0027] The purity of L-lysine acetate was determined by high performance liquid chromatography (HPLC) using the area normalization method. The chromatographic conditions were as follows:

[0028] The chromatographic column was a C18 reversed-phase column (e.g., Agilent ZORBAX SB-C18, 4.6 × 250 mm, 5 μm). Mobile phase A was 0.1% trifluoroacetic acid (TFA) aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution. A gradient elution program was used (0 min 5% B, 10 min 30% B, 15 min 95% B, 20 min 5% B), the flow rate was 1.0 mL / min, the detection wavelength was 210 nm, the column temperature was 30 °C, and the injection volume was 10 μL. The sample was filtered through a 0.22 μm filter before injection, and the purity was calculated by peak area normalization.

[0029] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0030] Example 1:

[0031] (1) Take 100 mL of fermentation broth, add 0.5 g of diatomaceous earth, stir at 50 °C for 30 min, centrifuge at 8000 rpm for 15 min, and obtain a clear liquid;

[0032] (2) The clarified liquid was first treated with a 0.22 μm microfiltration membrane and then with a 10 kDa ultrafiltration membrane, and the permeate was collected.

[0033] (3) Preheat the permeate to 50℃ and stir for 30 min. Add 0.1 g L-malic acid and then cool it down at a rate of 1℃ / min while stirring at 80 rpm. When the temperature drops to 45℃, maintain the temperature for 10 min. During the heat preservation section, use ultrasound assistance (40 kHz, 200 W) to promote crystal nucleus formation. Then, cool it down at a rate of 1℃ / min and maintain the temperature at 40℃ for 10 min. During the heat preservation section, use ultrasound assistance (40 kHz, 200 W) to promote crystal nucleus formation. Continue to perform pulsed gradient cooling treatment in the above cooling and heat preservation cycle for a total of 6 cycles until the temperature drops to 20℃. Then stop stirring and maintain the temperature for crystal growth until no new crystals are generated. The temperature growth time is about 2 h. Centrifuge and filter to separate the crystals from the mother liquor to obtain crude lysine acetate.

[0034] (4) The crude lysine acetate was dissolved in 5 times its weight of distilled water. The dissolved solution was then passed through an activated carbon column (5cm×20cm), a resin bed (D101 macroporous resin and AB-8 weakly polar resin were packed in a volume ratio of 3:1), and a MIPs column (prepared with L-histidine as the template molecule) at a flow rate of 1.5 BV / h to obtain a purified lysine acetate solution.

[0035] (5) The purified lysine acetate solution is recrystallized in the same way as in step (3) to prepare lysine acetate.

[0036] The test results showed that the yield of lysine acetate reached 68%, the purity of lysine acetate reached 99.83%, other amino acids: ND (<0.01%), heavy metals: <0.5ppm, solvent residue: <50ppm.

[0037] Example 2:

[0038] (1) Take 100 mL of fermentation broth, add 0.5 g of diatomaceous earth, stir at 50 °C for 30 min, centrifuge at 8000 rpm for 15 min, and obtain a clear liquid;

[0039] (2) The clarified liquid was first treated with a 0.22 μm microfiltration membrane and then with a 10 kDa ultrafiltration membrane, and the permeate was collected.

[0040] (3) Preheat the permeate to 50℃ and stir for 30 min. Add 0.1 g L-malic acid and then cool it down at a rate of 1℃ / min while stirring at 80 rpm. When the temperature drops to 45℃, maintain the temperature for 15 min. During the heat preservation section, use ultrasound assistance (40 kHz, 200 W) to promote crystal nucleus formation. Then, cool it down at a rate of 1℃ / min and maintain the temperature at 40℃ for 15 min. During the heat preservation section, use ultrasound assistance (40 kHz, 200 W) to promote crystal nucleus formation. Continue to perform pulsed gradient cooling treatment in the above cooling and heat preservation cycle for a total of 6 cycles until the temperature drops to 20℃. Then stop stirring and maintain the temperature for crystal growth until no new crystals are generated. The temperature growth time is about 2 h. Centrifuge and filter to separate the crystals from the mother liquor to obtain crude lysine acetate.

[0041] (4) The crude lysine acetate was dissolved in 5 times its weight of distilled water. The dissolved solution was then passed through an activated carbon column (5cm×20cm), a resin bed (D101 macroporous resin and AB-8 weakly polar resin were packed in a volume ratio of 3:1), and a MIPs column (prepared with L-histidine as the template molecule) at a flow rate of 1.6 BV / h to obtain a purified lysine acetate solution.

[0042] (5) The purified lysine acetate solution is recrystallized in the same way as in step (3) to prepare lysine acetate.

[0043] The test results showed that the yield of lysine acetate reached 70%, the purity of lysine acetate reached 99.68%, other amino acids: ND (<0.01%), heavy metals: <0.5ppm, solvent residue: <50ppm.

[0044] Comparative Example 1:

[0045] (1) Take 100 mL of fermentation broth, add 0.5 g of diatomaceous earth, stir at 50 °C for 30 min, centrifuge at 8000 rpm for 15 min, and obtain a clear liquid;

[0046] (2) The clarified liquid was first treated with a 0.22 μm microfiltration membrane and then with a 10 kDa ultrafiltration membrane, and the permeate was collected.

[0047] (3) Preheat the permeate to 50℃ and stir for 30 min. Add 0.1 g L-malic acid and then cool it at a rate of 2℃ / min while stirring at 80 rpm. When the temperature drops to 40℃, maintain the temperature for 10 min. During the heat preservation section, use ultrasound assistance (40 kHz, 200 W) to promote crystal nucleus formation. Then, cool it down at a rate of 2℃ / min to 30℃ and maintain the temperature for 10 min. During the heat preservation section, use ultrasound assistance (40 kHz, 200 W) to promote crystal nucleus formation. Continue to perform pulsed gradient cooling treatment in the above cooling and heat preservation cycle for a total of 3 cycles until the temperature drops to 20℃. Stop stirring and maintain the temperature for crystal growth until no new crystals are generated. The temperature growth time is about 6 h. Centrifuge and filter to separate the crystals from the mother liquor to obtain crude lysine acetate.

[0048] (4) The crude lysine acetate was dissolved in 5 times its weight of distilled water. The dissolved solution was then passed through an activated carbon column (5cm×20cm), a resin bed (D101 macroporous resin and AB-8 weakly polar resin were packed in a volume ratio of 3:1), and a MIPs column (prepared with L-histidine as the template molecule) at a flow rate of 1.5 BV / h to obtain a purified lysine acetate solution.

[0049] (5) The purified lysine acetate solution is recrystallized in the same way as in step (3) to prepare lysine acetate.

[0050] The test results showed that the yield of lysine acetate reached 62%, the purity of lysine acetate reached 98.21%, other amino acids: ND (<0.01%), heavy metals: <0.5ppm, solvent residue: <50ppm.

[0051] Comparative Example 2:

[0052] (1) Take 100 mL of fermentation broth, add 0.5 g of diatomaceous earth, stir at 50 °C for 30 min, centrifuge at 8000 rpm for 15 min, and obtain a clear liquid;

[0053] (2) The clarified liquid was first treated with a 0.22 μm microfiltration membrane and then with a 10 kDa ultrafiltration membrane, and the permeate was collected.

[0054] (3) Preheat the permeate to 50°C and stir for 30 min. Add 0.1 g L-malic acid and then cool it down at a rate of 1°C / min while stirring at 80 rpm. When the temperature drops to 45°C, keep it at a constant temperature for 10 min. Then cool it down at a rate of 1°C / min until it reaches 40°C and keep it at a constant temperature for 10 min. Continue to perform pulsed gradient cooling in the same way as the above cooling and holding sections. Repeat this process 6 times until the temperature drops to 20°C. Then stop stirring and keep the temperature constant for crystal growth until no new crystals are formed. The constant temperature crystal growth time is about 5 h. Separate the crystals from the mother liquor by centrifugation and filtration to obtain crude lysine acetate.

[0055] (4) The crude lysine acetate was dissolved in 5 times its weight of distilled water. The dissolved solution was then passed through an activated carbon column (5cm×20cm), a resin bed (D101 macroporous resin and AB-8 weakly polar resin were packed in a volume ratio of 3:1), and a MIPs column (prepared with L-histidine as the template molecule) at a flow rate of 1.5 BV / h to obtain a purified lysine acetate solution.

[0056] (5) The purified lysine acetate solution is recrystallized in the same way as in step (3) to prepare lysine acetate.

[0057] The test results showed that the yield of lysine acetate reached 59%, the purity of lysine acetate reached 99.16%, other amino acids: ND (<0.01%), heavy metals: <0.5ppm, solvent residue: <50ppm.

[0058] Comparative Example 3:

[0059] (1) Take 100 mL of fermentation broth, add 0.5 g of diatomaceous earth, stir at 50 °C for 30 min, centrifuge at 8000 rpm for 15 min, and obtain a clear liquid;

[0060] (2) The clarified liquid was first treated with a 0.22 μm microfiltration membrane and then with a 10 kDa ultrafiltration membrane, and the permeate was collected.

[0061] (3) Preheat the permeate to 50°C and stir for 30 min. Cool it down at 1°C / min with a stirring speed of 80 rpm until it reaches 45°C. Hold the temperature for 10 min and use ultrasound (40 kHz, 200 W) to promote crystal nuclei formation during the holding period. Then cool it down at 1°C / min until it reaches 40°C. Hold the temperature for 10 min and use ultrasound (40 kHz, 200 W) to promote crystal nuclei formation during the holding period. Continue to perform pulsed gradient cooling in the same way as the cooling and holding periods. Repeat this process 6 times until the temperature drops to 20°C. Then stop stirring and keep the temperature constant for crystal growth until no new crystals are formed. The constant temperature crystal growth time is about 5 h. Separate the crystals from the mother liquor by centrifugation and filtration to obtain crude lysine acetate.

[0062] (4) The crude lysine acetate was dissolved in 5 times its weight of distilled water. The dissolved solution was then passed through an activated carbon column (5cm×20cm), a resin bed (D101 macroporous resin and AB-8 weakly polar resin were packed in a volume ratio of 3:1), and a MIPs column (prepared with L-histidine as the template molecule) at a flow rate of 1.5 BV / h to obtain a purified lysine acetate solution.

[0063] (5) The purified lysine acetate solution is recrystallized in the same way as in step (3) to prepare lysine acetate.

[0064] The yield of lysine acetate was 64%, the purity of lysine acetate was 97.25% (HPLC area normalization method), other amino acids: ND (<0.01%), heavy metals: <0.5ppm, solvent residue: <50ppm.

[0065] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for purifying L-lysine acetate, characterized in that, Includes the following steps: S1. Take the fermentation broth containing L-lysine acetate, add diatomaceous earth for adsorption treatment, and centrifuge to obtain a clear liquid; S2. Pre-treat the clarified liquid sequentially using microfiltration and ultrafiltration membranes, and collect the permeate. S3. Preheat the permeate to 40~60℃. Add 0.08~0.15% L-malic acid to the permeate by mass ratio. Cool the solution at a rate of 4~6℃ / 5min while stirring at 60~100 rpm. After 5min, maintain the temperature at a constant temperature for 8~12min. During the temperature maintenance stage, use ultrasound to promote crystal nucleus formation. Continue the pulsed gradient cooling process by repeating the above cooling and temperature maintenance cycle for a total of 5~8 cycles until the temperature drops to 15~25℃. Then stop stirring and maintain the temperature at a constant temperature to grow crystals until no new crystals are formed. Separate the crystals from the mother liquor to obtain crude lysine acetate. S4. The crude lysine acetate was dissolved in water. The dissolved solution was then passed through an activated carbon column, a resin bed filled with D101 macroporous resin and AB-8 weakly polar resin, and a MIPs column prepared with L-histidine as the template molecule at a flow rate of 1~2 BV / h to obtain a purified lysine acetate solution. S5. The purified lysine acetate solution is recrystallized in the same way as in step (3) to prepare lysine acetate.

2. The refining method according to claim 1, characterized in that, The pulsed gradient cooling step is further preferably as follows: preheat the permeate to 45~55℃, add 0.08~0.12% L-malic acid to the permeate by mass ratio, and cool it at a rate of 4~6℃ / 5min while stirring at 80~100rpm. After 5min, maintain the temperature for 8~12min, and use ultrasound to promote crystal nucleation during the temperature maintenance stage. Continue the pulsed gradient cooling process in the above cooling and temperature maintenance cycle for a total of 5~7 cycles until the temperature drops to 18~22℃. Then stop stirring and maintain the temperature for crystal growth.

3. The refining method according to claim 1, characterized in that, In step S1, the amount of diatomaceous earth added is 0.1~1g / 100mL of fermentation broth.

4. The refining method according to claim 1, characterized in that, In step S1, centrifugation is performed at 6000~10000 rpm for 10~20 min.

5. The refining method according to claim 1, characterized in that, In step S2, the microfiltration membrane is a 0.22 μm microfiltration membrane.

6. The refining method according to claim 1, characterized in that, In step S2, the cutoff value of the ultrafiltration membrane is 5~15kDa.

7. The refining method according to claim 1, characterized in that, In step S3, the frequency of ultrasound assistance is 30~50kHz and the power is 150~250W.

8. The refining method according to claim 1, characterized in that, In step S4, D101 macroporous resin and AB-8 weakly polar resin are filled at a volume ratio of 2~4:1.