Method for extracting high-purity Ectoine from Ectoine mother liquor

Abstract

The invention relates to the technical field of biochemical engineering, in particular to a method for extracting high-purity Ectoine from Ectoine mother liquor. According to the method, the Ectoine mother liquor is purified into high-purity Ectoine solid through the steps of chromatographic separation, ion exchange process, concentration and decoloration and crystallization in sequence; chromatographic separation adopts a specific filler (silica gel bonded octadecylsilane (C18) or a strong cation exchange resin filler), and separation is carried out at room temperature by controlling the sample size and the flow rate; in the ion exchange process, strong-acid cation exchange resin and strong-base anion exchange resin are connected in series to remove cation and anion impurities; concentrating and decoloring through vacuum concentration and activated carbon decoloring; and adding low-molecular alcohol for cooling crystallization, and carrying out spinning filtration and drying to obtain high-purity Ectoine solid. According to the method, the purity of Ectoin can reach 99% or above, and the method has the advantages of being low in cost, environmentally friendly and suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, specifically to a method for extracting high-purity ectoine from ectoine mother liquor. Background Technology

[0002] Ectoin (tetrahydromethylpyrimidine carboxylic acid) is an important biocompatible compound with broad application prospects in cosmetics, pharmaceuticals, food and other fields. The market demand for high-purity ectoin is showing a continuous growth trend.

[0003] Ectoin preparation includes chemical synthesis and bio-fermentation. Chemical synthesis uses L-acetylasparagine as a raw material, involving condensation to form a lactam, protection, carbonyl reduction, amide hydrolysis, amino deprotection, hydrochlorination, and cyclization to obtain the ectoin molecule. This method has a long synthesis process, each step is difficult to monitor, the reaction conditions are harsh, and post-processing is cumbersome, resulting in low yields. Furthermore, the synthesis requires the use of highly toxic sodium sulfide, posing certain risks. Due to the limitations of chemical synthesis, most existing ectoin preparations utilize bio-fermentation. In bio-fermentation, traditional methods of fermenting halophilic bacteria have been largely abandoned, and fermentation of genetically engineered Escherichia coli is more commonly used. This yields higher concentrations of ectoin fermentation broth, which can be further purified through filtration and crystallization to obtain pure ectoin.

[0004] During the crystallization and purification of ectoine, the fermentation broth undergoes multiple crystallization extractions, resulting in a mother liquor containing a certain amount of ectoine. This mother liquor is also rich in impurities such as inorganic salts, organic acids, proteins, and pigments. Traditional methods are insufficient to efficiently recover ectoine from this mother liquor, leading to resource waste and increased production costs. Therefore, developing an efficient and economical method for ectoine mother liquor purification is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting high-purity ectoine from ectoine mother liquor, in order to solve the problem that ectoine is difficult to recover efficiently from existing ectoine mother liquor, resulting in resource waste and increased production costs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for extracting high-purity ectoine from ectoine mother liquor, comprising the following steps:

[0007] S1, Chromatographic separation

[0008] Take the mother liquor of ectoine crystallization and separate it by chromatographic column to obtain the separated solution;

[0009] The packing material in the chromatographic column includes sodium-type strong ion exchange resin;

[0010] S2, Intersection

[0011] The S1 separation solution was passed sequentially through a series of strongly acidic cation-weakly basic anion-strongly basic anion exchange resins to obtain the ion exchange solution.

[0012] S3, Concentrated Decolorization

[0013] Take the S2 ion exchange solution, and after vacuum concentration, active decolorization, and secondary concentration, obtain ectoine crystal slurry;

[0014] S4, Crystallization

[0015] Take S3 ectoine crystal slurry, add low molecular weight alcohol, cool, crystallize, filter, wash, and dry to achieve high-purity extraction of ectoine from ectoine crystallization mother liquor.

[0016] Furthermore, in S1, after adsorption by the chromatographic column, purified water is used for elution, and the trend of ectoine content in the eluent is tracked by optical rotation. The ectoine solution with an optical rotation greater than or equal to +1.0° and a conductivity less than 1 ms in the later stage is taken as the separation solution.

[0017] Furthermore, the sodium-type strong ion exchange resin is of type ZG106Na; the column height-to-diameter ratio is 30:1, the operating temperature is 30–50℃; the ectoine concentration in the feed is 200–300 g / L, the feed flow rate is 0.5–1 BV / h, and the feed volume is 0.2–0.4 BV; the elution purification water flow rate is 1–2 BV / h, and the elution purification water volume is 0.5–1.0 BV.

[0018] Furthermore, in S2, after the S1 separation liquid is adsorbed by a strong acid cation exchange resin, it is first washed with purified water and then eluted with ammonia to obtain an eluent; the eluent is then passed through a weak base anion exchange resin and a strong base anion exchange resin in sequence, and then eluted with ammonia to obtain an ion exchange solution.

[0019] Furthermore, the strongly acidic cation exchange resin includes model D001; the weakly basic anion exchange resin includes models D318, D301F, and D301; and the strongly basic anion exchange resin includes models D201 and D730.

[0020] The separation solution is passed sequentially through a cation exchange resin and anion exchange resin at a flow rate of 1–3 BV / h. After adsorption by the strongly acidic cation exchange resin, it is washed with purified water until the conductivity is below 300 μS / cm, and then eluted with 1–2% dilute ammonia. The eluent with an optical rotation greater than 0.2° is collected. After adsorption by a weakly basic anion-strong basic anion exchange resin, it is eluted with 1–2% dilute ammonia. The solution from the strong basic anion exchange resin is collected. The ion exchange ends when the optical rotation is below 0.2°, yielding the ion exchange solution.

[0021] Further, in step S3, the temperature for vacuum concentration is 40–60°C, the pressure is -0.08–-0.1 MPa, and the solution is concentrated to 1 / 5–1 / 10 of its original volume; the amount of activated carbon added is 0.5–2% of the mass of the concentrated solution; the decolorization temperature is 30–50°C; the stirring intensity is 100–200 rpm; the decolorization time is 30–60 min; after decolorization, the activated carbon is removed by filtration; and the solution is concentrated a second time until crystals precipitate to obtain ectoine crystal slurry.

[0022] Furthermore, in S4, the low molecular weight alcohol includes ethanol; the low molecular weight alcohol is added in a fast-flow manner, with the amount added being 0.2 to 0.5 times the volume of the ectoine crystal slurry, and the flow time being 0.5 to 1 hour.

[0023] Furthermore, after the low molecular weight alcohol is added, the mixture is kept at 50°C for 0.5 hours; under a stirring intensity of 50-100 rpm, the slurry is rapidly cooled to below 10°C and maintained for 2-4 hours; after crystallization, the mixture is filtered, washed with low molecular weight alcohol, and then vacuum dried to obtain high-purity ectoine.

[0024] The beneficial effects of this invention are:

[0025] 1. High purity: Through the synergistic effect of chromatographic separation, ion exchange and concentration decolorization, various impurities are effectively removed, and the purity of ectoine can reach more than 99%, meeting the demand of the high-end market for high-purity products.

[0026] 2. Low cost: It reduces the use of complex equipment and the consumption of chemical reagents, recovers ectoine from the mother liquor, reduces production costs, improves production efficiency, and is suitable for large-scale industrial production.

[0027] 3. Environmentally friendly: Compared with traditional processes, it reduces the generation of sewage and waste, making it more environmentally friendly. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0029] The technical solution of this invention is as follows:

[0030] Chromatographic separation: Ectoin mother liquor is separated by passing it through a chromatographic column filled with specific packing material, such as silica gel bonded octadecylsilane (C18) or strong cation exchange resin packing material, and separation is carried out at room temperature; by utilizing the difference in adsorption and desorption of ectoin and impurities by the chromatographic column, ectoin is initially separated, and most of the macromolecular impurities and some pigments are removed.

[0031] Ion exchange process: The chromatographically separated solution is introduced into an ion exchange column, which uses a strong acid cation exchange resin and a strong base anion exchange resin connected in series. The solution is passed through the cation exchange column at a flow rate of 1-3 BV / h to remove cationic impurities. Then it is passed through the anion exchange column at the same flow rate to remove anionic impurities. Through the ion exchange process, the purity of the ectoine solution is further improved.

[0032] Concentration and Decolorization: The ectoine solution after ion exchange is concentrated under reduced pressure at a temperature of 40-60℃ and a pressure of -0.08 to -0.1 MPa to 1 / 5 to 1 / 10 of its original volume. Then, 0.5-2% (mass fraction) of activated carbon is added to the concentrate, and the solution is stirred and decolorized at 30-50℃ for 30-60 minutes. After decolorization, the activated carbon is removed by filtration, and the solution is further concentrated to obtain a high-purity ectoine solution.

[0033] Crystallization: Add low molecular weight alcohol to a high-purity ectoine solution, cool to below 10°C after addition, and after crystallization, filter, wash with low molecular weight alcohol, and dry to obtain high-purity ectoine product.

[0034] The ectoine mother liquor used in the following embodiments and comparative examples in this application is the proprietary mother liquor of Henan Chengguo Transformation Biotechnology Co., Ltd.

[0035] Ectocin is detected using the following methods:

[0036] In the chromatographic separation and ion exchange process control, the polarimetry method is used to quickly and roughly determine the content of ectoine in the material. A polarimeter and a 10cm funnel-type polarimeter tube are used. The specific operation is as follows: First, clean the polarimeter tube to ensure that there are no impurities left inside and outside the polarimeter tube. After rinsing the inner wall of the polarimeter tube twice with the material solution, take an appropriate amount of material solution and put it into the polarimeter tube. The polarimeter tube is placed in the polarimeter and the optical rotation value A is displayed at room temperature. Then the content of ectoine in the material (g / L) = A / 0.14.

[0037] High-performance liquid chromatography (HPLC) can accurately detect ectoin content. The chromatographic column is a Yuexu LP-C18 column, and the volume ratio of the mobile phase components is acetonitrile:pure water = 20:80. An ultraviolet detector is used with a detection wavelength of 210 nm, a column temperature of 40 ℃, an injection volume of 10 μL, and a flow rate of 0.6 mL / min. The sample to be tested needs to be diluted to a suitable concentration and filtered through a 0.22 μm aqueous filter membrane before being tested.

[0038] Example 1

[0039] Take a chromatographic column with a height-to-diameter ratio of 30:1, and pack it with sodium-type chromatographic resin ZG106Na. The resin packing volume is 2L (i.e., 1BV). Set aside for later use.

[0040] Chromatographic separation: Take 500 mL (0.25 BV) of ectoine mother liquor with an ectoine content of 260 g / L; inject the mother liquor into the chromatographic column at a flow rate of 1 BV / h at 40℃. After injection, elute with purified water at a flow rate of 2 BV / h, with a volume of 1 BV; use polarimetry to quickly track the trend of the material, discard the waste liquid in the early stage, and collect the eluent containing ectoine (optical rotation greater than or equal to +1.0° and conductivity less than 1 ms).

[0041] Ion exchange process: The chromatographically separated collected solution is passed sequentially through 1L of strong acid cation exchange resin, 300mL of weak base anion exchange resin, and 200mL of strong base anion exchange resin at a flow rate of 1BV / h. After adsorption on the strong acid cation exchange resin column, the cation exchange column is rinsed with purified water until the conductivity of the effluent is below 300µs / cm, and then eluted with 1% ammonia. The eluent with an optical rotation greater than 0.2° is collected. The eluent is then sequentially passed through a weak base anion exchange resin column and a strong base anion exchange resin column. The content of the material is determined by polarimetry. Ion exchange ends when the optical rotation is below 0.2°, and the ion exchange solution is collected.

[0042] Concentration and decolorization: The ion-exchange solution was concentrated to 500 mL under reduced pressure at 50℃ and -0.09 MPa. 2% (mass fraction relative to the concentrate) of activated carbon was added to the concentrate, and the solution was stirred at 40℃ for 45 min at a stirring intensity of 150 rpm. After filtering to remove the activated carbon, the solution was further concentrated until crystals precipitated, yielding 327 mL of crystal slurry.

[0043] Crystallization: Add 100 mL of ethanol to the crystal slurry. The ethanol addition time is controlled at 40 min. After the addition is completed, keep it at 50 °C for 0.5 h. Then, under a stirring intensity of 100 rpm, quickly cool it to 5 °C and keep it at 2 h. Filter the mixture and wash the filter cake with ethanol to obtain 96.5 g of ectoine solid. The purity was tested to be 99.2%.

[0044] Example 2

[0045] Take a chromatographic column with a height-to-diameter ratio of 30:1, and pack it with sodium-type chromatographic resin ZG106Na. The resin packing volume is 2L (i.e., 1BV). Set aside for later use.

[0046] Chromatographic separation: Take 400 mL of ectoine mother liquor with an ectoine content of 300 g / L and inject the mother liquor into the chromatographic column at a flow rate of 0.5 BV / h. After injection, elute with purified water at a flow rate of 1 BV / h and a volume of purified water of 0.5 BV. Use polarimetry to quickly track the trend of the material, discard the waste liquid in the early stage, and collect the eluent containing ectoine.

[0047] Ion exchange process: The chromatographically separated collected solution is passed sequentially through 1L of strongly acidic cation exchange resin, 300mL of weakly basic anion exchange resin, and 200mL of strongly basic anion exchange resin at a flow rate of 2BV / h. After adsorption on the strongly acidic cation exchange resin column, the column is rinsed with purified water until the conductivity of the effluent is below 300µs / cm, and then eluted with 1.5% ammonia. The eluent with an optical rotation greater than 0.2° is collected. The eluent is then sequentially passed through a weakly basic anion exchange resin column and a strongly basic anion exchange resin column. The content of the material is determined by polarimetry. Ion exchange ends when the optical rotation is below 0.2°, and the ion exchange solution is collected.

[0048] Concentration and decolorization: The ion-exchange solution was concentrated to 300 mL under reduced pressure at 45℃ and -0.085 MPa. 1.5% (mass fraction relative to the concentrate) of activated carbon was added to the concentrate, and the solution was stirred at 35℃ for 50 min at a stirring intensity of 150 rpm. After filtering to remove the activated carbon, the solution was further concentrated until crystals precipitated, yielding 283 mL of crystal slurry.

[0049] Crystallization: Add 80 mL of ethanol to the crystal slurry. The ethanol addition time is controlled at 30 min. After the addition is completed, keep it at 50 °C for 0.5 h. Then, under a stirring intensity of 100 rpm, rapidly cool it to 5 °C and keep it at 3 h. Filter the mixture and wash the filter cake with ethanol to obtain 92.8 g of ectoine solid. The purity was tested to be 99.5%.

[0050] Comparative Example 1

[0051] In this comparative example, only strong acidic cation exchange resin and strong basic anion exchange resin were used, and other conditions were the same as in Example 1; the resulting ectoine solid purity was only 95%, and the yield was low.

[0052] Comparative Example 2

[0053] In this comparative example, the chromatographic separation step was omitted, and everything else was the same as in Example 1. The purity of the obtained ectoine solid was 97%, but it still contained a lot of impurities and could not meet the requirements of a high-purity product.

[0054] By comparing the examples and comparative examples, it is clear that the purification process of the present invention has significant advantages in improving the purity and yield of ectoine.

[0055] This invention is not limited to the preferred embodiments described above. Anyone can derive other forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. A method for extracting high-purity ectoine from ectoine mother liquor, characterized in that, Includes the following steps: S1, Chromatographic separation Take the mother liquor of ectoine crystallization and separate it by chromatographic column to obtain the separated solution; The packing material in the chromatographic column includes sodium-type strong ion exchange resin; S2, Intersection The S1 separation solution was passed sequentially through a series of strongly acidic cation-weakly basic anion-strongly basic anion exchange resins to obtain the ion exchange solution. S3, Concentrated Decolorization Take the S2 ion exchange solution, and after vacuum concentration, active decolorization, and secondary concentration, obtain ectoine crystal slurry; S4, Crystallization Take S3 ectoine crystal slurry, add low molecular weight alcohol, cool, crystallize, filter, wash, and dry to achieve high-purity extraction of ectoine from ectoine crystallization mother liquor.

2. The method for extracting high-purity ectoine from ectoine mother liquor according to claim 1, characterized in that: In S1, after adsorption by the chromatographic column, purified water is used for elution. The trend of ectoine content in the eluent is tracked by optical rotation. The ectoine solution with an optical rotation greater than or equal to +1.0° and a conductivity less than 1 ms in the later stage is taken as the separation solution.

3. The method for extracting high-purity ectoine from ectoine mother liquor according to claim 2, characterized in that: The sodium-type strong ion exchange resin is model ZG106Na; the column height-to-diameter ratio is 30:1, the operating temperature is 30–50℃; the concentration of ectoine in the feed is 200–300 g / L, the feed flow rate is 0.5–1 BV / h, and the feed volume is 0.2–0.4 BV; the elution purification water flow rate is 1–2 BV / h, and the elution purification water volume is 0.5–1.0 BV.

4. The method for extracting high-purity ectoine from ectoine mother liquor according to claim 1, characterized in that: In S2, after the S1 separation liquid is adsorbed by a strong acid cation exchange resin, it is first washed with purified water and then eluted with ammonia to obtain an eluent; the eluent is then passed through a weak base anion exchange resin and a strong base anion exchange resin in sequence, and then eluted with ammonia to obtain an ion exchange solution.

5. The method for extracting high-purity ectoine from ectoine mother liquor according to claim 4, characterized in that: The strongly acidic cation exchange resins include model D001; the weakly basic anion exchange resins include models D318, D301F, and D301; and the strongly basic anion exchange resins include models D201 and D730. The separation solution is passed sequentially through a cation exchange resin and anion exchange resin at a flow rate of 1–3 BV / h. After adsorption by the strongly acidic cation exchange resin, it is washed with purified water until the conductivity is below 300 μS / cm, and then eluted with 1–2% dilute ammonia. The eluent with an optical rotation greater than 0.2° is collected. After adsorption by a weakly basic anion-strong basic anion exchange resin, it is eluted with 1–2% dilute ammonia. The solution from the strong basic anion exchange resin is collected. The ion exchange ends when the optical rotation is below 0.2°, yielding the ion exchange solution.

6. The method for extracting high-purity ectoine from ectoine mother liquor according to claim 1, characterized in that: In step S3, the temperature for vacuum concentration is 40–60°C, the pressure is -0.08–-0.1 MPa, and the solution is concentrated to 1 / 5–1 / 10 of its original volume. The amount of activated carbon added is 0.5–2% of the mass of the concentrated solution. The decolorization temperature is 30–50°C, the stirring intensity is 100–200 rpm, and the decolorization time is 30–60 min. After decolorization, the activated carbon is removed by filtration. The solution is then concentrated a second time until crystals precipitate to obtain ectoine crystal slurry.

7. The method for extracting high-purity ectoine from ectoine mother liquor according to claim 1, characterized in that: In S4, the low molecular weight alcohol includes ethanol; the low molecular weight alcohol is added in a fast-flow manner, with the amount added being 0.2 to 0.5 times the volume of the ectoine crystal slurry, and the flow time being 0.5 to 1 hour.

8. The method for extracting high-purity ectoine from ectoine mother liquor according to claim 7, characterized in that: After the low molecular weight alcohol is added, the mixture is kept at 50°C for 0.5 hours; under a stirring intensity of 50-100 rpm, the crystal slurry is rapidly cooled to below 10°C and maintained for 2-4 hours; after crystallization, the mixture is filtered, washed with low molecular weight alcohol, and then vacuum dried to obtain high-purity ectoine.