阅读说明：本技术 一种鱼油中二十碳五烯酸的分离纯化方法 (Separation and purification method of eicosapentaenoic acid in fish oil ) 是由 王希 陶冶 陈强强 郁明钧 于 2021-08-27 设计创作，主要内容包括：本发明提供了一种鱼油中二十碳五烯酸的分离纯化方法。本发明的鱼油中二十碳五烯酸的分离纯化方法,包括如下步骤：1)将鱼油粗品进行溶解、过滤,得到鱼油溶液；2)将步骤1)得到的鱼油溶液上样到装有UniSil微球的层析柱中进行层析,采用纯水和有机溶剂作为流动相对鱼油溶液进行洗脱；3)分段收集经步骤2)层析、洗脱后的目的峰值的鱼油溶液,对符合要求的组份液进行汇总,得到纯化的二十碳五烯酸。本发明的鱼油中二十碳五烯酸的分离纯化方法,仅需一步层析分离纯化鱼油中EPA,不仅纯度高、收率高且稳定,而且操作简单方便,所用固定相可重复利用,所用流动相量少节约,大大降低了成本。(The invention provides a method for separating and purifying eicosapentaenoic acid in fish oil. The method for separating and purifying the eicosapentaenoic acid in the fish oil comprises the following steps: 1) dissolving and filtering the crude fish oil product to obtain a fish oil solution; 2) loading the fish oil solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting the fish oil solution by using pure water and an organic solvent as mobile phases; 3) collecting the fish oil solution of the target peak value after chromatography and elution in the step 2) in sections, and summarizing the component liquid meeting the requirements to obtain the purified eicosapentaenoic acid. The method for separating and purifying eicosapentaenoic acid in fish oil only needs one-step chromatographic separation and purification of EPA in fish oil, not only has high purity, high and stable yield, but also has simple and convenient operation, can repeatedly utilize the used stationary phase, saves a small amount of the used mobile phase and greatly reduces the cost.)

1. A separation and purification method of eicosapentaenoic acid in fish oil is characterized by comprising the following steps:

1) dissolving and filtering a fish oil crude product with 70-75% EPA purity to obtain a fish oil solution with 10-20mg/mL EPA concentration;

2) loading the fish oil solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting the fish oil solution by using pure water and an organic solvent as mobile phases, wherein the volume percentage of the organic solvent in the mobile phases is 89.5-90.5%;

3) collecting the fish oil solution of the target peak value after chromatography and elution in the step 2) in sections, and summarizing the component liquid meeting the requirements to obtain the purified eicosapentaenoic acid.

2. The separation and purification method according to claim 1, wherein in step 2), the UniSil microspheres are C18UniSil microspheres;

preferably, the particle size of the C18UniSil microspheres is 10-30 μm, preferably 20 μm.

3. The separation and purification method according to claim 1 or 2, wherein in the step 2), the organic solvent is one of methanol, ethanol or acetonitrile; methanol is preferred.

4. The separation and purification method according to any one of claims 1 to 3, wherein the amount of the mobile phase used in step 2) is 6 to 8 column volumes, based on 1 column volume of the packing contained in the chromatography column.

5. The separation and purification method according to any one of claims 1 to 4, wherein in step 2), the specific process of elution is as follows: eluting the fish oil solution by adopting a mobile phase with an organic solvent volume percentage of 89.5-90.5%, wherein the elution time is 50-70min, and the flow rate of the mobile phase is 70-80 mL/min.

6. The separation and purification method according to any one of claims 1 to 5, wherein the step 2) further comprises a step of pre-column treatment of the chromatography column before loading.

7. The separation and purification method according to claim 6, wherein the specific process of the pre-column treatment is as follows: and removing impurities from the chromatographic column by using methanol, and balancing the chromatographic column after the impurities are removed by using a flow phase with the methanol volume percentage of 89.5-90.5%.

8. The separation and purification method according to any one of claims 1 to 7, wherein in step 1), the filtration is performed with a filter membrane having a pore size of 0.3 to 0.5 μm.

9. The separation and purification method according to any one of claims 1 to 8, wherein the separation and purification method comprises the steps of:

1) dissolving a fish oil crude product with 70-75% EPA purity in 89.5-90.5% methanol solution, and filtering with a filter membrane with a pore size of 0.3-0.5 μm to obtain a fish oil solution with 10-20mg/mL EPA concentration;

2) removing impurities from a chromatographic column filled with C18UniSil microspheres by using methanol, balancing the chromatographic column after impurity removal by using a mobile phase with an organic solvent volume percentage of 89.5-90.5%, loading the fish oil solution obtained in the step 1) into the chromatographic column subjected to balancing treatment, eluting the fish oil solution by using a mobile phase with an organic solvent volume percentage of 89.5-90.5%, wherein the elution time is 50-70min, and the flow rate of the mobile phase is 70-80 mL/min;

3) collecting the fish oil solution of the target peak value after chromatography and elution in the step 2) in sections, and summarizing the component liquid meeting the requirements to obtain the purified eicosapentaenoic acid.

10. Eicosapentaenoic acid obtained by the separation and purification method as claimed in any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of separation and purification, relates to a separation and purification method of eicosapentaenoic acid, and particularly relates to a separation and purification method of eicosapentaenoic acid in fish oil.

Background

Eicosapentaenoic acid (Eicosapentaenoica)cid) is an organic substance of formula C₂₀H₃₀O₂The molecular weight of the product is 302.451, the product is colorless to light yellow transparent liquid at normal temperature, and the product is odorless and has a certain odor after oxidation, and is often used for health care.

Omega-3 fatty acids, including EPA, have a positive role in the treatment of lung diseases, kidney diseases, type 2 diabetes, large bowel ulcers and crohn's disease. Omega-3 fatty acids (including EPA) present in fish oils have been shown to reduce adverse immune responses and to be effective in treating inflammation caused by autoimmune deficiencies, such as rheumatoid arthritis. Omega-3 fatty acids have been shown to promote the health of the circulatory system and prevent the accumulation of cholesterol and fat in the arterial wall, and supplementation with fish oils also allows diabetic patients to reduce hypertension. Maintaining the omega-3 fatty acid content of the body in a properly balanced position is essential for normal growth and development. The nutrition specialist advises that the infant should absorb various types of omega-3 fatty acids from the daily diet and supplements. According to the requirements of these recommendations, infants should absorb less than 0.1% of EPA in their daily diet.

Wherein the structural formula of EPA is shown as follows:

CN107586259A discloses a method for purifying unsaturated fatty acids and eicosapentaenoic acid, which comprises providing ethyl esterified fish oil, and separating unsaturated fatty acids containing eicosapentaenoic acid and docosahexaenoic acid in the ethyl esterified fish oil by simulated moving bed chromatography, thereby obtaining unsaturated fatty acids containing eicosapentaenoic acid and docosahexaenoic acid with high purity. According to the method for purifying the unsaturated fatty acid, the unsaturated fatty acid containing the eicosapentaenoic acid and the docosahexaenoic acid is separated from the fish oil by using the simulated moving bed chromatography, so that the separation efficiency can be effectively improved, and the high-purity unsaturated fatty acid containing the eicosapentaenoic acid and the docosahexaenoic acid can be obtained. However, the purity of eicosapentaenoic acid of this method is to be further improved.

CN107586609A discloses fish oil containing high-concentration ethyl eicosapentaenoate and a purification method thereof, relating to the field of preparation of ethyl eicosapentaenoate. The purification method comprises the following steps: obtaining the crystallization point temperature range of each fatty acid according to the content of each fatty acid in the fish oil; and then, cooling the fish oil at a cooling rate of 1-DEG C/h within the temperature range of the crystallization point of each fatty acid by adopting a dynamic melt crystallization method, so that impurity fatty acids contained in the fish oil are crystallized and separated out and then removed. The method ensures that the impurity fatty acid which is easy to crystallize in the fish oil is continuously crystallized and separated out by setting a proper cooling rate in the temperature range of the crystallization point, and simultaneously ensures that the eicosapentaenoic acid ethyl ester in the fish oil mother liquor is continuously concentrated, and the separation efficiency is high. The fish oil has high content of eicosapentaenoic acid ethyl ester, good biological activity and wide medical application. However, the yield of eicosapentaenoic acid from this process is in need of further improvement.

Therefore, it is necessary to provide a method for efficiently separating and purifying eicosapentaenoic acid from fish oil with high purity and high yield.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for separating and purifying eicosapentaenoic acid in fish oil, which can meet the requirement of purity of more than 97 percent only by one-step chromatographic purification and has high and stable purification yield.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for separating and purifying eicosapentaenoic acid in fish oil comprises the following steps:

1) dissolving and filtering a fish oil crude product with 70-75% EPA purity to obtain a fish oil solution with 10-20mg/mL EPA concentration;

2) loading the fish oil solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting the fish oil solution by using pure water and an organic solvent as mobile phases, wherein the volume percentage of the organic solvent in the mobile phases is 89.5-90.5%;

3) collecting the fish oil solution of the target peak value after chromatography and elution in the step 2) in sections, and summarizing the component liquid meeting the requirements to obtain the purified eicosapentaenoic acid.

In the step 2), the model of the UniSil microspheres is C18UniSil microspheres. The C18UniSil microsphere has a particle size of 10-30 μm and a pore diameter ofThe polymer microsphere has strictly controlled particle size and pore diameter structure, is monodisperse and has a pore channel structure, and has good pertinence when used as chromatographic packing. Preferably, the particle size of the C18UniSil microspheres is 20 μm.

In the step 1), the EPA purity of the crude fish oil is 70-75%, such as 70%, 71%, 72%, 73%, 74%, 75% and the like; the EPA concentration in the fish oil solution is 10-20mg/mL, for example, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg/mL, 18mg/mL, 19mg/mL or 20mg/mL, etc.

In the step 2), the organic solvent is one of methanol, ethanol or acetonitrile; methanol is preferred.

In the step 2), the volume ratio of the pure water to the organic solvent in the mobile phase is (9.5-10.5): (89.5-90.5).

The invention adopts the organic solvent and the purified water as the mobile phase, and can finish the elution of the EPA only by 6 to 8 column volumes, so the mobile phase used in the process is safe, pollution-free and low in cost, and the used mobile phase has less amount and shorter purification period. Wherein, the volume of the filler in the invention is 490mL, namely the volume of one column is 490 mL.

In the step 2), the specific process of elution is as follows: eluting the fish oil solution by adopting a flowing phase with an organic solvent volume percentage of 89.5-90.5%, wherein the elution time is 50-70min, for example, the elution time is 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min, 60min, 61min, 62min, 63min, 64min, 65min, 66min, 67min, 68min, 69min or 70 min; the flow rate of the mobile phase is 70-80mL/min, such as 70mL/min, 71mL/min, 72mL/min, 73mL/min, 74mL/min, 75mL/min, 76mL/min, 77mL/min, 78mL/min, 79mL/min, or 80mL/min, and the like.

Step 2) before loading, the method also comprises a step of carrying out column pretreatment on the chromatographic column.

The specific process of the column pretreatment is as follows: and (3) removing impurities from the chromatographic column by adopting pure methanol, and balancing the chromatographic column after impurity removal by adopting a flow phase with the volume percentage of an organic solvent being 89.5-90.5%.

In step 1), the filtration is performed by using a filter membrane with a pore size of 0.3-0.5 μm, for example, the pore size of the filter membrane is 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm or 0.5 μm.

As a preferable scheme of the invention, the method for separating and purifying the eicosapentaenoic acid in the fish oil comprises the following steps:

1) dissolving a fish oil crude product with 70-75% EPA purity in 89.5-90.5% methanol solution, and filtering with a filter membrane with a pore size of 0.3-0.5 μm to obtain a fish oil solution with an EPA concentration of 10-20 mg/mL;

2) removing impurities from a chromatographic column filled with C18UniSil microspheres by using pure methanol, balancing the chromatographic column after impurity removal by using a mobile phase with an organic solvent volume percentage of 89.5-90.5%, loading the fish oil solution obtained in the step 1) into the chromatographic column subjected to balancing treatment, eluting the fish oil solution by using a mobile phase with an organic solvent volume percentage of 89.5-90.5%, wherein the elution time is 50-70min, and the flow rate of the mobile phase is 70-80 mL/min;

3) collecting the fish oil solution of the target peak value after chromatography and elution in the step 2) in sections, and summarizing the component liquid meeting the requirements to obtain the purified eicosapentaenoic acid.

The second object of the present invention is to provide eicosapentaenoic acid obtained by the separation and purification method according to the first object.

Compared with the prior art, the invention has the beneficial effects that:

the method for separating and purifying eicosapentaenoic acid in fish oil only needs one-step chromatography to separate and purify EPA in fish oil, not only has high purity, high and stable yield, but also has simple and convenient operation, the used stationary phase can be recycled, the used mobile phase is less and saved, the elution of EPA can be completed by using organic solvent and purified water as the mobile phase and only using 6 to 8 column volumes, so the mobile phase used in the process is safe, pollution-free and low in cost, the used mobile phase is less, the purification period is short, and the cost is greatly reduced. Specifically, the requirement of purity of more than 98% can be met only by one-step chromatographic purification, and the purification yield is more than 95%.

Drawings

FIG. 1 is a scanning electron micrograph of C18UniSil microspheres used in example 1 of the present invention;

FIG. 2 is a gas chromatographic analysis of eicosapentaenoic acid (EPA) in fish oil before purification of example 1 of the present invention;

FIG. 3 is a gas chromatographic analysis of eicosapentaenoic acid (EPA) in the purified fish oil of example 1 of the present invention.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached figures 1-3.

Unless otherwise specified, various starting materials of the present invention are commercially available or prepared according to conventional methods in the art.

Example 1

1) Taking a fish oil crude product with EPA purity of 73%, adding 89.5% methanol for dissolving, wherein the EPA content in the solution is 15 mg/mL; after the solution is clarified, filtering the solution by using a filter membrane with the aperture of 0.45 mu m, and collecting filtrate for later use;

2) A50X 250mm column, UniSilC18 (available from Suzhou Naichi micro-Tech Co., Ltd., particle size 20 μm, pore size)) The packed volume of the chromatographic column used as the chromatographic column packing is 490 mL; pre-column pretreatment is carried out on the chromatographic column, pure methanol is firstly used for removing impurities, and then the flow of which the volume percentage of methanol is 89.5 percent is used for carrying out the pretreatment on the chromatographic column after the impurities are relatively removedBalancing; then eluting for 60min by adopting a flow phase with the methanol volume percentage of 89.5 percent relative to the fish oil solution, and controlling the flow rate at 75 mL/min;

3) and collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by gas chromatography to obtain the eluent with the purity of 98.52% of EPA and the yield of 95.6%.

Fig. 1 is a scanning electron micrograph of the C18UniSil microspheres used in example 1, and it can be seen that the C18UniSil microspheres have a strictly controlled particle size and pore size structure, and are monodisperse microspheres having a pore structure. Fig. 2 shows the gas chromatographic analysis of eicosapentaenoic acid in the fish oil before purification, which was found to have some impurities. Fig. 3 shows the gas chromatographic analysis of eicosapentaenoic acid in the purified fish oil, showing very few impurities and very small peaks.

Example 2

1) Taking a fish oil crude product with the EPA purity of 73%, and adding a 90.5% methanol solution, wherein the EPA content in the solution is 15 mg/mL; after the solution is clarified, filtering the solution by using a filter membrane with the aperture of 0.45 mu m, and collecting filtrate for later use;

2) A50X 250mm column, UniSilC18 (available from Suzhou Naichi micro-Tech Co., Ltd., particle size 20 μm, pore size)) The packed volume of the chromatographic column used as the chromatographic column packing is 490 mL; pre-column pretreatment is carried out on the chromatographic column, impurity removal is carried out by using a high-concentration organic phase, and then the flow with the methanol volume percentage of 90.5 percent is used for balancing the chromatographic column after impurity removal; then eluting for 60min by using a flow phase with 90.5 percent of methanol volume percent relative to the fish oil solution, and controlling the flow rate at 75 ml/min;

3) and collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by gas chromatography to obtain the eluent with the purity of EPA of 98.05% and the yield of 96.1%.

Example 3

This example is different from example 1 in that the column pretreatment, that is, the impurity removal and the equilibration of the column were not performed, and the rest was the same as example 1.

And collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by gas chromatography to obtain the eluent with the purity of EPA of 98.33% and the yield of 95.4%.

Example 4

This example is different from example 1 in that the organic solvent used in the mobile phase for the equilibration and elution processes was ethanol, and the rest was the same as example 1.

And collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by gas chromatography to obtain the eluent with the purity of 98.09% of EPA and the yield of 95.3%.

Comparative example 1

This comparative example differs from example 1 in that the packed phase in the column was a commercial non-uniform particle size distribution of 15 μmC18, and the rest was the same as in example 1.

And collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by gas chromatography to obtain the eluent with the EPA purity of 96.7% and the yield of 90.8%.

Comparative example 2

The comparative example is a conventional EPA purification method, and a molecular rectification method is directly adopted.

The purity of EPA in the fraction was 85%.

Comparative example 3

This comparative example is different from example 1 in that the mobile phase used for the equilibration and elution treatments was pure methanol, and the rest was the same as example 1.

And collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by gas chromatography to obtain the eluent with the EPA purity of 90% and the yield of 96%.

Comparative example 4

This example is different from example 1 in that the mobile phase used in the equilibration and elution processes was 50% by volume of an organic solvent, and the rest was the same as example 1.

And collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by gas chromatography to obtain the eluent with the EPA purity of 98.5% and the yield of 80%.

Comparative example 5

This comparative example is different from example 1 in that the mobile phase used for the equilibration and elution treatments was 95% by volume of an organic solvent, and the rest was the same as example 1.

And collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by gas chromatography to obtain the eluent with the purity of EPA of 90.1% and the yield of 97%.

The method is used for deep purification of eicosapentaenoic acid (EPA) in the fish oil, can meet the requirement that the EPA purity is more than 98% only by one-step chromatographic purification, and has the purification yield of more than 95% and stability.

The present invention is illustrated by the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, i.e. it is not meant to imply that the present invention must rely on the above-mentioned detailed process equipment and process flow to be practiced. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.