CN107586609B

CN107586609B - Fish oil containing high-concentration ethyl eicosapentaenoate and purification method thereof

Info

Publication number: CN107586609B
Application number: CN201711021946.6A
Authority: CN
Inventors: 梁洪; 毛华良; 徐风行
Original assignee: Ningbo Yake Biotechnology Co ltd
Current assignee: Ningbo Yake Biotechnology Co ltd
Priority date: 2017-10-27
Filing date: 2017-10-27
Publication date: 2020-08-21
Anticipated expiration: 2037-10-27
Also published as: CN107586609A

Abstract

The invention provides fish oil containing high-concentration ethyl eicosapentaenoate and a purification method thereof, and relates 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 at a cooling rate of 1-3 ℃/hour within the temperature range of the crystallization point of each fatty acid by adopting a dynamic melting 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.

Description

Fish oil containing high-concentration ethyl eicosapentaenoate and purification method thereof

Technical Field

The invention relates to the field of preparation of eicosapentaenoic acid ethyl ester, and particularly relates to fish oil containing high-concentration eicosapentaenoic acid ethyl ester and a purification method thereof.

Background

Eicosapentaenoic acid (EPA), an essential fatty acid which cannot be synthesized by human body, belongs to omega-3 type long chain polyunsaturated fatty acid and is mainly derived from fish oil. It has effects in regulating endocrine, enhancing immunity, dilating blood vessel, reducing serum cholesterol and low density lipoprotein level, reducing platelet aggregation, relieving inflammation, and resisting cancer. The high-purity EPA can be used for preventing and treating diseases such as atherosclerosis, coronary heart disease, hypertension, inflammatory diseases, tumor, senile dementia and the like, and has development prospect in the field of pharmaceutical and pharmaceutical industry. Because the physiological functions of various fatty acids are not completely the same, and some impurities are harmful to human bodies, the development of high-purity EPA monomers is of great significance.

At present, the methods for separating and purifying fish oil reported in the prior art mainly comprise: molecular distillation, rectification, low-temperature crystallization, urea inclusion, lipase, supercritical fluid extraction, silver resin chromatography, silver nitrate complexation, and the like. Wherein, the products usually prepared by a molecular distillation method, a low-temperature crystallization method, a urea inclusion method, a lipase method and a supercritical fluid extraction method are the mixture of eicosapentaenoic acid ethyl ester (EPA-EE) and docosahexaenoic acid ethyl ester (DHA-EE), and the purity of the EPA-EE monomer is hardly over 80 percent. Silver resin chromatography and silver nitrate complexation also have the risks of causing pollution and bringing impurities.

Disclosure of Invention

The first purpose of the invention is to provide a method for purifying fish oil containing high-concentration ethyl eicosapentaenoate, which enables impurity fatty acid easy to crystallize in the fish oil to be crystallized and separated out continuously by setting proper gradient temperature, and enables the ethyl eicosapentaenoate in the fish oil mother liquor to be concentrated continuously, thereby having high separation efficiency.

The second purpose of the invention is to provide the fish oil containing high-concentration eicosapentaenoic acid ethyl ester prepared by the method, wherein the content of the eicosapentaenoic acid ethyl ester in the fish oil is high, and the application is wide.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a method for purifying fish oil containing high-concentration ethyl eicosapentaenoate, which 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 at a cooling rate of 1-3 ℃/hour within the temperature range of the crystallization point of each fatty acid by adopting a dynamic melting crystallization method, so that impurity fatty acids contained in the fish oil are crystallized and separated out and then removed.

The fish oil containing high-concentration ethyl eicosapentaenoate prepared by the purification method.

Compared with the prior art, the beneficial effects of the invention comprise:

according to the method for purifying the fish oil containing the high-concentration eicosapentaenoic acid ethyl ester, provided by the invention, the impurity fatty acid easy to crystallize in the fish oil is continuously crystallized and precipitated by setting a proper cooling rate in the temperature range of the crystallization point through a mode of combining dynamic melting crystallization and program cooling, meanwhile, the eicosapentaenoic acid ethyl ester in the fish oil mother liquor is continuously concentrated, the separation of the eicosapentaenoic acid ethyl ester and other impurity fatty acids in a eutectic form is avoided, the separation efficiency is high, and the product yield is high. The content of the eicosapentaenoic acid ethyl ester in the obtained fish oil product is greatly increased (from 20-50 wt% in the raw materials to 60-90 wt%), and meanwhile, the adverse phenomena that fatty acid in the fish oil is oxidized at high temperature to generate trans-fatty acid, the bioactivity is reduced and the like can be avoided.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a trend chart of the crystallization point of the ethyl linoleate in the raw fish oil changing along with the change of the mass fraction of the ethyl linoleate, which is drawn according to experimental data (the abscissa is the number of times of measuring samples and the ordinate is the mass fraction);

FIG. 2 is a schematic structural diagram of a dynamic crystallization apparatus provided in an embodiment;

FIG. 3 is a chart of the DSC differential scanning calorimeter analyzing the melting point and crystallization point of the raw material components of the Chilean anchovy oil in example 1;

FIG. 4 is a gas chromatography spectrum of the fish oil containing high concentration of EPA-EE prepared in example 1.

Reference numbers: 110-a crystallizer; 111-refrigerant accommodating space; 112-an insulating layer; 113-a refrigerant inlet; 114-refrigerant outlet; 115-feed port; 116-a discharge port; 117-upper baffle; 118-a lower baffle; 119-a crystallization tube; 120-mother liquor recycle tank; 130-a collection tank; 140-a circulation pump; 150-refrigerating and heating equipment.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The inventor researches and discovers that fatty acid in fish oil is easy to generate oxidation reaction at the temperature of more than 60 ℃, decomposition reaction at the temperature of more than 130 ℃, and trans-EPA-EE is formed by eicosapentaenoic acid ethyl ester (EPA-EE) at higher temperature. The inventor finds in a molecular distillation experiment that when the set temperature reaches 140 ℃, the peroxide value of the fish oil sample is obviously increased, and a coking phenomenon occurs. Various trans fatty acids (such as trans EPA-EE) and peroxidation products can cause harm to human health. Therefore, the size of the peroxide value and the content of trans-fatty acids are of great importance for the quality of fish oil containing eicosapentaenoic acid ethyl ester.

In order to prevent the fatty acid in the fish oil from generating oxidation, decomposition and other reactions in the separation and purification process, generating trans-fatty acid and reducing the bioactivity, the inventor proposes the following technical scheme:

the present embodiment provides a method for purifying fish oil containing high-concentration ethyl eicosapentaenoate, comprising:

step S1, obtaining the crystallization point temperature range of each fatty acid according to the content of each fatty acid in the fish oil;

the fish oil is a complex molten mixture consisting of a plurality of fatty acids, wherein the phenomenon of eutectic melting is generated in most fatty acid components when the temperature is changed, so that the components of a crystallized product and a mother solution after static crystallization are approximately the same, the crystallization time is long, and the separation effect is not obvious.

Therefore, in order to avoid eutectic melting of eicosapentaenoic acid ethyl ester and other impurity fatty acids during the separation and purification process, it is necessary to obtain the crystallization point temperature range of each fatty acid according to the content of each fatty acid component in the fish oil raw material.

However, due to the complex eutectic and eutectic phenomena among the fatty acid components in the fish oil, the melting point and the crystallization point of a single fatty acid in the fish oil are changed along with the change of the content of each component, for example, the crystallization point temperature of the ethyl linolenate in the fish oil raw material is gradually reduced along with the reduction of the mole fraction of the ethyl linolenate, as shown in fig. 1, when the mass fraction of the ethyl linolenate in the mother liquor is 2.1%, the crystallization temperature is about-60 ℃; when the mass fraction of the ethyl linolenate in the mother liquor is reduced to 1.45 percent, the crystallization temperature is about-75 ℃. Therefore, it is difficult to measure a specific value of the crystallization point temperature of a single fatty acid in practical operation.

Meanwhile, the fish oil raw materials from different sources and the fish oil raw materials from the same source have different contents of each fatty acid component, which also causes difficulty in finding the crystallization point of each fatty acid component. Through experiments, the crystallization points of the components in the fish oil and data in literature are greatly different.

Therefore, in order to overcome the problem of finding out the crystallization point of the impurity fatty acid, the inventor adopts a thermal analysis method to analyze the sample, for example, adopts differential scanning calorimetry to estimate the crystallization point range of each component in the fish oil sample, and uses the crystallization point range as a reference for setting the gradient temperature in the subsequent separation process.

And step S2, cooling at a cooling rate of 1-3 ℃/h within the temperature range of the crystallization point of each fatty acid by adopting a dynamic melting crystallization method, so that impurity fatty acids contained in the fish oil are crystallized and separated out, and then the impurity fatty acids are removed, thereby obtaining the fish oil containing high-concentration ethyl eicosapentaenoate.

The dynamic melting crystallization is a new separation technique, it is according to the difference of freezing point among the substances to be separated, make the liquid supplies reach the partial crystallization to realize through reducing the temperature of the liquid supplies progressively in the course of flowing, the solid phase that is crystallized out has different chemical compositions with surplus mother liquor, thus achieve the goal of separating and purifying. The driving force of the dynamic melting crystallization process is the supersaturation degree or supercooling degree of a certain component in the molten liquid in the flowing state, and the crystallization process is that the certain component is in the supersaturation state in the molten liquid in the process of gradually reducing the temperature of the molten liquid in the flowing state, starts nucleation and gradually grows into crystals.

The separation of each impurity fatty acid from the mother liquor is realized by the temperature decrease in the crystallization point temperature range in a targeted manner based on the crystallization point temperature range of each fatty acid obtained in step S1. The impurity fatty acids contained in fish oil are mainly: arachidonic acid ethyl ester, linolenic acid ethyl ester and oleic acid ethyl ester, and other fatty acids in small quantities, wherein the crystallization point temperatures of the impurity fatty acids are different and are all far higher than that of the target product EPA-EE. The inventor experiments prove that no crystallization phenomenon of EPA-EE occurs at the temperature below-120 ℃. Thus, in step S2, fatty acids as other impurities in the raw material mother liquor are removed from the mother liquor by crystallization, whereby a tail liquor containing EPA-EE at a high concentration is obtained. Meanwhile, crystals of a certain fatty acid monomer in different crystallization point temperature ranges, such as arachidonic acid ethyl ester, linolenic acid ethyl ester, oleic acid ethyl ester and the like, can be obtained, and high-quality byproducts can be obtained through simple refining.

Further, the dynamic melt crystallization method comprises: the fish oil is circulated through the crystallization vessel and the crystallization vessel is cooled within the temperature range of the crystallization point.

Wherein, circulating the fish oil through the crystallization vessel means: introducing fish oil from one end of a crystallization container, crystallizing the fish oil in the crystallization container in the flowing process to obtain a first crystal and a first tail liquid, and then introducing the first tail liquid into the crystallization container again to perform the circulation continuously until each impurity fatty acid in the fish oil is crystallized and separated in the temperature range of the crystallization point of the impurity fatty acid to obtain the tail liquid of high-concentration EPA-EE. Preferably, the crystallization vessel is a vertical falling film crystallizer.

Furthermore, the temperature reduction rate of the temperature reduction is 1-3 ℃/hour, namely the temperature reduction of the crystallization container is carried out within the temperature range of the crystallization point of each impurity fatty acid at the rate of 1-3 ℃ per hour, so that the impurity fatty acid at the crystallization point temperature is crystallized and precipitated. The cooling rate is 1-3 ℃/h, or 2.5-2.5 ℃/h, or 1.7-2.3 ℃/h, or 1.8-2.2 ℃/h, or 1.9-2.1 ℃/h, or 2 ℃/h.

Further, the flow rate of the fish oil circulating through the crystallization container is 100-200L/h, or 120-180L/h, or 130-170L/h, or 140-160L/h, or 145-155L/h, or 150L/h. The flow rate of the fish oil circulating through the crystallization container is not too large or too small, the flow rate is too large, and the crystallization is not sufficient; and the flow velocity is too small, the crystallization time is long, and the separation efficiency is low.

Furthermore, the impurity fatty acid in the fish oil comprises arachidonic acid ethyl ester, the content of the arachidonic acid ethyl ester is 2-4 wt%, and the crystallization point temperature range of the arachidonic acid ethyl ester is-25 to-70 ℃. Namely, when the content of the arachidonic acid ethyl ester in the fish oil is 2-4 wt%, the crystallization point temperature range of the arachidonic acid ethyl ester monomer is-25 to-70 ℃, and in the dynamic melting crystallization process, the temperature of a crystallization container is firstly reduced from room temperature to-25 ℃, and then is reduced at the temperature reduction rate of 1-3 ℃/hour between-25 ℃ and-70 ℃, so that the arachidonic acid ethyl ester is crystallized and precipitated. Preferably, during the dynamic melt crystallization, the composition of the circulating mother liquor is monitored in real time until no ethyl arachidonic acid is detected in the circulating mother liquor, indicating that the ethyl arachidonic acid has been completely precipitated from the mother liquor. Usually, the arachidonic acid ethyl ester can be rapidly crystallized and separated out in the temperature range of the crystallization point, and the whole crystallization process does not exceed 1 h.

Or when the content of the arachidonic acid ethyl ester is 2-3 wt%, the crystallization point temperature range of the arachidonic acid ethyl ester is-30 to-70 ℃.

Or when the content of the arachidonic acid ethyl ester is 3-4 wt%, the crystallization point temperature range of the arachidonic acid ethyl ester is-25 to-60 ℃.

Furthermore, the impurity fatty acid in the fish oil comprises ethyl linolenate, the content of the ethyl linolenate is 0.5-5 wt%, and the crystallization point temperature range of the ethyl linolenate is-50 to-85 ℃.

Or when the content of the ethyl linolenate is 0.5-2.5 wt%, the crystallization point temperature range of the ethyl linolenate is-55 to-85 ℃;

or when the content of the ethyl linolenate is 2.5-5 wt%, the crystallization point temperature of the ethyl linolenate is-50 to-80 ℃;

furthermore, the impurity fatty acid in the fish oil comprises ethyl oleate, the content of the ethyl oleate is 1-25 wt%, and the crystallization point temperature range of the ethyl oleate is-65 to-100 ℃.

Or the content of the ethyl oleate is 1-10 wt%, and the crystallization point temperature range of the ethyl oleate is-70 to-100 ℃.

Or the content of the ethyl oleate is 10-25 wt%, and the crystallization point temperature range of the ethyl oleate is-65 to-90 ℃.

The present embodiment also provides a cyclic dynamic crystallization apparatus, as shown in fig. 2, including: the crystallizer 110, the mother liquor circulating tank 120 and the circulating pump 140, wherein an upper baffle 117 and a lower baffle 118 are arranged in the crystallizer 110, a refrigerant accommodating space 111 is defined by the upper baffle 117, the lower baffle 118 and the inner wall of the crystallizer 110, a plurality of crystallization tubes 119 are arranged between the upper baffle 117 and the lower baffle 118 in parallel, the upper end openings of the crystallization tubes 119 are communicated with a feed inlet 115 of the crystallizer 110, the lower end openings of the crystallization tubes 119 are communicated with a discharge outlet 116 of the crystallizer 110, the crystallizer 110 is connected with the mother liquor circulating tank 120 through a pipeline, and under the action of the circulating pump 140, materials circularly flow between the crystallization tubes 119 and the mother liquor circulating tank 120.

The circulating dynamic crystallization device further comprises a refrigerating and heating device 150, the refrigerating and heating device 150 is connected with a refrigerant containing space 111 in the crystallizer 110 through a pipeline, the refrigerant is refrigerated (or heated) in the refrigerating and heating device 150, enters a cooling space from a refrigerant inlet 113 at the bottom end of the crystallizer 110, cools (or heats) the crystallization tube 119, and then flows out of a refrigerant outlet 114 at the upper end of the crystallizer 110 to the refrigerating and heating device 150 to circulate in sequence, wherein the use temperature range of the refrigerant is-125-50 ℃.

In addition, the outer peripheral wall of the mold 110 is further provided with an insulating layer 112; the discharge port 116 of the crystallizer 110 is connected to the collecting tank 130 through a pipe.

In operation, firstly, appropriate parameters are set on the cooling and heating device 150, and the refrigerant reaching the set temperature is conveyed into the refrigerant accommodating space 111, so that the refrigerant is distributed on the outer wall of the crystallization tube 119, and the temperature change in the crystallization tube 119 is controlled by heat exchange between the refrigerant and the crystallization tube 119. Then the crude fish oil raw material is put into a mother liquor circulating tank 120, diluted to a certain proportion by a proper solvent, and filled with nitrogen for protection. The material is fed into the feed inlet 115 at the top of the crystallizer 110 by the circulating pump 140, is uniformly distributed by the liquid distributor arranged at the top of the crystallizer 110, flows in the form of liquid film along the inner wall of the crystallization tube 119 in the crystallizer 110, the component reaching the crystallization point is crystallized in the crystallization tube 119 and attached to the inner wall of the crystallization tube 119, and the component which is not crystallized flows into the bottom mother liquid circulating tank 120 in the form of falling film for repeated circulation. By recycling and crystallizing the crystallizer temperature, the crystals formed by the impurity fatty acids (secondary products) are continuously enriched in the crystallization tube 119, while the content of the target product (i.e., EPA-EE) is concentrated in the uncrystallized matter in the mother liquor circulation tank 120. After the crystallization is completed, the temperature is gradually increased to melt the crystals of the by-products such as oleic acid, linolenic acid, arachidonic acid, etc. in the crystallization tube 119, respectively, and the melted crystals flow into the collection tank 130, thereby obtaining a high-value by-product.

Further, still include: and (3) carrying out liquid chromatography separation on tail liquid obtained by the fish oil through a dynamic melt crystallization method.

On the basis of primary concentration of the crude fish oil by a dynamic melt crystallization technology, a liquid chromatography process is further adopted for separation and purification, so that the EPA-EE content in the fish oil can be improved to a level of more than 96-99%, wherein the yield of the product with the EPA-EE content of 96% can reach 80%. The oxidation value of the product is lower than 3meq/kg, the anisidine value is lower than 6meq/kg, and the DHA-EE content is less than 0.3 percent (if any); trans EPA-EE content less than 0.5% (if any). The high-quality EPA-EE product has higher biological activity and can provide high-quality raw materials for the pharmaceutical industry.

The inventor finds that various fillers of the liquid chromatography column have great difference on the separation effect of the fish oil fatty acid, and even the fillers with the same material have different retention time of each component peak due to different parameters such as particle size and pore diameter. Based on the consideration of separation effect, unit time output value and the like, the invention finally adopts C18 reverse chromatographic packing with the grain diameter of 10 mu m and the pore diameter of

Loaded on DAC-50 x 250mm dynamic axial compression column. And the specific mobile phase is prepared by utilizing the mutual matching of solvents such as methanol, ethanol, acetonitrile, water and the like, and the flow rate, the sample loading amount and the sampling time are repeatedly verified, so that the purity and the yield of the product are considerable, the process stability is high, and the yield in unit time has great advantages.

In addition, the embodiment also provides an application of the fish oil containing high-concentration EPA-EE in preparing a medicament for treating cardiovascular related diseases.

Cardiovascular-related diseases include: acute myocardial ischemia, acute myocardial infarction, angina pectoris, arrhythmia, atrial fibrillation, atherosclerosis, atrial fibrillation, cardiac insufficiency, chronic heart failure, chronic stable angina pectoris, congestive heart failure, coronary artery disease, coronary heart disease, deep vein thrombosis, diabetes, diabetic neuropathy, diastolic dysfunction, edema, essential hypertension, pulmonary embolism, fatty liver, homozygous family hypercholesterolemia (HoFH), homozygous family sitosterolemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, unstable angina, and ischemic complications in myocardial infarction, hypotension, metabolic syndrome, mixed dyslipidemia, platelet aggregation, pulmonary hypertension, recurrent hemodynamically unstable Ventricular Tachycardia (VT), recurrent ventricular arrhythmia, myocardial ischemia, recurrent Ventricular Fibrillation (VF), ruptured aneurysm, stroke, supraventricular tachycardia, symptomatic atrial fibrillation/flutter, tachycardia, type ii diabetes, venous thromboembolism, ventricular arrhythmia.

The features and properties of the present invention are further described in detail below with reference to examples:

example 1

The embodiment provides a fish oil containing high-concentration ethyl eicosapentaenoate, which is obtained by separating and purifying a raw material of the fish oil, wherein the content of each fatty acid in the fish oil is determined by the following weight percentage:

the weight percentage of eicosapentaenoic acid ethyl ester is 40.66%, the weight percentage of arachidonic acid ethyl ester is 3.99%, the weight percentage of linolenic acid ethyl ester is 4.45%, and the weight percentage of oleic acid ethyl ester is 20.43%.

The purification method of the fish oil comprises the following steps:

the DSC differential scanning calorimeter was used to analyze the sample of the tail fish oil, and the results are shown in fig. 3, and the DSC chart shows the temperature ranges of the crystallization points of the fatty acids in the tail fish oil:

the arachidonic acid ethyl ester is-32 to-50 ℃, the linolenic acid ethyl ester is-55 to-75 ℃, and the oleic acid ethyl ester is-70 ℃ to-85 ℃.

And (3) a crystallization separation process: after the Chilean anchovy fish oil raw material is properly diluted, fish oil mother liquor is obtained, the fish oil mother liquor is introduced into a crystallizer, under the action of a circulating pump, the fish oil mother liquor circularly flows between the crystallizer and a mother liquor collecting tank positioned outside the crystallizer at the flow rate of 130L/h, and the crystallizer is cooled at the same time, wherein the cooling procedure is as follows:

first-stage crystallization: firstly, reducing the temperature of a crystallizer from room temperature to-32 ℃, then reducing the temperature of the crystallizer to-50 ℃ at a cooling rate of 2 ℃/hour, completing the first-stage crystallization, crystallizing and separating out the arachidonic acid ethyl ester in the mother solution, and circulating the residual tail solution;

and (3) second-stage crystallization: reducing the temperature of the crystallizer from minus 50 ℃ to minus 55 ℃, reducing the temperature of the crystallizer to minus 75 ℃ at the cooling rate of 2 ℃/hour, completing the second stage of crystallization, separating out the linolenic acid ethyl ester crystals in the mother liquor, and circulating the residual tail liquor (in the stage, when the temperature of the crystallizer is between minus 70 ℃ and minus 75 ℃, partial linolenic acid ethyl ester and oleic acid ethyl ester are simultaneously separated out in a eutectic form);

and (3) third-stage crystallization: and continuously reducing the temperature of the crystallizer from-75 ℃ to-85 ℃ at the cooling rate of 2 ℃/h to finish the third stage of crystallization, so that the ethyl oleate in the mother liquor is crystallized and separated out to obtain tail liquor.

According to the detection method, the content of eicosapentaenoic acid ethyl ester in tail liquid is 83%; concentrating the tail liquid to obtain fish oil containing high-concentration ethyl eicosapentaenoate; the fish oil is refined by liquid chromatography to obtain product with eicosapentaenoic acid ethyl ester purity of 97%, and the gas chromatogram of the product is shown in FIG. 4.

Example 2

The embodiment provides a fish oil containing high-concentration ethyl eicosapentaenoate, which is obtained by separating and purifying anchovy oil serving as a raw material, wherein the anchovy oil is determined to contain the following fatty acids in percentage by mass:

60.2 percent of eicosapentaenoic acid ethyl ester, 3.2 percent of arachidonic acid ethyl ester, 0.75 percent of linolenic acid ethyl ester and 3 percent of oleic acid ethyl ester.

The purification method of the fish oil comprises the following steps:

analyzing an anchovy oil sample by adopting a DSC differential scanning calorimeter, and acquiring the crystallization point temperature ranges of all fatty acids in the anchovy oil according to a DSC chart, wherein the temperature ranges are respectively as follows:

the arachidonic acid ethyl ester is-45 to-65 ℃, the linolenic acid ethyl ester is-65 to-80 ℃, and the oleic acid ethyl ester is-79 to-95 ℃.

And (3) a crystallization separation process: after the anchovy oil raw material is properly diluted, fish oil mother liquor is obtained, the fish oil mother liquor is introduced into a crystallizer, under the action of a circulating pump, the fish oil mother liquor circularly flows between the crystallizer and a mother liquor collecting tank positioned outside the crystallizer at the flow rate of 200L/h, and the crystallizer is cooled at the same time, wherein the cooling procedure is as follows:

first-stage crystallization: firstly, reducing the temperature of a crystallizer from room temperature to minus 45 ℃, then reducing the temperature of the crystallizer to minus 65 ℃ at the cooling rate of 3 ℃/hour, completing the first-stage crystallization, crystallizing and separating out the arachidonic acid ethyl ester in the mother solution, and circulating the residual tail solution;

and (3) second-stage crystallization: reducing the temperature of the crystallizer from-65 ℃ to-80 ℃ at a cooling rate of 1 ℃/hour, completing the second stage of crystallization, separating out the linolenic acid ethyl ester crystals in the mother liquor, and circulating the residual tail liquor (in the stage, when the temperature of the crystallizer is between-79 ℃ and-80 ℃, part of linolenic acid ethyl ester and oleic acid ethyl ester are simultaneously separated out in a eutectic form);

and (3) third-stage crystallization: and (4) continuously reducing the temperature of the crystallizer from minus 80 ℃ to minus 95 ℃ at the cooling rate of 1.5 ℃/hour, completing the crystallization of the third stage, and crystallizing and separating out the ethyl oleate in the mother liquor to obtain tail liquor.

According to the detection method, the content of eicosapentaenoic acid ethyl ester in tail liquid is 81%; concentrating the tail liquid to obtain fish oil containing high-concentration ethyl eicosapentaenoate; the fish oil is refined by a liquid chromatography technology to obtain a product with the purity of eicosapentaenoic acid ethyl ester reaching 98 percent.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. A method for purifying fish oil containing high-concentration ethyl eicosapentaenoate, which is characterized by comprising the following steps:

cooling at a cooling rate of 1-3 ℃/hour within the temperature range of the crystallization point of each fatty acid by adopting a dynamic melting crystallization method, so that impurity fatty acids contained in the fish oil are crystallized and separated out and then removed; the dynamic melt crystallization method comprises: circulating the fish oil through a crystallization container, and cooling the crystallization container within the crystallization point temperature range;

the content of the eicosapentaenoic acid ethyl ester in the high-concentration eicosapentaenoic acid ethyl ester fish oil is 20-50 wt%.

2. The method for purifying fish oil according to claim 1, wherein the cooling rate of the cooling is 1.5-2.5 ℃/hr.

3. The method for purifying fish oil of claim 1, wherein the flow rate of the fish oil circulating through the crystallization vessel is 100 to 200L/h.

4. The method of purifying fish oil of claim 1, wherein the method of obtaining the crystallization point temperature range for each fatty acid comprises thermal analysis.

5. The method for purifying fish oil according to claim 1, further comprising: and (3) carrying out liquid chromatography separation on the tail liquid obtained by the dynamic melt crystallization method on the fish oil.

6. The method for purifying fish oil according to claim 1, wherein the fatty acid impurity in the fish oil comprises arachidonic acid ethyl ester, the content of the arachidonic acid ethyl ester is 2-4 wt%, and the crystallization point temperature of the arachidonic acid ethyl ester is in the range of-25 to-70 ℃.

7. The method for purifying fish oil as claimed in claim 1, wherein the impurity fatty acid in the fish oil comprises ethyl linolenate, the content of the ethyl linolenate is 0.5-5 wt%, and the crystallization point temperature range of the ethyl linolenate is-50 to-85 ℃.

8. The method for purifying fish oil as claimed in claim 1, wherein the fatty acid impurities in the fish oil comprise ethyl oleate, the content of the ethyl oleate is 1-25 wt%, and the crystallization point temperature of the ethyl oleate is-65-100 ℃.

9. A fish oil containing eicosapentaenoic acid ethyl ester at a high concentration in an amount of 60 to 90 wt% obtained by the purification method as claimed in any one of claims 1 to 8.