CN103396310B - Method for separating and purifying eicosapentaenoic acid ester and docosahexenoic acid ester from micro-algal oil or fish oil - Google Patents

Abstract

The invention discloses a method for separating and purifying eicosapentaenoate and docosahexaenoate from microalgae oil or fish oil. In the method, a non-polar solvent is used as a raw material solvent, and a polar solvent, Ionic liquids, binary mixed solvents composed of ionic liquids and polar organic solvents are used as extraction agents, and high-purity eicosapenta is obtained by separating and preparing high-purity eicosapenta from microalgae oil or fish oil containing fatty acid esters with different degrees of saturation by using fractional distillation extraction technology Ester (EPA ester) and docosahexaenoate (DHA ester), the method is simple to operate, high separation efficiency, less solvent consumption, high product purity, high yield, easy industrial production, etc.

Description

A method for separating and purifying eicosapentaenoate and docosahexaenoate from microalgae oil or fish oil

technical field

The invention relates to the technical field of chemical separation, and specifically designs a method for separating and purifying eicosapentaenoate (EPA ester) and docosahexaenoate (DHA ester) from microalgae oil or fish oil.

Background technique

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) both belong to the omega-3 family of unsaturated fatty acids and are essential fatty acids for the human body. Microalgae, which are planktonic marine microorganisms containing chlorophyll, can "eat" carbon dioxide and produce oil through photosynthesis, which is rich in polyenoic acid fatty acids. In addition, deep-sea fish contains a large amount of high-value high unsaturated fatty acids, especially EPA and DHA. Both microalgae oil and fish oil are the main raw materials for preparing high-purity EPA ester and DHA ester.

EPA and DHA play a vital role in human health and have various physiological effects. EPA has the reputation of "vascular scavenger", which mainly acts on the cardiovascular system, and can reduce blood fat, blood pressure and cholesterol, prevent arteriosclerosis; reduce thrombosis, prevent cardiovascular and cerebrovascular diseases; prevent senile dementia; fight cancer and inhibit tumors; Prevention and treatment of diabetes, anti-inflammation, anti-aging and other effects. And DHA, commonly known as "brain gold", can pass through the blood-brain barrier, enter the brain, and act on the nervous system. In addition to all the physiological functions of EPA, DHA also has the functions of protecting the retina, improving vision, promoting the intellectual development of infants, and improving memory etc.

In methyl ester type microalgae oil or ethyl ester type fish oil after initial extraction, EPA ester and DHA ester coexist with other low unsaturation and saturated fatty acid esters. To obtain high-purity EPA ester and DHA ester, you need Use proper separation methods to separate EPA and DHA esters from other fatty acid esters. Due to the similar structure and physicochemical properties of fatty acid esters, it is difficult to separate EPA esters and DHA esters from ester-type microalgae oil or fish oil to obtain high-purity EPA esters and DHA esters. The separation methods reported in patents and literature mainly include low-temperature crystallization, molecular distillation, urea inclusion, supercritical fluid extraction, _AgNO3 complexation, adsorption, etc.

CN200910159368.1 discloses a method for extracting DHA from dinoflagellate fermentation broth. The fermentation broth is treated to obtain crude algae oil, and then the crude algae oil is subjected to 5-stage continuous molecular distillation to obtain DHA content of 40%-50%. Products, the product purity is relatively low.

Patent CN1263145 discloses the use of urea inclusion method to enrich and produce refined fish oil rich in polyenoic acid esters from crude fish oil. This process can obtain products with a total content of EPA esters and DHA esters greater than or equal to 75%. CN200810052840.7 and CN201210247842.8 disclose a method for separating and preparing docosapentaenoic acid and docosahexaenoic acid mixed fatty acids from microalgae oil by urea inclusion method. The cost of this process is relatively low, and it is suitable for Industrial application, but this method (urea inclusion method) consumes a large amount of organic solvent and urea, and the product purity is relatively low.

Patent CN1478875A discloses a technology for the separation of fish oil esterification products using the _AgNO3 aqueous solution method, which can prepare products with a total content of EPA ester + DHA ester greater than 95% and a DHA ester content greater than 95%. However, this method will bring heavy metal Ag into the product, and AgNO ₃ is expensive, which is not suitable for industrial production.

CN200810052839.4 discloses a method of separating and preparing high-purity docosapentaenoic acid methyl ester and docosahexaenoic acid methyl ester by using silver ion column chromatography. Although this method can obtain docosahexaenoic acid methyl ester with higher purity Pentaenoic acid methyl ester and docosahexaenoic acid methyl ester, but use expensive silver, and the processing amount is small, and the solvent consumption is large, which is easy to cause the loss of silver and the heavy metal pollution of the product.

CN1634852A discloses a method for separating and preparing high-purity EPA esters and DHA esters using supercritical fluid chromatography to obtain EPA esters and DHA esters with a purity greater than 90%, but the processing capacity is quite small and the equipment cost is relatively high. JP09263787 uses liquid-liquid extraction to separate and prepare highly unsaturated fatty acid esters. This method uses acetonitrile-water mixture as the extraction agent, but due to the high hydrophobicity of fatty acid esters, its extraction capacity and yield are very low.

In order to obtain EPA esters and DHA esters with higher purity, the combined use of two or more separation methods is also a commonly used method. CN102285880A discloses a semi-preparative/preparative high-performance liquid chromatography-mass spectrometry technology to separate and prepare EPA esters and DHA esters, and obtain EPA esters and DHA esters with a preliminary reading greater than 99%. Although this method can obtain high-purity EPA esters and DHA esters, the separation selectivity is better, but the processing capacity is small, the cost is high, and more toxic solvents such as tetrahydrofuran or acetonitrile are used. CN1986515A discloses a technique of combining supercritical extraction and low-temperature crystallization to directly enrich omega-3 unsaturated fatty acids in soft-shelled turtle oil, and the total content of EPA and DHA in the obtained soft-shelled turtle oil extract is 25%-30% . JP09157684A has reported the separation and preparation of high-purity highly unsaturated fatty acid esters using the coupling technology of supercritical extraction and chromatographic separation. Although this coupling technology can obtain high-purity single products, the processing capacity of chromatography is small and consumes Lots of organic solvents.

Contents of the invention

The invention provides a method for separating and purifying eicosapentaenoate and docosahexaenoate from microalgae oil or fish oil. The preparation process is simple, the product yield and purity are high, and the cost is low.

A method for separating and purifying eicosapentaenoate and docosahexaenoate from microalgae oil or fish oil, comprising the following steps:

(1) Dissolve ester-type microalgae oil or ester-type fish oil in a non-polar solvent to prepare a raw material solution, use a polar solvent or ionic liquid or a binary mixed solvent composed of an ionic liquid-polar solvent as an extraction agent, and mix with the raw material The same solvent as the solution is a detergent, fractionated extraction is carried out, and the extract is collected;

(2) Back-extracting the extract with the non-polar solvent, concentrating the back-extract in vacuum, washing with water and drying to obtain a mixture of eicosapentaenoate and docosahexaenoate. The fractional distillation extraction is divided into an extraction section and a washing section. The extractant enters the fractionation extraction system from the first stage of the extraction section, the raw material liquid enters the fractionation extraction system from the last stage of the extraction section, and the detergent enters the fractionation extraction system from the first stage of the washing section. The system is mixed with the raw material liquid at the last stage of the extraction section, and enters the extraction section together, and the extraction phase and the washing phase are in multi-stage countercurrent contact. The extract rich in eicosapentaenoate (EPA ester) and docosahexaenoate (DHA ester) flows out from the first stage of the washing section, and the extract is collected; the outflow from the first stage of the extraction section is rich in Low unsaturated and saturated fatty acid ester raffinate, collect the raffinate.

For fractional distillation extraction, the core of innovation and technical difficulty lies in the design and optimization of extractant. The extractant must be able to recognize the slight structural differences between EPA esters and DHA esters and other fatty acid esters to achieve selective extraction and separation. At the same time, the extractant must have a high extraction capacity for fatty acid esters. However, since fatty acid esters are a class of lipid compounds and lack polar groups in their structure, their solubility in polar extractants is low, resulting in low extraction capacity of the extractant, which is not conducive to large-scale application.

Ionic liquids are substances that are liquid at room temperature or close to room temperature and are a new type of green separation medium composed of anions and cations. The anion and cation structures of ionic liquids can be adjusted, aiming at the slight differences in the structure and properties of target compounds and impurities, by designing the anion and cation structures of ionic liquids to adjust the interaction mode and strength of ionic liquids and solutes, and achieve specific separation effects . The ionic liquid extractant designed in the invention has high extraction capacity and high selectivity.

Most ionic liquids have low viscosity and can be used directly for extraction. Some ionic liquids have high viscosity and are difficult to use directly for extraction. The study found that adding polar solvents such as dimethyl sulfoxide, dimethylformamide, acetonitrile, and methanol to the ionic liquid to form an ionic liquid-polar solvent composite extractant also has high separation selectivity and extraction capacity. At the same time the viscosity is low.

The ionic liquid is composed of two parts: cation M ⁺ and anion ^N- :

The cation M ⁺ is choline cation with single or multiple substituents, imidazolium cation with single or multiple substituents, pyridinium cation with single or multiple substituents, quaternary ammonium cation with single or multiple substituents and one of the benzimidazolium cations with single or multiple substituents; the substituents are alkyl groups with 1 to 8 carbon atoms. When there are multiple substituents, each substituent can be the same or not same.

The anion ^N- is one of fatty acid radicals, thiocyanate radicals, dicyanamide radicals, tricyanomethane radicals, tetracyanoborate radicals, halide ions, tetrafluoroborate radicals and phenols with 1-18 carbon atoms.

The anion N ^- is one of fatty acid, thiocyanate, dicyanamide, tricyanomethane, tetracyanoborate, halide, tetrafluoroborate and phenol with 1 to 18 carbon atoms .

Fatty acid radicals with 1-18 carbon atoms are preferred as the anion of the ionic liquid. This type of ionic liquid has a high extraction capacity for EPA ester and DHA ester due to its long non-polar carbon chain.

The non-polar solvent is one of n-hexane, n-heptane, n-octane and petroleum ether.

The strong polar solvents are monohydric alcohols with 1 to 4 carbon atoms, polyols with 1 to 4 carbon atoms, furfural, acetonitrile, dimethylformamide (DMF), dimethyl sulfoxide One of (DMSO), N-methylpyrrolidone (NMP), and sulfolane.

The mass fraction of the ionic liquid in the binary mixed solvent is 5-70%. When using a binary mixed solvent as the extractant and forming a two-phase extraction system with a non-polar organic solvent, the separation selectivity of the extraction system for EPA esters and DHA esters can be significantly improved, especially when the viscosity of the ionic liquid used is relatively high , effectively reduce the mass transfer resistance of the two-phase system, improve the extraction efficiency, and facilitate large-scale industrial production.

The ester-type microalgae oil is a methyl ester type, and the total mass fraction of eicosapentaenoate and docosahexaenoate in the ester-type microalgae oil is 5% to 50%; The type fish oil is an ethyl ester type, and the total mass fraction of eicosapentaenoate and docosahexaenoate in the ester type fish oil is 5% to 80%.

The total concentration of the fatty acid ester in the raw material liquid is preferably 10-200 g/L, more preferably 20-150 g/L.

If the total concentration of fatty acid esters in the raw material liquid is too high, the viscosity of the raw material liquid is too high, which is not conducive to the extraction and mass transfer process, and will reduce the separation selectivity; The disadvantages are small amount, large solvent consumption, and reduced process economy.

The temperature of the fractional extraction is 20-40°C. If the temperature is too low, the two-phase mass transfer rate will decrease, and it will take a long time to reach extraction equilibrium, which is not conducive to production operations; if the temperature is too high, the solvent will volatilize seriously, and polyunsaturated fatty acid esters are heat-sensitive substances, which are very easy to extract at high temperatures. Oxidation, and reduce the partition coefficient and selectivity of the extraction.

The flow ratio of the raw material liquid, extractant and detergent is 0.2-0.8:0.3-0.8:0.8-3.

The steps of vacuum concentration, water washing and drying of the stripping solution are routine operations.

The extraction devices used in the fractionation extraction process are common extraction devices such as packed towers, sieve tray towers, rotating disk towers, mixing and settling tanks, and centrifugal extractors.

In a separation process, the raw material liquid solvent, washing agent and extractant during stripping are all the same solvent.

The present invention uses gas chromatography (GC) analysis, and the specific GC analysis conditions are: CP7489 (100m x 2.5mm ID, film thickness 0.2μm), FID detector, injector temperature 250°C, detector temperature 280°C, column temperature Adopt temperature program: the initial temperature is 80°C, keep it for 4 minutes, increase the temperature to 220°C at a rate of 10°C/min, and keep it for 30 minutes. The carrier gas is N ₂ , the flow rate is 1ml/min, and the split ratio is 50.

The computing method of yield and purity among the present invention is as follows:

Yield=the quality of EPA ester and DHA ester in the product/the quality of EPA ester and DHA ester in the raw material×100%;

Absolute purity=the mass of EPA ester and DHA ester in the product/the total mass of product×100%;

Compared with prior art, the present invention has following advantage:

1. The present invention adopts the binary mixed solvent-nonpolar solvent extraction system of polar solvent-nonpolar solvent, ionic liquid-nonpolar solvent, ionic liquid and polar solvent, to the various fatty acid esters of similar structure It has high selective separation ability and large extraction capacity.

2. The present invention adopts fractional distillation extraction technology, which has the advantages of simple operation, simple process, high yield, high product purity, large processing capacity and easy industrial application.

3. The inventive method adopts optimized conditions, the purity of EPA ester+DHA ester can reach 90-99%, and the rate of recovery of EPA ester+DHA ester can reach more than 90%.

4. The ionic liquid extractant used in the inventive method has the characteristics of high stability, non-flammable and non-explosive, etc., which improves the safety of the separation and extraction process; the ionic liquid is a kind of environmentally friendly solvent at the same time, so as the extractant, it can It reduces environmental pollution and has broad application prospects; the recovery of ionic liquid is relatively easy, and it is convenient for the repeated use of extractant.

Detailed ways

In the following implementations, gas chromatography (GC) was used for quantitative analysis of EPA esters and DHA esters. All the raw materials used are commercially available.

The process of fractional distillation extraction is:

The extractant enters the fractionation extraction system from the first stage of the extraction section, the raw material liquid enters the fractionation extraction system from the last stage of the extraction section, and the detergent enters the fractionation extraction system from the first stage of the washing section, and is mixed with the raw material liquid at the last stage of the extraction section , and enter the extraction section together, and the extraction phase and the washing phase are in multistage countercurrent contact. The extract rich in eicosapentaenoate (EPA ester) and docosahexaenoate (DHA ester) flows out from the first stage of the washing section, and the extract is collected; the outflow from the first stage of the extraction section is rich in Low unsaturated and saturated fatty acid ester raffinate, collect the raffinate.

Example 1

Dissolve the mixture of fatty acid ethyl esters (ester-type fish oil) in n-hexane to make a raw material solution with a total concentration of fatty acid ethyl esters of 40 g/L, in which the mass fraction of EPA ethyl ester + DHA ethyl ester is about 5%. Use N,N-dimethylformamide as the extractant and n-hexane as the detergent. The flow ratio of the raw material solution, the extractant and the detergent is 0.4:0.5:1.2. Fractional extraction is carried out at 30°C, and the extracted liquid and raffinate. The extract was back-extracted with n-hexane and concentrated in vacuo to obtain a mixture of EPA ethyl ester + DHA ethyl ester. Among them, the purity of EPA ethyl ester+DHA ethyl ester is 91.1%, and the yield is 94.6%.

Example 2

Dissolve the mixture of fatty acid methyl esters (ester-type microalgae oil) in n-octane to prepare a raw material solution with a total concentration of fatty acid methyl esters of 60g/L, in which the content of EPA methyl ester + DHA methyl ester is about 6%. Using dimethyl sulfoxide as the extractant and n-octane as the detergent, the flow ratio of the raw material solution, the extractant, and the detergent is 0.2:0.4:2, fractional extraction is carried out at 35°C, and the extract and raffinate. The extract was back-extracted with n-octane and concentrated in vacuo to obtain a mixture of EPA methyl ester + DHA methyl ester. Among them, the purity of EPA methyl ester+DHA methyl ester is 94.1%, and the yield is 86.5%.

Example 3

Dissolve the mixture of fatty acid esters (ester-type microalgae oil) in petroleum ether to prepare a raw material solution with a total concentration of fatty acid methyl esters of 80g/L, in which the content of EPA methyl ester + DHA methyl ester is about 25%. Using 1-butyl-3-methylimidazolium thiocyanate ionic liquid as extractant and petroleum ether as detergent, the flow ratio of raw material liquid, extractant and detergent is 0.4:0.6:2.5 at 25°C The fractional distillation extraction was carried out, and the extract and raffinate were collected. The extract was back-extracted with petroleum ether and concentrated in vacuo to obtain a mixture of EPA methyl ester + DHA methyl ester. Among them, the purity of EPA methyl ester+DHA methyl ester is 96.8%, and the yield is 83.2%.

Example 4

Dissolve the mixture of fatty acid ethyl esters (ester-type fish oil) in n-hexane to prepare a raw material solution with a total concentration of fatty acid ethyl esters of 40g/L, in which the content of EPA ethyl ester + DHA ethyl ester is about 30%. With 1-butyl-3-methylimidazole dinitrile amine salt ionic liquid as extractant, n-hexane as detergent, the flow ratio of raw material liquid, extractant and detergent is 0.2:0.4:1.8, at 30 Fractional extraction was carried out at ℃ to collect the extract and raffinate. The extract was back-extracted with n-hexane and concentrated in vacuo to obtain a mixture of EPA ethyl ester + DHA ethyl ester. Among them, the purity of EPA ethyl ester+DHA ethyl ester is 94.8%, and the yield is 87.4%.

Example 5

Dissolve the mixture of fatty acid esters (ester-type microalgae oil) in n-hexane to prepare a raw material solution with a total concentration of fatty acid methyl esters of 20 g/L, in which the content of EPA methyl ester + DHA methyl ester is about 50%. N-butylpyridinium acetate ionic liquid was used as extractant, n-hexane was used as detergent, the flow ratio of raw material liquid, extractant and detergent was 0.5:0.5:3, fractional extraction was carried out at 20°C, Collect the extract and raffinate. The extract was back-extracted with n-hexane and concentrated in vacuo to obtain a mixture of EPA methyl ester + DHA methyl ester. Among them, the purity of EPA methyl ester+DHA methyl ester is 93.5%, and the yield is 89.1%.

Example 6

Dissolve the mixture of fatty acid ethyl esters (ester-type fish oil) in n-hexane to prepare a raw material solution with a total concentration of fatty acid ethyl esters of 40 g/L, in which the content of EPA ethyl ester + DHA ethyl ester is about 80%. With 1-ethyl-3-methylimidazole trinitrile carbon salt ionic liquid as extractant, n-hexane as detergent, the flow ratio of raw material liquid, extractant and detergent is 0.8:0.6:2, at 40 Fractional extraction was carried out at ℃, and the extract and raffinate were collected. The extract was back-extracted with n-hexane and concentrated in vacuo to obtain a mixture of EPA ethyl ester + DHA ethyl ester. Among them, the purity of EPA ethyl ester+DHA ethyl ester is 96.2%, and the yield is 89.2%.

Example 7

Dissolve the mixture of fatty acid methyl esters (ester-type microalgae oil) in n-hexane to prepare a raw material solution with a total concentration of fatty acid methyl esters of 150g/L, in which the content of EPA methyl ester + DHA methyl ester is about 35%. With 1-butyl-3-methylimidazolium tetrafluoroborate ionic liquid-N,N-dimethylformamide binary mixed solvent (1-methyl-3-octylimidazolium tetrafluoroboric acid in mixed solvent The mass fraction of the salt ionic liquid is 20%) as the extractant, n-hexane as the detergent, the flow ratio of the raw material liquid, the extractant, and the detergent is 0.5:0.4:1.9, fractional extraction is carried out at 30°C, and the collected Extract and raffinate. The extract was back-extracted with n-hexane and concentrated in vacuo to obtain a mixture of EPA methyl ester + DHA methyl ester. Among them, the purity of EPA methyl ester+DHA methyl ester is 96.0%, and the yield is 91.8%.

Example 8

Dissolve the mixture of fatty acid ethyl esters (ester-type fish oil) in n-heptane to prepare a raw material solution with a total concentration of fatty acid ethyl esters of 180g/L, in which the content of EPA ethyl ester + DHA ethyl ester is about 50%. Using tetraethylamine-dicyanamide salt ionic liquid-dimethyl sulfoxide binary mixed solvent (the mass fraction of tetraethylamine-dicyanamide salt ionic liquid in the mixed solvent is 50%) as the extraction agent and n-heptyl Alkanes are used as detergent, and the flow ratio of raw material liquid, extractant, and detergent is 0.4:0.4:1.3. Fractional extraction is carried out at 30°C, and the extract and raffinate are collected. The extract was back-extracted with n-heptane and concentrated in vacuo to obtain a mixture of EPA ethyl ester + DHA ethyl ester. Among them, the purity of EPA ethyl ester+DHA ethyl ester is 91.9%, and the yield is 83.1%.

Example 9

Dissolve the mixture of fatty acid methyl esters (ester-type microalgae oil) in n-hexane to prepare a raw material solution with a total concentration of fatty acid methyl esters of 100g/L, in which the content of EPA methyl ester + DHA methyl ester is about 15%. Use choline laurate ionic liquid-N,N-dimethylformamide binary mixed solvent (the mass fraction of choline laurate ionic liquid in the mixed solvent is 70%) as the extraction agent, and n-hexane as the detergent , the flow ratio of raw material liquid, extractant, and detergent is 0.4:0.6:1, fractional extraction is carried out at 30°C, and the extract and raffinate are collected. The extract was back-extracted with n-hexane and concentrated in vacuo to obtain a mixture of EPA methyl ester + DHA methyl ester. Among them, the purity of EPA methyl ester+DHA methyl ester is 98.5%, and the yield is 84.3%.

Claims (7)

1. the method for separation and purification eicosa-pentaenoic acid esters and docosahexenoic acid ester from ester type micro-algae oil or fish oil, is characterized in that, comprise the following steps:

(1) micro-for ester type algae oil or ester type fish oil are dissolved in non-polar solvent preparation raw material liquid, the binary mixed solvent formed with ionic liquid or ionic liquid-polar solvent is for extraction agent, with the solvent identical with material solution for washing composition, carry out fractionation extraction, collect extraction liquid;

Described ionic liquid is by positively charged ion M ⁺and anion N ^-two portions are formed:

Described positively charged ion M ⁺for the one that there is single or multiple substituent choline cation, there is single or multiple substituting group glyoxaline cation, there is single or multiple substituent pyridine type positively charged ion, there is single or multiple substituting group quaternary ammonium cation and have in single or multiple substituting group benzimidazolium;

Anion N ^-for the one that carbonatoms is in the fatty acid radical of 1-18, thiocyanate ion, dicyanamide root, three cyanogen methane roots, four cyanogen borates, halide-ions, tetrafluoroborate and phenol;

Described polar solvent to be carbon atom number be 1 ~ 4 monohydroxy-alcohol, carbon atom number be one in the polyvalent alcohol of 1 ~ 4;

The massfraction of described binary mixed solvent intermediate ion liquid is 5 ~ 70%;

(2) described extraction liquid is stripped through described non-polar solvent, strip liquor is obtained after vacuum concentration, washing also drying the mixture of eicosa-pentaenoic acid esters and docosahexenoic acid ester.

2. method according to claim 1, is characterized in that, described substituting group to be carbonatoms be 1 ~ 8 alkyl.

3. method according to claim 1, it is characterized in that, described non-polar solvent is the one in normal hexane, normal heptane, octane and sherwood oil.

4. method according to claim 1, it is characterized in that, the micro-algae oil of described ester type is methyl esters type, and in the micro-algae oil of ester type, the total mass mark of eicosa-pentaenoic acid esters and docosahexenoic acid ester is 5% ~ 50%; Described ester type fish oil is ethyl ester type, and in ester type fish oil, the total mass mark of eicosa-pentaenoic acid esters and docosahexenoic acid ester is 5% ~ 80%.

5. method according to claim 1, it is characterized in that, in described stock liquid, the total concn of fatty acid ester is 10 ~ 200g/L.

6. method according to claim 1, it is characterized in that, the temperature of described fractionation extraction is 20 ~ 40 DEG C.

7. method according to claim 1, is characterized in that, described stock liquid, heating up in a steamer than being 0.2 ~ 0.8:0.3 ~ 0.8:0.8 ~ 3 between extraction agent and washing composition three.