CN111979051A

CN111979051A - Method for extracting polyunsaturated fatty acid from microalgae algal oil

Info

Publication number: CN111979051A
Application number: CN202010874054.6A
Authority: CN
Inventors: 许正双; 俞威; 张奔; 李洋; 张涛
Original assignee: Bio Pharm Science & Technology Co ltd; Peking University Shenzhen Graduate School
Current assignee: Bio Pharm Science & Technology Co ltd; Peking University Shenzhen Graduate School
Priority date: 2020-08-26
Filing date: 2020-08-26
Publication date: 2020-11-24

Abstract

The invention provides a method for extracting polyunsaturated fatty acid from microalgae algal oil, which comprises 3 steps: sequentially saponifying, acidifying and extracting the algae oil to obtain crude fat; esterifying the crude fat to obtain a fatty acid fat mixture; and separating the fatty acid ester mixture to obtain the polyunsaturated fatty acid ester, namely obtaining the finished product. The beneficial technical effects are as follows: the invention aims at a process for extracting unsaturated polyene fatty acid with different purities and suitable for various purposes from microalgae algal oil by utilizing a simple and efficient method.

Description

Method for extracting polyunsaturated fatty acid from microalgae algal oil

Technical Field

The invention belongs to the field of extraction processes, and particularly relates to a method for extracting polyunsaturated fatty acids from microalgae algal oil.

Background

Unsaturated fatty acids are essential fatty acids for human body, some polyunsaturated fatty acids can be synthesized by enzyme function, while some polyunsaturated fatty acids cannot be synthesized by themselves or in sufficient quantity, and are not sufficient for intake in the normal dietary structure in quantities that meet the needs of the body, such as omega-3-eicosapentaenoic acid (EPA) and omega-3-docosahexaenoic acid (DHA). On the other hand, these polyunsaturated fatty acids play an irreplaceable health and therapeutic role in the body, for example EPA has the function of clearing the waste (cholesterol and triglycerides) in the blood vessels, colloquially known as "vascular scavenger"; DHA has the efficacies of softening blood vessels, strengthening brain, benefiting intelligence and improving eyesight, and is commonly called as brain gold. In recent years, the single application of polyunsaturated fatty acids and the mechanism of action thereof have been studied intensively, for example, EPA ethyl ester (trade name) is approved by FDA in 12 months in 2019

(or Icosapentethyl, IPE)) for use in clinical reducing the risk of cardiovascular disease. Physiological functions of polyunsaturated fatty acids include:

1) unsaturated fatty acid can regulate blood lipid, clear thrombus, and prevent cardiovascular system diseases

Polyunsaturated fatty acids have a significant impact on arterial thrombosis and platelet function. The intake of linoleic acid has a strong correlation with the linoleic acid content in plasma phospholipids, cholesterol esters and triglycerides, and the total linoleic acid, alpha-linolenic acid, arachidonic acid, EPA and DHA of platelets have a significant correlation with the fatty acid concentration in plasma triglycerides, phospholipids and adipose tissue. In 12 months of 2019, polyunsaturated fatty acids are FDA approved for the treatment of cardiovascular diseases, and are currently the only FDA approved for clinical treatment of high triglycerides and patients with concurrent metabolic disease, preventing and treating cardiovascular diseases.

2) Polyunsaturated fatty acids and cell growth

Studies have shown that polyunsaturated fatty acids have an effect on brain, retina and neural tissue development. DHA and arachidonic acid are the two major polyunsaturated fatty acids in the retina of the brain, with significant effects particularly in fetal infants.

3) Polyunsaturated fatty acid and anticancer agent

A large number of experiments show that DHA and EPA have good anticancer effect, and the anticancer mechanism of DHA and EPA has four main aspects: A) omega-3 fatty acids interfere with the formation of omega-6 polyunsaturated fatty acids and reduce the concentration of arachidonic acid, reducing the amount of interleukin that promotes the production of PGE2, and thus reducing the production of PGE2 believed to have a promoting effect on carcinogenesis; B) the membrane synthesis of cancer cells requires a large amount of cholesterol, and omega-3 fatty acids lower cholesterol levels, thereby inhibiting cancer cell growth; C) DHA and EPA in immune cells generate more substances with beneficial physiological effects, participate in regulation and control of cell gene expression, improve the body immunocompetence and reduce tumor necrosis factors; D) EPA and DHA greatly increase the fluidity of cell membranes, are beneficial to cell metabolism and repair, and prevent abnormal proliferation of tumor cells.

4) Polyunsaturated fatty acids and immunomodulation

Polyunsaturated fatty acids such as arachidonic acid, EPA and DHA can affect different functions of various cells. Among them, omega-3 fatty acids are particularly potent: regulating the generation of eicosanoids by cells of an immune system, particularly reducing the generation of proinflammatory factors PGE2 and leukotriene B4; adjusting the fluidity of the film; ③ Regulation of the cellular Signal transduction pathways, in particular with lipid mediators, protein kinases C and Ca²⁺Mobilize the relevant pathways; regulating the expression of gene related to the production of cell factor, the proliferation of peroxisome and the assembly of fatty acid oxidized lipoprotein.

5) Others

The polyunsaturated fatty acid can also prevent skin aging, delay aging, prevent anaphylaxis, and promote hair growth.

Sources of polyunsaturated fatty acids include two major classes of algal oils and deep sea fish oils. Wherein the production of polyunsaturated fatty acids by microalgae has the following advantages:

the microalgae can grow and reproduce quickly, synthesize and enrich polyunsaturated fatty acids with high concentration by itself and are less influenced by the environment. The content of polyunsaturated fatty acid in some microalgae bodies is as high as 5-6% of the dry weight of cells, and the relative content of the polyunsaturated fatty acid is far higher than that of the polyunsaturated fatty acid in fish bodies;

the polyunsaturated fatty acid is purified from the algae body, the extraction process from the fish oil is simpler, and the fish oil has no fishy smell and can be used as a food additive; the fish oil does not contain cholesterol, avoids the defect that a large amount of cholesterol is taken when the fish oil is eaten, and can be used as a medicine;

some microalgae can be directly eaten, so that oxidative decomposition in the purification process is reduced; some algae contain simple PUFA species, and single component separation and purification are relatively easy to perform;

the existing separation and purification technology of polyunsaturated fatty acid comprises an adsorption separation method, a crystallization method, a urea inclusion method, a molecular distillation method, a supercritical extraction method and the like:

(1) an adsorption separation method: the method utilizes an adsorbent to selectively adsorb and separate the polyunsaturated fatty acid. Typically, silver ions form complexes with the carbon-carbon double bonds of unsaturated fatty acids, the more double bonds the more complex the more stable the complex. The disadvantage of this process is the high cost of silver salts, which do not have a degree of separation for other polyunsaturated fatty acids and can only be used to separate saturated fatty acids from less unsaturated fatty acids, resulting in a mixture of polyunsaturated fatty acids.

(2) Low-temperature crystallization method: also called solvent fractionation, which utilizes different fatty acids or fatty acid salts to be separated and purified by their different solubilities in organic solvents at low temperatures. The solubility of fatty acids in organic solvents decreases with increasing carbon chain length and increases with increasing number of double bonds, and this solubility difference appears more pronounced with decreasing temperature. Therefore, the mixed fatty acid is dissolved in the organic solvent, and the saturated and low unsaturated fatty acids are separated out as crystals by cooling to-40 ℃ or below and keeping the temperature for a period of time, while the polyunsaturated fatty acids remain in the solvent. The desired polyunsaturated fatty acids are obtained by removing a large amount of saturated fatty acids and a part of the low unsaturated fatty acids by filtration and then evaporating the filtrate from the solvent. The content of EPA and DHA can reach 73-79% by a multistage crystallization method. The low-temperature crystallization method has simple process and convenient operation, the active ingredients are not easy to generate denaturation reactions such as oxidation, polymerization, isomerization and the like, and the method is suitable for small and medium-sized enterprises and has certain practical significance. The disadvantage is the high energy consumption and still the inefficient separation of polyunsaturated fatty acids, the resulting product being a mixture of polyunsaturated fatty acids.

(3) The urea inclusion method comprises the following steps: the basic feature of the more common separation method of polyunsaturated fatty acids is that fatty acid mixtures can be separated according to the difference in the degree of unsaturation of the fatty acids. The principle is that urea molecules can form a stable crystal inclusion compound with saturated fatty acid or monounsaturated fatty acid to be separated out in the crystallization process, and polyunsaturated fatty acid has certain spatial configuration due to more double bonds and bent carbon chains and is not included by urea, so that the saturated fatty acid, the monounsaturated fatty acid and the urea are removed by a filtration method to form the stable inclusion compound, and the polyunsaturated fatty acid with higher purity can be obtained. The urea inclusion method has the advantages of low cost, simple equipment, cheap reagent and simple and convenient operation. However, the urea inclusion method requires detailed optimization of the process and determination of specific parameters according to the components of the crude fat. Compared with the former two methods, the method can not effectively separate polyunsaturated fatty acid, and the process requires low-temperature crystallization operation and has larger energy consumption.

(4) Molecular distillation method: is a special liquid-liquid separation technology, and is separated by utilizing the difference of the volatility of mixture components. The process is generally carried out under a high vacuum at an absolute pressure of from 1.33Pa to 0.0133 Pa. Under the condition, the intermolecular attraction of the fatty acid is reduced, the volatility is improved, and the distillation temperature is greatly reduced compared with the atmospheric distillation. During the fractional distillation, saturated fatty acid and monounsaturated fatty acid are first distilled out, while unsaturated fatty acid with more double bonds is finally distilled out. The product obtained by molecular distillation has good appearance and color, can effectively prevent polyunsaturated fatty acid from being oxidized and decomposed by heating, and has the defects of large investment of high-vacuum equipment, higher energy consumption and low productivity.

(5) Supercritical C02 extraction: supercritical fluid extraction is a new separation technology developed in recent decades, and the basic principle is that the solubility of each component of raw materials in supercritical fluid is greatly changed by adjusting temperature and pressure to achieve the separation purpose. In addition, carbon dioxide (critical temperature 31.3 ℃, critical pressure 7.374MPa) and other substances with low critical temperature and chemical inertness are often selected as the extracting agent for supercritical fluid extraction; therefore, the method is particularly suitable for separating heat-sensitive substances and easily-oxidized substances. The supercritical extraction can effectively separate fatty acids with large chain length difference, but if the fatty acids with similar carbon chain length are separated, other separation technologies must be combined, and the equipment investment is also high.

Disclosure of Invention

In view of the above-mentioned shortcomings in the background art, the present invention provides a method for extracting polyunsaturated fatty acids from microalgae algal oil, which comprises the following steps:

a method for extracting polyunsaturated fatty acid from microalgae algal oil comprises the following steps:

step 1: the algae oil is sequentially saponified, acidified and extracted to obtain crude fat.

Step 2: and (3) esterifying the crude fat to obtain a fatty acid fat mixture.

And step 3: and separating the fatty acid ester mixture to obtain the polyunsaturated fatty acid ester, namely obtaining the finished product.

Further, in step 1, saponification is carried out with an alcohol solution of an alkali metal hydroxide. The alkali metal hydroxide is sodium hydroxide, lithium hydroxide or potassium hydroxide, preferably sodium hydroxide and potassium hydroxide, most preferably potassium hydroxide.

Further, the alcohol solution is methanol, ethanol or isopropanol, preferably methanol or ethanol.

Further, in step 1, the temperature of the saponification reaction of algal oil is 0 to 45 degrees, preferably 25 to 35 degrees. The saponification reaction time is 12-36 hours, preferably 18-30 hours, and the reaction time is controlled by analytical monitoring means.

Further, in the step 1, the acidification is carried out by using dilute hydrochloric acid or dilute sulfuric acid under a low-temperature condition, and the pH value is between 1 and 3.

Further, in step 1, the extraction is performed using a mixed solvent, and a halogenated hydrocarbon-alcohol system, an alkane-ester system, and an ether-alkane or ether-ester system are used as organic phases. Preferably an alkane-ester system solvent. Wherein the halogenated hydrocarbon is selected from chloroform, dichloromethane and dichloroethane. The alcohol is selected from methanol, ethanol, and isopropanol. The alkane is selected from C4-C8 alkane or mixed alkane. The ester is selected from ethyl acetate or methyl acetate. The ether is selected from the group consisting of diethyl ether, tetrahydrofuran, 1, 4-dioxane, and methyl tert-butyl ether. And removing the solvent from the solution obtained by the extraction in the step by using a rotary evaporator to obtain crude ester.

Further, in step 2, the esterification process is carried out by using an acid-catalyzed esterification method: the crude ester is completely converted into fatty acid ester mixture by using ethanol as solvent and heating for 16 hours under the catalysis of concentrated sulfuric acid. The fatty acid ester mixture is an ethyl ester mixture.

Further, in step 3, the separation comprises a pre-separation and a further separation 2.

The fatty acid ester mixture is pre-separated with 1-3 times by weight of crude silica gel.

The further separation adopts column chromatography diatomite and silica gel mixed filler with the volume ratio of fatty acid ester being 1:3-8 times.

Further, in the preliminary separation in step 3, it is preferably 2 times by weight. The eluent is mobile phase A + mobile phase B. The pre-separation realizes the purpose of removing pigment and impurities with larger polarity. The resulting fatty acid ester mixture was a bright yellow solution.

Furthermore, in the further separation in step 3, the filler with a ratio of 5-6 times by volume is preferred, wherein the diatomite accounts for 0-10% (by volume) of the total volume of the separation filler, and the preferred ratio is 4-6% by volume. The elution solvent is mobile phase A + mobile phase B. Wherein the mobile phase A is halogenated hydrocarbon, including chloroform, dichloromethane or dichloroethane. The mobile phase B is alkane selected from C4-C8 alkane or mixed alkane.

The eluates were collected separately according to time and the composition and purity were determined by gas chromatography-mass spectrometry.

Advantageous technical effects

The invention aims at a process for extracting unsaturated polyene fatty acid with different purities and suitable for various purposes from microalgae algal oil by utilizing a simple and efficient method.

Drawings

FIG. 1 shows the GCMS spectrum of the crude ester of example 1-EE.

FIG. 2 is the GCMS spectrum of the end product of example 1-EE.

FIG. 3 shows the GCMS spectrum of the crude ester of example 2-EE.

FIG. 4 is the GCMS spectrum of the end product of example 2-EE.

FIG. 5 is a GCMS analysis spectrum of the example 3-EE component.

FIG. 6 is a GCMS analysis spectrum of the example 3-EE component.

FIG. 7 is a GCMS analysis spectrum of the example 4-EE component.

FIG. 8 shows the EE high resolution spectrum ([ M + Na ] +, calculated 353.2451, found 353.2451).

FIG. 9 shows an EE NMR spectrum (upper hydrogen spectrum, lower carbon spectrum).

FIG. 10 is a process flow diagram of the present invention.

Detailed Description

The structural features of the present invention will be described in detail with reference to the accompanying drawings.

Referring to fig. 10, a method for extracting polyunsaturated fatty acids from microalgae algal oil comprises the following steps:

Step 2: and (3) esterifying the crude fat to obtain a fatty acid fat mixture.

Example 1

19.2 g of algal oil (crude extract of microalgae) was weighed, added with lithium hydroxide-methanol solution (300 ml, 0.4 mol/l), and stirred at room temperature overnight. Water (100 ml) was added and methanol was recovered by rotary evaporator. The residue was neutralized to pH1 with a hydrochloric acid solution (1 mol/l) with an alkane (300 ml) added to the ice bath. After filtration the filtrate separated and the aqueous phase was extracted with further alkane (400 ml). The combined organic phases were dried over anhydrous sodium sulfate and spin dried to give 8.6 g of crude oil.

8.6 g of the crude oil was dissolved in ethanol (100 ml), concentrated sulfuric acid (4%) was added, and the mixture was heated to 75 ℃ for reaction for 2.5 hours. After cooling to room temperature, saturated sodium chloride solution (100 ml) was added and excess ethanol was removed by rotary evaporation. The residue was extracted with alkane or ester (200 ml x 3). After extraction, the organic phases were combined, dried over anhydrous sodium sulfate and the filtrate was spin-dried to give 8.2 g of crude ester. The EPA Ethyl Ester (EE) content of the product was found to be 38% by GC analysis (see FIG. 1 for GCMS analysis).

The EE separation process using packed column chromatography was as follows: no component when alkane is used for elution; eluting with 1-10% of mobile phase A + mobile phase B to obtain organic fatty acid component, but no EE is eluted; the component containing EE can be obtained by using 10 to 20 percent of mobile phase A + mobile phase B for continuous elution. A total of 3.0 g of yellow oily product were obtained using 2.4 l of eluent, and the result of GCMS analysis showed an EE content of 93% (see FIG. 2 for GCMS analysis) and a product yield of 90%. The polarity of the mobile phase is increased continuously without EE component flowing out.

Example 2

16.6 g of algal oil was weighed, added with potassium hydroxide-methanol solution (300 ml, 0.4 mol/l) and stirred at room temperature overnight. Water (100 ml) was added and methanol was recovered by rotary evaporator. The residue was neutralized to pH1 with an alkane (300 ml) added to an ice bath using HCl solution (1 mol/l). Suction filtration, separation of layers and extraction of the aqueous phase with alkane (400 ml). The combined organic phases were dried over anhydrous sodium sulfate and spin dried to give 8.94 g of crude oil.

8.94 g of the crude oil was dissolved in ethanol (100 ml), concentrated sulfuric acid (4%) was added, and the mixture was heated to 75 ℃ for reaction for 2.5 hours. After cooling to room temperature, saturated sodium chloride solution (100 ml) was added and alkane extracted (200 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate and the filtrate was rotary dried to give 9.01 g of crude ester. EE content was 31% (see FIG. 3 for the GCMS analysis).

The EE separation process using packed column chromatography was as follows: continued elution with 20% mobile phase a + mobile phase B gave a fraction containing EE. The first 1.2L of eluent obtains 2.1 g of organic matters and no EE product; the second batch of 1.2 liters of eluent obtains 0.5 g of organic matters, and the EE content in GCMS analysis is 42.5 percent; the third 1.2 l of eluate yielded 2.7 g of organics with an EE content of 95% by GCMS analysis (see FIG. 4 for GCMS analysis); the fourth batch of eluent was free of EE product. The total yield of the target product is more than 99 percent.

Example 3

20 g of algal oil (crude extract of microalgae) was weighed, added with potassium hydroxide-methanol solution (300 ml, 0.4 mol/l), and stirred at room temperature overnight. Water (100 ml) was added and methanol was recovered by rotary evaporator. The residue was neutralized to pH1 with an alkane (300 ml) added to an ice bath using HCl solution (1 mol/l). Suction filtration, separation of layers and extraction of the aqueous phase with alkane (400 ml). The combined organic phases were dried over anhydrous sodium sulfate and spin dried to give 13.47 g of crude oil.

13.47 g of the crude oil was dissolved in ethanol (150 ml), concentrated sulfuric acid (4%) was added, and the mixture was heated to 75 ℃ for reaction for 2.5 hours. After cooling to room temperature, saturated sodium chloride (100 ml) was added and alkane extracted (200 ml × 3). The combined organic phases were dried over anhydrous sodium sulfate and the filtrate was rotary dried to give 11.10 g of crude ester. The EE content was 30%.

The EE separation process using packed column chromatography was as follows: by continuing the elution with mobile phase a + mobile phase B, a fraction containing EE can be obtained. The first 10% mobile phase a + mobile phase B was eluted, the earlier mobile phase was recovered by rotary evaporator and monitored by GCMS and TLC to give 6.2 g of organic material without EE product. After the EE peak appears in GCMS, 10% of mobile phase A + mobile phase B is used for elution, the mobile phase in the early stage is recycled by a rotary evaporator, the fraction before the EE purity reaches 98% is collected by GCMS, and the solvent is recycled by the rotary evaporator to obtain 1.30 g of organic matter with 64% of EE content (the GCMS analysis spectrum is shown in figure 5). After the EE purity is higher than 98%, 30% of mobile phase A + mobile phase B is used for elution, and the mobile phase at the early stage is recycled by a rotary evaporator to obtain 2.48 g of EE product with the purity of more than 98% (a GCMS analysis spectrum is shown in figure 6). The total yield of the product is 98 percent.

Example 4

26 g of algal oil (crude extract of microalgae) was weighed, added with potassium hydroxide-methanol solution (300 ml, 0.4 mol/l), and stirred at room temperature overnight. Water (100 ml) was added and methanol was recovered by rotary evaporator. The residue was neutralized to pH1 with an alkane (300 ml) added to an ice bath using HCl solution (1 mol/l). Suction filtration, separation of layers and extraction of the aqueous phase with alkane (400 ml). The combined organic phases were dried over anhydrous sodium sulfate and spin dried to give 13.60 g of crude oil.

13.60 g of the crude oil was dissolved in ethanol (150 ml), concentrated sulfuric acid (4%) was added, and the mixture was heated to 75 ℃ to react for 2.5 hours. After cooling to room temperature, saturated sodium chloride (100 ml) was added and alkane extracted (200 ml × 3). The combined organic phases were dried over anhydrous sodium sulfate and the filtrate was rotary dried to give 12.61 g of crude ester with an EE content of 35.49% in the crude product.

The EE separation process using packed column chromatography was as follows: the fraction containing EE can be obtained by continuous elution with mobile phase A + mobile phase B and follow-up detection by GCMS and TLC. The first 13% mobile phase a + mobile phase B was eluted, the earlier mobile phase was recovered by rotary evaporator and monitored by GCMS and TLC to give 6.2 g of organic material without EE product. After the GCMS has an EE peak, eluting by using 30 percent of mobile phase A + mobile phase B, recycling the mobile phase through a rotary evaporator, collecting all fractions of the GCMS showing the EE product, and recycling the solvent by using the rotary evaporator. A total of 4.45 g of EE-containing product was obtained with a purity of 94% (see FIG. 7 for the GCMS analysis). The total yield of the product is 93 percent.

Example 5

Referring to FIGS. 8 and 9, 500 g of algal oil was dissolved in a solution of potassium hydroxide-methanol (0.4 mol/l, 5 l) and mechanically stirred at room temperature for 21 hours. After the reaction was complete, water (2 l) was added and methanol was recovered using a rotary evaporator. The residue was taken up in n-hexane (2 l) and the system was cooled to 10 ℃ by passing through a cold water bath, and the pH was adjusted to 1 with dilute hydrochloric acid (1 mol/l). Alkane (4L x2) was added for extraction, the combined organic phases were dried and concentrated to recover the solvent. The aqueous phase was concentrated to 2-3 l by rotary evaporation, extracted with ethyl acetate (3.5 l x2), the combined organic phases dried and concentrated to recover the solvent. The crude oil obtained was concentrated by two extractions, 380 g.

380 g of crude oil are added to a solution of ethanol (2.5 l), concentrated sulfuric acid (4%) is added dropwise with stirring and the mixture is heated to 80 ℃ for reaction overnight. After the reaction was complete, the excess ethanol was removed by rotary evaporator, saturated sodium chloride solution (1.2 l) was added and extracted with alkane (2 l x 3). The organic phases were combined and dried over anhydrous sodium sulfate and the solvent was removed on a rotary evaporator to yield 370 g of crude black. The crude product was coarsely fractionated with silica gel (855 g) to remove pigments and more polar impurities, yielding 204 g of a golden yellow oil. The content of EE is 31 percent according to GCMS detection.

The EE separation process using packed column chromatography was as follows: the fraction containing EE can be obtained by continuous elution with mobile phase A + mobile phase B and follow-up detection by GCMS and TLC. The first 13% mobile phase a + mobile phase B eluted, and the earlier mobile phase was recovered by rotary evaporator for reuse and monitored by GCMS and TLC. After the GCMS has an EE peak, eluting with 30% of mobile phase A + mobile phase B, recycling the mobile phase through a rotary evaporator, collecting a solution with the EE content of less than 60%, removing the solvent by the rotary evaporator to obtain 52 g of a product with the EE content of 50%, continuously eluting with 30-50% of mobile phase A + mobile phase B until no EE product exists, and removing the solvent by the rotary evaporator to obtain 36.2 g of a product with the EE content of more than 95%. The total yield of the product is 97%.

Claims

1. A method for extracting polyunsaturated fatty acid from microalgae algal oil is characterized by comprising the following steps: the method comprises the following steps:

step 1: sequentially saponifying, acidifying and extracting the algae oil to obtain crude fat;

step 2: esterifying the crude fat to obtain a fatty acid fat mixture;

2. The method of claim 1, wherein the method comprises the steps of: in step 1, saponifying with an alcohol solution of an alkali metal hydroxide; the alkali metal hydroxide is sodium hydroxide, lithium hydroxide or potassium hydroxide.

3. The method of claim 2, wherein the method comprises the steps of: the alcohol solution is methanol, ethanol or isopropanol.

4. The method of claim 1, wherein the method comprises the steps of: in the step 1, the saponification reaction temperature is 0-45 ℃; the saponification reaction time is 12-36 hours.

5. The method of claim 1, wherein the method comprises the steps of: in the step 1, the acidification is carried out by using dilute hydrochloric acid or dilute sulfuric acid under a low-temperature condition, and the pH value is between 1 and 3.

6. The method of claim 1, wherein the method comprises the steps of: in the step 1, the extraction is carried out by adopting a mixed solvent, and a halogenated hydrocarbon-alcohol system, an alkane-ester system and an ether-alkane or ether-ester system are adopted as organic phases; and removing the solvent from the solution obtained by the extraction in the step by using a rotary evaporator to obtain crude ester.

7. The method of claim 1, wherein the method comprises the steps of: in step 2, the esterification process is carried out by adopting an acid catalysis esterification method: using ethanol as a solvent, and heating and reacting for 16 hours under the catalysis of concentrated sulfuric acid to completely convert fatty acid in the crude ester into a fatty acid ester mixture; the fatty acid ester mixture is an ethyl ester mixture.

8. The method of claim 1, wherein the method comprises the steps of: in step 3, the separation comprises a pre-separation and a further separation 2;

pre-separating the fatty acid ester mixture by using crude silica gel with the weight ratio of 1-3 times;

9. The method of claim 8, wherein the step of extracting the polyunsaturated fatty acids from the microalgae oil comprises: in the preliminary separation of step 3, preferably 2 times by weight; the eluent is a mobile phase A + a mobile phase B; the purpose of removing pigment and impurities with larger polarity is realized by pre-separation; the resulting fatty acid ester mixture was a bright yellow solution.

10. The method of claim 8, wherein the step of extracting the polyunsaturated fatty acids from the microalgae oil comprises: in the further separation in the step 3, the filler with the proportion of 5-6 times of volume ratio is preferably selected, wherein the proportion of the diatomite in the total volume of the separation filler is 0-10%; the elution solvent is mobile phase A + mobile phase B; wherein the mobile phase A is halogenated hydrocarbon, including chloroform, dichloromethane or dichloroethane; the mobile phase B is alkane selected from C4-C8 alkane or mixed alkane;