Method for separating saturated and unsaturated fatty acids and application thereof

Abstract

The invention relates to a method for separating saturated and unsaturated fatty acids and application thereof. The residual amount of the dioxin in the unsaturated fatty acid is reduced through ethanol washing, reduced pressure distillation, molecular cycle fractionation, ethyl eicosapentaenoate and ethyl docosahexaenoate extraction, silicic acid gel adsorption and a glycerol density fractional crystallization system, and the residual amount of the dioxin in the unsaturated fatty acid is at an extremely low level and reaches below 0.005 ng / kg of TEQ; the total mass fraction of EPA and DHA in the unsaturated fatty acid is increased through an enzymatic ester exchange reaction, a urea inclusion reaction and a glycerol density fractional crystallization system, the total mass fraction of EPA and DHA in the unsaturated fatty acid is at a high level, and the purity reaches 99% or above.

Description

Technical Field

[0001] The invention relates to the technical field of unsaturated fatty acid extraction, in particular to a method for separating saturated and unsaturated fatty acids and application thereof. Background Art

[0002] Fatty acids include saturated fatty acids and unsaturated fatty acids. Unsaturated fatty acids further include monounsaturated fatty acids and polyunsaturated fatty acids. Polyunsaturated fatty acids, especially DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), can improve cardiovascular and immune functions, reduce cancer, diabetes and hypertension, and play an important role in human health. High-purity DHA and EPA have a wide range of application needs in the fields of food and beverages, medicine and health products.

[0003] How to separate saturated and unsaturated fatty acids and improve the purity of unsaturated fatty acids, especially DHA and EPA, is one of the main tasks of scientific researchers. At present, the methods for separating saturated and unsaturated fatty acids mainly include chromatography, supercritical extraction, crystallization, etc. However, chromatography and supercritical extraction are costly, complicated to operate, and have limited processing capacity, while crystallization has low purity.

[0004] Microalgae crude oil is a crude oil extracted from microalgae cells. Due to its sustainability and high yield, microalgae crude oil is considered to be one of the important sources of oil in the future. Microalgae crude oil is rich in a variety of fatty acids, including saturated fatty acids and unsaturated fatty acids, especially DHA and EPA, and is an ideal raw material for preparing high-purity DHA and EPA.

[0005] Dioxins are a class of chlorine-containing organic pollutants that are extremely toxic and environmentally persistent. They are highly chemically stable and fat-soluble, difficult to degrade, and easily accumulate in adipose tissue and oil, posing a serious threat to human health and the ecosystem. In recent years, due to the intensification of environmental pollution, the content of dioxins in microalgae crude oil has increased. Since dioxins are lipophilic, it is difficult to remove dioxins from microalgae crude oil. Commonly used methods for removing dioxins from oils and fats include supercritical fluid extraction and membrane separation. The equipment cost of supercritical fluid extraction is high, and its large-scale application is limited. The membrane of the membrane separation method is easily contaminated and needs to be cleaned or replaced regularly, which adds additional costs.

[0006] Therefore, it is of great value to establish a method for separating saturated and unsaturated fatty acids to improve the purity of unsaturated fatty acids while reducing the dioxin content. Summary of the invention

[0007] In order to solve the above problems, the first aspect of the present invention provides a method for separating saturated and unsaturated fatty acids, comprising the following steps:

[0008] Step S1, acid refining and degumming: heating the microalgae crude oil to 50°C to 55°C, adding citric acid solution to the microalgae crude oil, stirring evenly, then adding purified water, stirring evenly; heating to 65°C to 70°C, centrifuging and collecting the oil phase liquid to prepare oil A;

[0009] Step S2, ethanol washing: adding ethanol solution to oil A to wash oil A; after washing, centrifuging and collecting the oil phase liquid to prepare oil B;

[0010] Step S3, vacuum distillation: placing the oil B in a distiller for vacuum distillation, collecting the heavy fraction, and preparing the oil C;

[0011] Step S4, enzymatic transesterification with immobilized lipase: after adding ethanol to the oil C, add immobilized lipase to carry out enzymatic transesterification reaction; filter to remove the lipase to prepare oil D;

[0012] Step S5, molecular fractionation: placing the oil D in a molecular fractionator for fractionation, collecting the heavy fraction, and preparing the oil E;

[0013] Step S6, treatment of ethyl eicosapentaenoate and ethyl docosahexaenoate: adding extractant I to oil E, mixing evenly, to prepare oil F; the extractant I is a mixture of ethyl eicosapentaenoate and ethyl docosahexaenoate;

[0014] Step S7, molecular circulation fractionation: placing oil F in a molecular fractionator for fractionation, collecting the heavy fraction to prepare oil G; collecting the distillate to prepare extractant II; adding extractant II to oil G, mixing evenly, and repeating step S7 to perform molecular circulation fractionation;

[0015] Step S8, silica gel adsorption: adding silica gel to the oil G, stirring and adsorbing; after adsorption, filtering to remove the silica gel, to prepare oil H;

[0016] Step S9, oil ethanolysis: adding ethanol and sodium hydroxide to the oil H to perform oil ethanolysis to prepare oil I;

[0017] Step S10, purified water extraction: add purified water to the oil I, mix evenly, extract, centrifuge and take the oil phase; repeat the extraction 1 to 3 times to prepare oil J;

[0018] Step S11, adding urea inclusion seed crystals: urea is dissolved in ethanol to prepare a urea solution, and the mass ratio of urea to ethanol is 1:(2-4); taking oil J, adding it to the urea solution, and then adding urea inclusion seed crystals to carry out inclusion reaction; filtering the reaction liquid, collecting the precipitated crystals and the filtrate respectively; the precipitated crystals are dispersed and extracted with purified water to prepare oil K; the filtrate is extracted with purified water to prepare oil L;

[0019] Step S12, glycerol density fractional crystallization: using a glycerol aqueous solution with a mass concentration of 50% as a centrifugal medium, adding the oil L into the centrifugal medium, performing ultracentrifugation, and collecting the liquid zone, which is the oil M;

[0020] Step S13, distillation: distill the oil M, collect the distillate and the heavy distillation at the bottom of the tower; the distillate is the polyunsaturated fatty acid ethyl ester.

[0021] The extractant II in step S7 can be reused.

[0022] As a preferred embodiment, the method for separating saturated and unsaturated fatty acids further comprises step S14 and step S15; in step S14, immobilized lipase is used to treat the heavy fractionation: immobilized lipase is added to the heavy fractionation at the bottom of the tower for enzymatic reaction; lipase is removed by filtration to produce oil N;

[0023] Step S15, molecular fractionation: placing the oil N in a molecular fractionator for fractionation, collecting the distillate, and preparing very long-chain polyunsaturated fatty acid ethyl esters.

[0024] As a preferred embodiment, the mass concentration of the citric acid solution in step S1 is 50%, the mass ratio of the citric acid solution to the microalgae crude oil is 0.2:100; the stirring time is 20 minutes to 40 minutes; and the mass ratio of the purified water to the microalgae crude oil is 2:100.

[0025] As a preferred embodiment, the ethanol solution in step S2 is an ethanol aqueous solution with a volume ratio of 50% to 70%; and the washing temperature is 8°C to 10°C.

[0026] As a preferred embodiment, the temperature of the vacuum distillation in step S3 is 60°C to 100°C, the pressure of the vacuum distillation is 5Pa to 10Pa, and the time of the vacuum distillation is 3 hours to 5 hours.

[0027] As a preferred embodiment, the immobilized lipase in step S4 is Novozym435; the mass ratio of ethanol to oil C is (0.8-1):1; the mass ratio of oil C to lipase Novozym435 is 1:(0.1-0.2); the reaction temperature is 37°C-40°C, and the reaction time is 18 hours-20 hours.

[0028] As a preferred solution, the flow rate of oil D during fractionation in step S5 is 100 kg / h / m 2 ~120kg / h / m 2 The distillation temperature is 150℃~180℃, and the distillation pressure is 20Pa~50Pa.

[0029] As a preferred embodiment, in step S6, the mass ratio of eicosapentaenoic acid ethyl ester to docosahexaenoic acid ethyl ester is 0.1-1; the mass ratio of extractant I to oil E is (0.05-0.07):1.

[0030] As a preferred solution, the flow rate of the oil F during fractionation in step S7 is 100 kg / h / m 2 ~120kg / h / m 2 The distillation temperature is 150℃~180℃, and the distillation pressure is 20Pa~50Pa.

[0031] As a preferred embodiment, the adsorption temperature in step S8 is 20°C to 30°C; the adsorption time is 3 hours to 5 hours; and the mass ratio of silica gel to oil G is (0.1 to 0.2):1.

[0032] As a preferred embodiment, the mass ratio of ethanol to oil H in step S9 is (1-6):1, the mass ratio of sodium hydroxide to oil H is (0.2-0.6):1, the reaction temperature is 70°C-80°C, and the reaction time is 1 hour to 3 hours.

[0033] As a preferred embodiment, the inclusion reaction temperature in step S11 is 5° C. to 7° C., the inclusion reaction time is 8 hours to 10 hours; and the mass ratio of the oil J to the urea solution is 1:(3-5).

[0034] In step S11, the fat K is saturated and monounsaturated fatty acids.

[0035] The present invention has no special requirements for urea inclusion seed crystals, and any crystal precipitate precipitated during the urea inclusion reaction can be used as urea inclusion seed crystals. Urea inclusion seed crystals can be prepared by a special urea inclusion reaction test, or a portion of urea inclusion crystal precipitate can be taken as urea inclusion seed crystals during the oil preparation process. It is preferred to use the urea inclusion reaction conditions of the present invention to prepare urea inclusion seed crystals.

[0036] As a preferred embodiment, the centrifugal force in step S12 is 1000000 g, the centrifugal time is 20 hours to 24 hours; the centrifugal temperature is 1° C. to 3° C.; and the mass ratio of the centrifugal medium to the oil L is (2 to 4):1.

[0037] As a preferred solution, the distillation temperature in step S13 is 180° C. to 185° C., and the pressure is 5 Pa to 10 Pa.

[0038] As a preferred embodiment, the immobilized lipase in step S14 is Lipozyme 435; the reaction temperature is 60°C to 80°C, the reaction time is 30 hours to 40 hours; and the mass ratio of the bottom heavy fraction to the lipase Lipozyme 435 is 20:(1-2).

[0039] As a preferred solution, the oil flow rate N during fractionation in step S15 is 60 kg / h / m 2 ~80kg / h / m 2 The distillation temperature is 170℃~180℃, and the distillation pressure is 10Pa~20Pa.

[0040] The second aspect of the present invention provides an unsaturated fatty acid, which is prepared by the above-mentioned method for separating saturated and unsaturated fatty acids.

[0041] In a third aspect of the present invention, the unsaturated fatty acids are applied to infant formula milk powder.

[0042] Through the above technical solutions, the present invention produces the following technical effects:

[0043] (1) The dioxin residue in oils and fats was reduced by ethanol washing, vacuum distillation, molecular circulation fractionation, extraction of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid, silica gel adsorption, and glycerol density fractionation crystallization. The dioxin residue in oils and fats was at an extremely low level, reaching TEQ 0.005ng / kg.

[0044] (2) The total mass fraction of EPA and DHA in the oil was increased by enzymatic ester exchange reaction, urea inclusion reaction, and glycerol density fractional crystallization. The total mass fraction of EPA and DHA in the oil was at a high level and the purity reached 99%.

[0045] (3) During the urea inclusion reaction, by adding urea inclusion seed crystals, the induction period of crystallization is significantly shortened, the reaction process is accelerated, and the reaction time is shortened.

[0046] (4) During glycerol density fractional crystallization, water is added to the centrifugal medium glycerol to increase the water solubility of the centrifugal medium. The water solubility gradient will stimulate the formation of lipid nano-micelles and thus shorten the equilibrium time. Adding water can also reduce the viscosity of the centrifugal medium and shorten the centrifugation time.

[0047] (5) The heavy fractions remaining in the process are fully utilized to prepare very long-chain polyunsaturated fatty acid ethyl esters, wherein the polyunsaturated fatty acid ethyl esters with carbon atoms greater than or equal to 24 have high purity and a total mass fraction of 90%; and the total cholesterol residue is low, only 0.2 mg / g. DETAILED DESCRIPTION

[0048] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the applicant first explains and analyzes through test examples and embodiments.

[0049] Definitions in this application:

[0050] Fatty acids in the present application include free fatty acids, fatty acid esters, and fatty acid chain derivatives.

[0051] Polyunsaturated fatty acids are fatty acids that have two or more double bonds in their molecules.

[0052] Very long chain polyunsaturated fatty acids are unsaturated fatty acids with more than 24 carbon atoms.

[0053] Long-chain polyunsaturated fatty acids are unsaturated fatty acids with more than 20 carbon atoms.

[0054] Medium chain fatty acids are fatty acids with 8-18 carbon numbers.

[0055] Short-chain fatty acids are fatty acids with 6 or fewer carbon atoms.

[0056] EPA is eicosapentaenoic acid and DHA is docosahexaenoic acid.

[0057] TEQ (Toxic Equivalent Quantity) is a standardized method for evaluating the toxicity of dioxin compounds, including polychlorinated dibenzo-p-dioxins (PCDDs). Due to the large variety of dioxin compounds and the huge differences in toxicity, TEQ simplifies toxicity assessment and comparison by standardizing the toxicity of various compounds relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

[0058] Test Example 1, dioxin removal test by ethanol washing:

[0059] 1. Preparation of test samples:

[0060] Preparation of Grease B1:

[0061] Acid refining and degumming: heating the crude microalgae oil to 50°C, adding citric acid solution to the crude microalgae oil, stirring evenly, then adding purified water, stirring evenly; heating to 65°C, centrifugally collecting the oil phase liquid to prepare oil A; the mass concentration of the citric acid solution is 50%, the mass ratio of the citric acid solution to the crude microalgae oil is 0.2:100; the mass ratio of the purified water to the crude microalgae oil is 2:100; the stirring time is 20 minutes;

[0062] Ethanol washing: adding ethanol solution to oil A to wash oil A; collecting the oil phase liquid by centrifugation after washing to prepare oil B1; the ethanol solution is 50% ethanol water by volume; the washing temperature is 8°C.

[0063] Preparation of oil B2: Use purified water for washing instead of ethanol, and other conditions are the same as those for preparation of oil B1.

[0064] 2. Test items:

[0065] The dioxin contents of oil A, oil B1 and oil B2 were measured according to the document "Dioxins and polychlorinated biphenyls in fish oil dietary supplements and licensed medicines" (Food Surveillance Information Sheet, Vol. 106, June 1997, MAFF, London).

[0066] 3. Results and analysis:

[0067] The test results are shown in the table below. The results show that ethanol washing can reduce the dioxin residue in oils and fats. Dioxins are highly fat-soluble. The inventors use ethanol solution instead of water to wash oils and fats, and extract dioxins from oils and fats through the fat-soluble properties of ethanol, thereby achieving the purpose of reducing dioxins in oils and fats.

[0068] Table 1 Ethanol washing test results

[0069] sample Processing conditions Dioxin residue (TEQng / kg) Grease A / 3.59 Oil B1 Ethanol washing 2.98 Oil B2 Water washing 3.60

[0070] Test Example 2, vacuum distillation test:

[0071] 1. Preparation of test samples:

[0072] Preparation of Grease C:

[0073] Vacuum distillation: Place the oil B1 in a distiller for vacuum distillation, collect the heavy fraction, and prepare oil C; the vacuum distillation temperature is 60°C, the vacuum distillation pressure is 5Pa, and the vacuum distillation time is 3 hours.

[0074] 2. The test items are the same as those in Test Example 1.

[0075] 3. Results and analysis:

[0076] The test results are shown in the table below. The results show that vacuum distillation can reduce the residual dioxin in oil. Dioxin is a general term for persistent organic pollutants with similar chemical structures and biological activities. Due to the heterogeneity of their molecular structures, they have different volatility, different fat solubility, and different binding tightness with oil.

[0077] The inventors utilized the heterogeneity of dioxin molecules to reduce the dioxin content in oils and fats while removing volatile substances, free fatty acids and other impurities through reduced pressure distillation.

[0078] Table 2 Vacuum distillation test results

[0079] sample Processing conditions Dioxin residue (TEQng / kg) Oil B1 Initial Value 2.98 Grease C Vacuum distillation 1.51

[0080] Test Example 3, immobilized lipase enzymatic transesterification test:

[0081] 1. Preparation of test samples:

[0082] Preparation of Grease E1:

[0083] Immobilized lipase enzymatic ester exchange: After adding ethyl acetate to oil C, immobilized lipase Novozym435 is added to carry out enzymatic reaction; the mass ratio of ethyl acetate to oil C is 0.8:1; the mass ratio of oil C to lipase Novozym435 is 1:0.1; the reaction temperature is 37°C, and the reaction time is 18 hours; the lipase is removed by filtration to prepare oil D.

[0084] Molecular fractionation: Place oil D in a molecular fractionator for fractionation, collect the heavy fraction, and make oil E1; the flow rate of oil D during fractionation is 100kg / h / m 2 , the distillation temperature is 150℃ and the distillation pressure is 20Pa.

[0085] Preparation of Grease E2:

[0086] Molecular fractionation: Place oil C in a molecular fractionator for fractionation, collect the heavy fraction, and make oil E2; the flow rate of oil C during fractionation is 100kg / h / m 2 , the distillation temperature is 150℃ and the distillation pressure is 20Pa.

[0087] 2. Test items:

[0088] The total mass fraction of EPA and DHA in Oil C, Oil E1 and Oil E2 was measured.

[0089] The dioxin residues of Grease C, Grease E1 and Grease E2 were measured using the same detection method as in Test Example 1.

[0090] 3. Results and analysis:

[0091] The test results are shown in the table below.

[0092] Compared with the oil E2 that was not subjected to the enzymatic transesterification reaction, the total mass fraction of EPA and DHA in the oil E1 was significantly improved. The total mass fraction of EPA and DHA can be significantly improved by the reaction conditions of the immobilized lipase enzymatic transesterification of the present invention. During the lipase enzymatic transesterification reaction, the reaction rate of short-chain fatty acid esters is greater than that of long-chain fatty acid esters, and the reaction rate of fatty acid esters with higher saturation is greater than that of fatty acid esters with lower saturation. EPA and DHA belong to polyunsaturated fatty acids with lower saturation, and the enzymatic transesterification reaction rate is slow. The inventors used this characteristic to replace the short-chain fatty acids, medium-chain fatty acids, saturated fatty acids, and monounsaturated fatty acids in the glyceride structure in the oil C from the glyceride through the transesterification reaction to form short-chain fatty acid ethyl esters, medium-chain fatty acid ethyl esters, saturated fatty acid ethyl esters, and monounsaturated fatty acid ethyl esters; while long-chain fatty acids such as EPA and DHA are retained in the glyceride structure. The volatility difference between the above-mentioned ethyl esters and glycerides is then used to separate them through molecular fractionation to increase the mass fractions of EPA and DHA in the oil.

[0093] Compared with the oil E2 that did not undergo enzymatic transesterification, the dioxin residue in the oil E1 was significantly reduced. During molecular fractionation, the medium-chain fatty acid ethyl esters, saturated fatty acid ethyl esters, and monounsaturated fatty acid ethyl esters that were transesterified from the glycerides were distilled with some dioxins, further reducing the dioxin residue in the heavy fraction.

[0094] Table 3 Enzymatic transesterification test results

[0095]

[0096] Test Example 4, test of eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester:

[0097] 1. Preparation of test samples:

[0098] Preparation of Grease G1:

[0099] Treatment of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid: add extractant I to oil E1, mix well, and prepare oil F; the extractant I is a mixture of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid, and the mass ratio of ethyl eicosapentaenoic acid to ethyl docosahexaenoic acid is 0.1; the mass ratio of extractant I to oil E is 0.05:1;

[0100] Molecular distillation: Place oil F in a molecular fractionator for fractionation, collect the heavy fraction and make oil G1; during the fractionation, the flow rate of oil F is 100kg / h / m2, the distillation temperature is 150℃, and the distillation pressure is 20Pa.

[0101] Preparation of Grease G2:

[0102] Molecular fractionation: oil E1 is placed in a molecular fractionator for fractionation, and the heavy fraction is collected to prepare oil G2; during fractionation, the flow rate of oil E1 is 100kg / h / m2, the fractionation temperature is 150°C, and the fractionation pressure is 20Pa.

[0103] 2. Test items:

[0104] The dioxin residues of Grease E1, Grease G1 and Grease G2 were measured using the same detection method as in Test Example 1.

[0105] 3. Results and analysis:

[0106] The test results are shown in the table below. Compared with oil G2 which was not treated with ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid, the dioxin residue in oil G1 was significantly reduced.

[0107] Some dioxins can bind to EPA and DHA molecules, and molecular fractionation makes it difficult to separate dioxins from EPA and DHA. The EPA and DHA content in oil E1 is relatively high, and it carries dioxins bound to EPA and DHA molecules. After adding ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid, the dioxins bound to EPA and DHA in glycerides are extracted through the effect of similar dissolution, and then the volatility and boiling point differences between glycerides and ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid are used to separate glycerides from ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid through molecular fractionation, thereby achieving the purpose of removing dioxins.

[0108] Table 4 Eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester test results

[0109]

[0110] Test Example 5, silica gel adsorption test:

[0111] 1. Preparation of test samples:

[0112] Preparation of Grease H:

[0113] Silicic acid gel adsorption: add silica gel to oil G1, stir and adsorb; filter and remove silica gel after adsorption to prepare oil H; the adsorption temperature is 20°C, the adsorption time is 3 hours, and the mass ratio of silica gel to oil G is 0.1:1.

[0114] 2. Test items:

[0115] The dioxin residues of Grease G1 and Grease H were measured using the same detection method as in Test Example 1.

[0116] 3. Results and analysis:

[0117] The test results are shown in the table below. After adsorption by silica gel, the dioxin residue in oil H was significantly reduced.

[0118] Silicic acid gel has a three-dimensional network structure with a large number of micropores, and the pore size is usually at the nanometer level. The surface of silica gel is rich in silanol groups, which can form hydrogen bonds or van der Waals forces with chlorine atoms or oxygen atoms in dioxin molecules to achieve chemical adsorption and achieve the purpose of removing dioxins.

[0119] Table 5 Silicic acid gel adsorption test results

[0120] sample Processing conditions Dioxin residue (TEQng / kg) Grease G1 Initial Value 0.26 Grease H Silicic acid gel adsorption 0.10

[0121] Test Example 6, adding urea to encapsulate seed crystals:

[0122] 1. Preparation of test samples:

[0123] Preparation of Grease L1:

[0124] Oil ethanolysis: add ethanol and sodium hydroxide to oil H to carry out oil ethanolysis to prepare oil I; the mass ratio of ethanol to oil H is 1:1, the mass ratio of sodium hydroxide to oil H is 0.2:1, the reaction temperature is 70°C, and the reaction time is 1 hour;

[0125] Purified water extraction: add purified water to oil I, mix well, extract, centrifuge and take the oil phase; repeat the purified water extraction once to make oil J;

[0126] Adding urea inclusion seed crystals: urea is dissolved in ethanol to prepare a urea solution, and the mass ratio of urea to ethanol is 1:2; taking oil J, adding it to the urea solution, and then adding urea inclusion seed crystals to carry out inclusion reaction; filtering the reaction liquid, and collecting the precipitate and the filtrate respectively; the inclusion reaction temperature is 5°C, and the inclusion reaction time is 8 hours; the mass ratio of oil J to urea solution is 1:3; the mass ratio of oil J to urea inclusion seed crystals is 1:0.1; the precipitate is dissolved and extracted with purified water to prepare oil K; the filtrate is extracted with purified water to prepare oil L1.

[0127] Preparation of oil L2: No urea inclusion seed was added during the inclusion reaction. Other process parameters were the same as those for preparation of oil L1.

[0128] 2. Test items:

[0129] The total mass fraction of EPA and DHA of Oil H, Oil J, Oil L1 and Oil L2 was measured.

[0130] The dioxin residues of Grease H, Grease J, Grease L1, and Grease L2 were measured using the same detection method as in Test Example 1.

[0131] 3. Results and analysis:

[0132] The test results are shown in the table below.

[0133] Dioxin residues can be reduced by ethanol hydrolysis, purified water extraction, and urea complexation. However, since dioxins are insoluble in water, ethanol hydrolysis and purified water extraction have limited effects on the reduction. Urea complexation can significantly reduce the dioxin residues of polyunsaturated fatty acid ethyl esters while removing saturated fatty acid ethyl esters and monounsaturated fatty acid ethyl esters.

[0134] Compared with oil L2 without urea inclusion seeds, under the same process parameters, especially when the inclusion reaction time was 8 hours, the total mass fraction of EPA and DHA in oil L1 was significantly increased.

[0135] During the urea inclusion reaction, the formation of seed crystals is relatively slow, and the inclusion reaction time required is relatively long. Only with sufficient reaction time can the fatty acid ethyl ester-urea inclusion complex precipitation be effectively formed. After adding seed crystals, the seed crystals provide ready-made crystal nuclei, promote the initiation of crystallization, reduce the randomness and uncertainty of spontaneous nucleation, significantly shorten the induction period of crystallization, accelerate the crystallization process, and improve the crystallization yield.

[0136] In this experiment, since urea inclusion seeds were not added to oil L2, a longer crystallization time was required. When the inclusion reaction lasted for 8 hours, the inclusion reaction was not sufficient, and the separation degree between saturated fatty acid ethyl esters and monounsaturated fatty acid ethyl esters and polyunsaturated fatty acid ethyl esters was low, which ultimately led to a low total mass fraction of EPA+DHA. However, oil L1 added urea inclusion seeds, which significantly shortened the induction period of crystallization. The inclusion reaction of 8 hours was sufficient for the inclusion reaction to be sufficient, and the separation degree between saturated fatty acid ethyl esters and monounsaturated fatty acid ethyl esters and polyunsaturated fatty acid ethyl esters was high, and the total mass fraction of EPA+DHA was high.

[0137] Table 6 Test results of adding urea inclusion seed

[0138]

[0139] The present invention has no special requirements for urea inclusion seed crystals, and any crystal precipitate precipitated during the urea inclusion reaction can be used as urea inclusion seed crystals. Urea inclusion seed crystals can be prepared by a special urea inclusion reaction test, or a portion of urea inclusion crystal precipitate can be taken as urea inclusion seed crystals during the oil preparation process. It is preferred to use the urea inclusion reaction conditions of the present invention to prepare urea inclusion seed crystals.

[0140] Test Example 7, glycerol density fractional crystallization test:

[0141] 1. Preparation of test samples:

[0142] Preparation of polyunsaturated fatty acid ethyl esters 1:

[0143] Glycerol density fractional crystallization: using a glycerol aqueous solution with a mass concentration of 50% as the centrifugal medium, adding the oil L1 into the centrifugal medium, performing ultracentrifugation, and collecting the liquid zone, which is the oil M; the centrifugal force is 1000000g, the centrifugation time is 20 hours; the centrifugation temperature is 1°C; the mass ratio of the centrifugal medium to the oil L1 is 2:1;

[0144] Distillation: distill the oil M, collect the distillate and the heavy fraction at the bottom of the tower; the distillate is the polyunsaturated fatty acid ethyl ester 1; the distillation temperature is 180°C and the pressure is 5Pa.

[0145] Preparation of polyunsaturated fatty acid ethyl ester 2: Oil L1 was directly distilled without glycerol density fractionation crystallization.

[0146] 2. Test items:

[0147] The total mass fraction of EPA and DHA in oil L1, oil M, polyunsaturated fatty acid ethyl ester 1, and polyunsaturated fatty acid ethyl ester 2 was measured.

[0148] The dioxin residues of oil L1, oil M, polyunsaturated fatty acid ethyl ester 1, and polyunsaturated fatty acid ethyl ester 2 were measured using the same detection method as in Test Example 1.

[0149] 3. Results and analysis:

[0150] The test results are shown in the table below. Through glycerol density fractional crystallization, the total mass fraction of EPA+DHA can be increased by utilizing the difference in crystallinity and density between EPA+DHA and other fatty acids; the residual dioxin content of oil M can be reduced by utilizing the difference in density between EPA+DHA and dioxins.

[0151] Adding water to the centrifugal medium glycerol increases the water solubility of the centrifugal medium. The water solubility gradient stimulates the formation of lipid nano-micelles, thereby shortening the equilibrium time. Adding water can also reduce the viscosity of the centrifugal medium and shorten the centrifugation time.

[0152] Table 7 Glycerol density fractional crystallization test results

[0153]

[0154] Test Example 8, Immobilized Lipase Treatment Test:

[0155] 1. Preparation of test samples:

[0156] Preparation of Very Long Chain Polyunsaturated Fatty Acid Ethyl Esters 1:

[0157] Immobilized lipase treatment of heavy fractionation: adding immobilized lipase Lipozyme 435 to the heavy fractionation at the bottom of the tower to carry out ester exchange reaction; the reaction temperature is 60° C., the reaction time is 30 hours; the mass ratio of the heavy fractionation at the bottom of the tower to the lipase Lipozyme 435 is 20:1; filtering to remove the lipase to prepare oil N;

[0158] Molecular fractionation: Place the oil N in a molecular fractionator for fractionation, collect the distillate, and prepare very long chain polyunsaturated fatty acid ethyl ester 1; the oil N flow rate during fractionation is 60 kg / h / m 2 , the distillation temperature is 170℃ and the distillation pressure is 10Pa.

[0159] Preparation of Very Long Chain Polyunsaturated Fatty Acid Ethyl Esters 2: The bottom heavy fractionation was not treated with immobilized lipase and was directly subjected to molecular fractionation.

[0160] 2. Test items:

[0161] The mass fraction of polyunsaturated fatty acid ethyl esters with carbon atoms greater than or equal to 24 in heavy fractionation, very long chain polyunsaturated fatty acid ethyl ester 1, and very long chain polyunsaturated fatty acid ethyl ester 2 was measured.

[0162] The total cholesterol residues of heavy fractions, very long chain polyunsaturated fatty acid ethyl esters 1, and very long chain polyunsaturated fatty acid ethyl esters 2 were measured according to AOAC Official Method 994.1.

[0163] 3. Results and analysis:

[0164] The test results are shown in the table below.

[0165] Compared with the very long chain polyunsaturated fatty acid ethyl ester 2 which has not been treated with immobilized lipase, the mass fraction of polyunsaturated fatty acid ethyl esters with 24 carbon atoms or more in the very long chain polyunsaturated fatty acid ethyl ester 1 is significantly improved. The volatility difference between cholesterol and very long chain polyunsaturated fatty acid ethyl ester is small, and the separation difficulty during molecular fractionation is relatively large. However, the volatility difference between cholesterol ester and very long chain polyunsaturated fatty acid ethyl ester is large, and the separation difficulty during molecular fractionation is relatively small. Through the immobilized lipase treatment conditions formulated by the present invention, free cholesterol can be converted into cholesterol ester, while very long chain polyunsaturated fatty acid ethyl ester rarely has a glycerol structure and still maintains an ethyl ester state. The volatility difference between cholesterol ester and very long chain polyunsaturated fatty acid ethyl ester is then utilized to remove cholesterol ester through molecular fractionation, thereby increasing the mass fraction of very long chain polyunsaturated fatty acid ethyl ester and reducing the total cholesterol residue.

[0166] The heavy fraction contains high levels of glycerol and free cholesterol, and has low commercial value, so it is generally discarded. However, this residue contains a large number of polyunsaturated fatty acids with 24 carbon atoms or more. Studies have shown that very long-chain polyunsaturated fatty acids have important physiological effects and are beneficial to eye and brain tissue function and male fertility. High-purity very long-chain polyunsaturated fatty acids are of great value to scientific research and dietary health.

[0167] Table 8 Immobilized lipase treatment test results

[0168]

[0169] Embodiment 1:

[0170] Step S1, acid refining and degumming: heating the microalgae crude oil to 50° C., adding citric acid solution to the microalgae crude oil, stirring evenly, then adding purified water, stirring evenly; heating to 65° C., centrifugally collecting the oil phase liquid to prepare oil A; the mass concentration of the citric acid solution is 50%, and the mass ratio of the citric acid solution to the microalgae crude oil is 0.2:100; the stirring time is 20 minutes; the mass ratio of the purified water to the microalgae crude oil is 2:100;

[0171] Step S2, ethanol washing: adding ethanol solution to oil A to wash oil A; collecting the oil phase liquid by centrifugation after washing to prepare oil B; the ethanol solution is 50% ethanol water by volume; the washing temperature is 8°C;

[0172] Step S3, vacuum distillation: placing oil B in a distiller for vacuum distillation, collecting heavy fractions, and preparing oil C; the vacuum distillation temperature is 60° C., the vacuum distillation pressure is 5 Pa, and the vacuum distillation time is 3 hours;

[0173] Step S4, enzymatic transesterification with immobilized lipase: after adding ethanol to oil C, add immobilized lipase to carry out enzymatic transesterification reaction; the immobilized lipase is Novozym435; the mass ratio of ethanol to oil C is 0.8:1; the mass ratio of oil C to lipase Novozym435 is 1:0.1; the reaction temperature is 37° C., and the reaction time is 18 hours; the lipase is removed by filtration to prepare oil D;

[0174] Step S5, molecular fractionation: placing oil D in a molecular fractionator for fractionation, collecting the heavy fraction to prepare oil E; the flow rate of oil D during fractionation is 100 kg / h / m 2 , the fractionation temperature is 150℃, and the fractionation pressure is 20Pa;

[0175] Step S6, treatment of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid: adding extractant I to oil E, mixing evenly, to prepare oil F; the extractant I is a mixture of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid; the mass ratio of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid is 0.1; the mass ratio of extractant I to oil E is 0.05:1;

[0176] Step S7, molecular circulation fractionation: placing oil F in a molecular fractionator for fractionation, collecting the heavy fraction to prepare oil G; collecting the distillate to prepare extractant II; during fractionation, the oil F flow rate is 100 kg / h / m2, the fractionation temperature is 150°C, and the fractionation pressure is 20 Pa;

[0177] Add extractant II to oil G, mix well, and repeat step S7 once;

[0178] Step S8, silica gel adsorption: adding silica gel to the oil G, stirring for adsorption; filtering to remove the silica gel after adsorption to prepare oil H; the adsorption temperature is 20°C; the adsorption time is 3 hours; the mass ratio of silica gel to oil G is 0.1:1;

[0179] Step S9, oil ethanolysis: adding ethanol and sodium hydroxide to the oil H to carry out oil ethanolysis to prepare oil I; the mass ratio of ethanol to oil H is 1:1, the mass ratio of sodium hydroxide to oil H is 0.2:1, the reaction temperature is 70°C, and the reaction time is 1 hour;

[0180] Step S10, purified water extraction: add purified water to oil I, mix evenly, extract, centrifuge and take the oil phase; repeat the extraction once to prepare oil J;

[0181] Step S11, adding urea inclusion seed crystals: urea is dissolved in ethanol to prepare a urea solution, and the mass ratio of urea to ethanol is 1:2; taking oil J, adding it to the urea solution, and then adding urea inclusion seed crystals to carry out inclusion reaction; filtering the reaction liquid, collecting the precipitated crystals and the filtrate respectively; the inclusion reaction temperature is 5°C, and the inclusion reaction time is 8 hours; the mass ratio of oil J to urea solution is 1:3; the precipitated crystals are dispersed and extracted with purified water to prepare oil K; the filtrate is extracted with purified water to prepare oil L;

[0182] Step S12, glycerol density fractional crystallization: using a glycerol aqueous solution with a mass concentration of 50% as a centrifugal medium, adding the oil L into the centrifugal medium, performing ultracentrifugation, and collecting the liquid zone, which is the oil M; the centrifugal force is 1000000g, the centrifugation time is 20 hours; the centrifugation temperature is 1°C; the mass ratio of the centrifugal medium to the oil L is 2:1;

[0183] Step S13, distillation: distill the oil M, collect the distillate and the heavy fraction at the bottom of the tower; the distillate is the polyunsaturated fatty acid ethyl ester; the distillation temperature is 180°C and the pressure is 5Pa;

[0184] Step S14, treating the heavy fraction with immobilized lipase: adding immobilized lipase to the heavy fraction at the bottom of the tower for enzymatic reaction; the immobilized lipase is Lipozyme 435; the reaction temperature is 60° C., the reaction time is 30 hours; the mass ratio of the heavy fraction at the bottom of the tower to the lipase Lipozyme 435 is 20:1; filtering to remove the lipase to produce oil N;

[0185] Step S15, molecular fractionation: placing the oil N in a molecular fractionator for fractionation, collecting the distillate, and preparing very long chain polyunsaturated fatty acid ethyl esters; the oil N flow rate during fractionation is 60 kg / h / m 2 , the distillation temperature is 170℃ and the distillation pressure is 10Pa.

[0186] Embodiment 2:

[0187] Step S1, acid refining and degumming: heating the microalgae crude oil to 52° C., adding citric acid solution to the microalgae crude oil, stirring evenly, then adding purified water, stirring evenly; heating to 67° C., centrifugally collecting the oil phase liquid to prepare oil A; the mass concentration of the citric acid solution is 50%, and the mass ratio of the citric acid solution to the microalgae crude oil is 0.6:100; the stirring time is 30 minutes; the mass ratio of the purified water to the microalgae crude oil is 2:100;

[0188] Step S2, ethanol washing: adding ethanol solution to oil A to wash oil A; collecting the oil phase liquid by centrifugation after washing to prepare oil B; the ethanol solution is 60% ethanol water by volume; the washing temperature is 9°C;

[0189] Step S3, vacuum distillation: placing oil B in a distiller for vacuum distillation, collecting heavy fractions, and preparing oil C; the vacuum distillation temperature is 80° C., the vacuum distillation pressure is 7 Pa, and the vacuum distillation time is 4 hours;

[0190] Step S4, enzymatic transesterification with immobilized lipase: after adding ethanol to oil C, add immobilized lipase to carry out enzymatic transesterification reaction; the immobilized lipase is Novozym435; the mass ratio of ethanol to oil C is 0.9:1; the mass ratio of oil C to lipase Novozym435 is 1:0.15; the reaction temperature is 38°C, and the reaction time is 19 hours; filter to remove lipase to prepare oil D;

[0191] Step S5, molecular fractionation: placing oil D in a molecular fractionator for fractionation, collecting the heavy fraction, and preparing oil E; the flow rate of oil D during fractionation is 110 kg / h / m 2 , the fractionation temperature is 160℃, and the fractionation pressure is 30Pa;

[0192] Step S6, treatment of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid: adding extractant I to oil E, mixing evenly, to prepare oil F; the extractant I is a mixture of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid; the mass ratio of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid is 0.5:1; the mass ratio of extractant I to oil E is 0.06:1;

[0193] Step S7, molecular circulation fractionation: placing oil F in a molecular fractionator for fractionation, collecting the heavy fraction to prepare oil G; collecting the distillate to prepare extractant II; during fractionation, the oil F flow rate is 110 kg / h / m2, the fractionation temperature is 170°C, and the fractionation pressure is 30 Pa;

[0194] Add extractant II to oil G, mix well, and repeat step S7 twice;

[0195] Step S8, silica gel adsorption: adding silica gel to the oil G, stirring for adsorption; filtering to remove the silica gel after adsorption to prepare oil H; the adsorption temperature is 25° C.; the adsorption time is 4 hours; the mass ratio of silica gel to oil G is 0.15:1;

[0196] Step S9, oil ethanolysis: adding ethanol and sodium hydroxide to the oil H to carry out oil ethanolysis to prepare oil I; the mass ratio of ethanol to oil H is 3:1, the mass ratio of sodium hydroxide to oil H is 0.4:1, the reaction temperature is 75°C, and the reaction time is 2 hours;

[0197] Step S10, purified water extraction: add purified water to oil I, mix evenly, extract, centrifuge and take the oil phase; repeat the extraction twice to prepare oil J;

[0198] Step S11, adding urea inclusion seed crystals: urea is dissolved in ethanol to prepare a urea solution, and the mass ratio of urea to ethanol is 1:3; taking oil J, adding it to the urea solution, and then adding urea inclusion seed crystals to carry out inclusion reaction; filtering the reaction liquid, collecting the precipitated crystals and the filtrate respectively; the inclusion reaction temperature is 6°C, and the inclusion reaction time is 9 hours; the mass ratio of oil J to urea solution is 1:4; the precipitated crystals are dispersed and extracted with purified water to prepare oil K; the filtrate is extracted with purified water to prepare oil L;

[0199] Step S12, glycerol density fractional crystallization: using a glycerol aqueous solution with a mass concentration of 50% as a centrifugal medium, adding the oil L into the centrifugal medium, performing ultracentrifugation, and collecting the liquid zone, which is the oil M; the centrifugal force is 1000000g, the centrifugation time is 22 hours; the centrifugation temperature is 2°C; the mass ratio of the centrifugal medium to the oil L is 3:1;

[0200] Step S13, distillation: distill the oil M, collect the distillate and the heavy fraction at the bottom of the tower; the distillate is the polyunsaturated fatty acid ethyl ester; the distillation temperature is 183° C. and the pressure is 7 Pa;

[0201] Step S14, treating the heavy fraction with immobilized lipase: adding immobilized lipase to the heavy fraction at the bottom of the tower for enzymatic reaction; the immobilized lipase is Lipozyme 435; the reaction temperature is 70° C., the reaction time is 35 hours; the mass ratio of the heavy fraction at the bottom of the tower to the lipase Lipozyme 435 is 20:1.5; filtering to remove the lipase to produce oil N;

[0202] Step S15, molecular fractionation: placing the oil N in a molecular fractionator for fractionation, collecting the distillate, and preparing very long chain polyunsaturated fatty acid ethyl esters; the oil N flow rate during fractionation is 70 kg / h / m 2 , the distillation temperature is 175℃ and the distillation pressure is 15Pa.

[0203] Embodiment 3:

[0204] Step S1, acid refining and degumming: heating the microalgae crude oil to 55° C., adding citric acid solution to the microalgae crude oil, stirring evenly, then adding purified water, stirring evenly; heating to 70° C., centrifugally collecting the oil phase liquid to prepare oil A; the mass concentration of the citric acid solution is 50%, and the mass ratio of the citric acid solution to the microalgae crude oil is 0.2:100; the stirring time is 40 minutes; the mass ratio of the purified water to the microalgae crude oil is 2:100;

[0205] Step S2, ethanol washing: adding ethanol solution to oil A to wash oil A; collecting the oil phase liquid by centrifugation after washing to prepare oil B; the ethanol solution is 70% ethanol water solution by volume; the washing temperature is 10°C;

[0206] Step S3, vacuum distillation: placing oil B in a distiller for vacuum distillation, collecting heavy fractions, and preparing oil C; the vacuum distillation temperature is 100° C., the vacuum distillation pressure is 10 Pa, and the vacuum distillation time is 5 hours;

[0207] Step S4, immobilized lipase enzymatic transesterification: after adding ethanol to the oil C, add immobilized lipase to carry out enzymatic transesterification reaction; the immobilized lipase is Novozym435; the mass ratio of ethanol to oil C is 1:1; the mass ratio of oil C to lipase Novozym435 is 1:0.2; the reaction temperature is 40°C, and the reaction time is 20 hours; filter to remove the lipase to prepare oil D;

[0208] Step S5, molecular fractionation: placing oil D in a molecular fractionator for fractionation, collecting the heavy fraction to prepare oil E; the flow rate of oil D during fractionation is 120 kg / h / m 2 , the fractionation temperature is 180℃, and the fractionation pressure is 50Pa;

[0209] Step S6, treatment of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid: adding extractant I to oil E, mixing evenly, to prepare oil F; the extractant I is a mixture of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid; the mass ratio of ethyl eicosapentaenoic acid and ethyl docosahexaenoic acid is 1; the mass ratio of extractant I to oil E is 0.07:1;

[0210] Step S7, molecular circulation fractionation: placing oil F in a molecular fractionator for fractionation, collecting the heavy fraction to prepare oil G; collecting the distillate to prepare extractant II; the flow rate of oil F during fractionation is 120 kg / h / m 2 , the fractionation temperature is 180℃, and the fractionation pressure is 50Pa;

[0211] Add extractant II to oil G, mix well, and repeat step S7 three times;

[0212] Step S8, silica gel adsorption: adding silica gel to the oil G, stirring for adsorption; filtering to remove the silica gel after adsorption to prepare oil H; the adsorption temperature is 30° C.; the adsorption time is 5 hours; the mass ratio of silica gel to oil G is 0.2:1;

[0213] Step S9, oil ethanolysis: adding ethanol and sodium hydroxide to the oil H to carry out oil ethanolysis to prepare oil I; the mass ratio of ethanol to oil H is 6:1, the mass ratio of sodium hydroxide to oil H is 0.6:1, the reaction temperature is 80°C, and the reaction time is 3 hours;

[0214] Step S10, purified water extraction: add purified water to oil I, mix evenly, extract, centrifuge and take the oil phase; repeat the extraction 3 times to prepare oil J;

[0215] Step S11, adding urea inclusion seed crystals: urea is dissolved in ethanol to prepare a urea solution, and the mass ratio of urea to ethanol is 1:4; taking oil J, adding it to the urea solution, and then adding urea inclusion seed crystals to carry out inclusion reaction; filtering the reaction liquid, collecting the precipitated crystals and the filtrate respectively; the inclusion reaction temperature is 7° C., and the inclusion reaction time is 10 hours; the mass ratio of oil J to urea solution is 1:5; the precipitated crystals are dispersed and extracted with purified water to prepare oil K; the filtrate is extracted with purified water to prepare oil L;

[0216] Step S12, glycerol density fractional crystallization: using a glycerol aqueous solution with a mass concentration of 50% as a centrifugal medium, adding the oil L into the centrifugal medium, performing ultracentrifugation, and collecting the liquid zone, which is the oil M; the centrifugal force is 1000000g, the centrifugation time is 24 hours; the centrifugation temperature is 3°C; the mass ratio of the centrifugal medium to the oil L is 4:1;

[0217] Step S13, distillation: distill the oil M, collect the distillate and the heavy fraction at the bottom of the tower; the distillate is polyunsaturated fatty acid ethyl ester; the distillation temperature is 185°C and the pressure is 10Pa;

[0218] Step S14, treating the heavy fraction with immobilized lipase: adding immobilized lipase to the heavy fraction at the bottom of the tower for enzymatic reaction; the immobilized lipase is Lipozyme 435; the reaction temperature is 80° C., the reaction time is 40 hours; the mass ratio of the heavy fraction at the bottom of the tower to the lipase Lipozyme 435 is 20:2; filtering to remove the lipase to produce oil N;

[0219] Step S15, molecular fractionation: placing the oil N in a molecular fractionator for fractionation, collecting the distillate, and preparing very long chain polyunsaturated fatty acid ethyl esters; the oil N flow rate during fractionation is 80 kg / h / m 2 , the distillation temperature is 180℃ and the distillation pressure is 20Pa.

[0220] The dioxin residues and the total mass fraction of EPA+DHA of the polyunsaturated fatty acid ethyl esters of Examples 1-3 were detected respectively.

[0221] The total cholesterol residue of the very long chain polyunsaturated fatty acid ethyl esters of Examples 1-3 and the total mass fraction of polyunsaturated fatty acid ethyl esters with carbon atoms greater than or equal to 24 were detected respectively.

[0222] The test results are shown in the table below. The polyunsaturated fatty acid ethyl ester prepared by the technical solution of the present invention has extremely low dioxin residues and a high total mass fraction of EPA+DHA; the very long chain polyunsaturated fatty acid ethyl ester prepared has a low total cholesterol residue and a high total mass fraction of polyunsaturated fatty acid ethyl esters with carbon atoms greater than or equal to 24.

[0223] Table 9 Example 1-3 polyunsaturated fatty acid ethyl ester detection results

[0224]

[0225] Table 10 Test results of very long chain polyunsaturated fatty acid ethyl esters of Example 1-3

[0226]

Claims

1. A method for separating saturated and unsaturated fatty acids, characterized in that The following steps are involved: Step S1, acid refining and degumming: adding citric acid solution to the microalgae crude oil, and then adding purified water, and stirring evenly; The temperature was raised to 65°C to 70°C, and the oil phase liquid was collected by centrifugation to prepare oil A; Step S2, ethanol washing: adding ethanol solution to oil A to wash oil A; after washing, centrifuging and collecting the oil phase liquid to prepare oil B; Step S3, vacuum distillation: placing the oil B in a distiller for vacuum distillation, collecting the heavy fraction, and preparing the oil C; Step S4, enzymatic transesterification with immobilized lipase: after adding ethanol to the oil C, add immobilized lipase to carry out enzymatic transesterification reaction; filter to remove the lipase to prepare oil D; Step S5, molecular fractionation: placing the oil D in a molecular fractionator for fractionation, collecting the heavy fraction, and preparing the oil E; Step S6, treatment of ethyl eicosapentaenoate and ethyl docosahexaenoate: adding extractant I to oil E, mixing evenly, to prepare oil F; the extractant I is a mixture of ethyl eicosapentaenoate and ethyl docosahexaenoate; Step S7, molecular circulation fractionation: placing oil F in a molecular fractionator for fractionation, collecting the heavy fraction to prepare oil G; collecting the distillate to prepare extractant II; adding extractant II to oil G, mixing evenly, and repeating step S7 to perform molecular circulation fractionation; Step S8, silica gel adsorption: adding silica gel to the oil G, stirring and adsorbing; after adsorption, filtering to remove the silica gel, to prepare oil H; Step S9, oil ethanolysis: adding ethanol and sodium hydroxide to the oil H to perform oil ethanolysis to prepare oil I; Step S10, purified water extraction: add purified water to the oil I, mix evenly, extract, centrifuge and take the oil phase; repeat the extraction 1 to 3 times to prepare oil J; Step S11, adding urea inclusion seed crystals: urea is dissolved in ethanol to prepare a urea solution; taking oil J, adding it to the urea solution, and then adding urea inclusion seed crystals to carry out an inclusion reaction; The reaction liquid is filtered, and the precipitated crystals and the filtrate are collected respectively; the precipitated crystals are dispersed and extracted with purified water to obtain oil K; the filtrate is extracted with purified water to obtain oil L; Step S12, glycerol density fractional crystallization: using a glycerol aqueous solution with a mass concentration of 50% as a centrifugal medium, adding the oil L into the centrifugal medium, performing ultracentrifugation, and collecting the liquid zone, which is the oil M; Step S13, distillation: distill the oil M, collect the distillate and the heavy fraction at the bottom of the tower; the distillate is the unsaturated fatty acid.

2. The method according to claim 1, characterized in that: In the step S1, the mass concentration of the citric acid solution is 50%, the mass ratio of the citric acid solution to the microalgae crude oil is 0.2:100; the stirring time is 20 minutes to 40 minutes; and the mass ratio of the purified water to the microalgae crude oil is 2:

100.

3. The method according to claim 2, characterized in that The ethanol solution in step S2 is an ethanol aqueous solution with a volume ratio of 50% to 70%; the washing temperature is 8°C to 10°C.

4. The method according to claim 3, characterized in that: The temperature of the vacuum distillation in step S3 is 60° C. to 100° C., the pressure of the vacuum distillation is 5 Pa to 10 Pa, and the vacuum distillation time is 3 hours to 5 hours.

5. The method according to claim 4, characterized in that The immobilized lipase in step S4 is Novozym435; the mass ratio of ethanol to oil C is (0.8-1):1; the mass ratio of oil C to lipase Novozym435 is 1:(0.1-0.2); the reaction temperature is 37°C-40°C, and the reaction time is 18 hours-20 hours.

6. The method according to claim 5, characterized in that The flow rate of oil D during fractionation in step S5 is 100 kg / h / m 2 ~120kg / h / m 2 The distillation temperature is 150℃~180℃, and the distillation pressure is 20Pa~50Pa.

7. The method according to claim 6, characterized in that In step S6, the mass ratio of eicosapentaenoic acid ethyl ester to docosahexaenoic acid ethyl ester is 0.1-1; the mass ratio of extractant I to oil E is (0.05-0.07):

1.

8. An unsaturated fatty acid, characterized in that The unsaturated fatty acid is prepared by the method according to claim 1.

9. Use of the unsaturated fatty acid according to claim 8 in infant formula milk powder.

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