A method for preparing a high-content EPA and low-content DHA fatty acid product by enzymatic method

Abstract

The present invention discloses a method for preparing a high-content eicosapentaenoic acid (EPA) and low-content docosahexaenoic acid (DHA) fatty acid product by an enzymatic method. The present invention uses EPA-rich oil as a raw material, utilizes the difference in the degree of difficulty of combining 1,2-propylene glycol / tert-butyl alcohol with different fatty acids, improves the selectivity of the enzyme, and simultaneously regulates the balance of the enzymatic hydrolysis reaction of the system to improve the difference in the content between EPA and DHA in the fatty acid. The present invention controls the water content of the reaction system to ensure a certain degree of hydrolysis reaction, and jointly regulates the balance of the hydrolysis reaction with 1,2-propylene glycol / tert-butyl alcohol to ensure the product yield. The lipase used in the present invention has a high degree of reusability, can significantly reduce production costs, and meet the requirements of large-scale production. EPA>70wt%, DHA<4wt% prepared by the method of the present invention.

Description

Technical Field

[0001] The invention relates to a method for preparing a fatty acid product with high content of eicosapentaenoic acid (EPA) and low content of docosahexaenoic acid (DHA) by enzymatic method. Background Art

[0002] n-3 polyunsaturated fatty acids are nutrients that participate in many physiological processes of the body and play an important role in human metabolism. EPA and DHA are the most common long-chain n-3 polyunsaturated fatty acids with a wide range of physiological effects. EPA, as a "vascular clearing agent", can arbitrarily lower blood lipids and cholesterol, prevent cardiovascular diseases, and prevent the formation of blood clots. It is often used in the middle-aged and elderly population. DHA, as "brain gold", can enhance memory and promote the development of the brain and visual system of infants and young children. It is often used in infants and young children.

[0003] EPA and DHA are mainly derived from marine organisms, such as tuna and sardines. There are certain differences in the content of EPA and DHA from different sources, but the relative content difference between the two is relatively low. For example, EPA is 6% and DHA is 22% in tuna oil; EPA is 16% and DHA is 9% in sardine oil; EPA is 22% and DHA is 9% in anchovy oil; EPA is 7% and DHA is 11% in salmon oil; EPA is 10% and DHA is 10% in cod; EPA is 14% and DHA is 8% in herring oil. Many studies have found that EPA alone and a mixture of EPA and DHA have different degrees of influence on the physiological functions of organisms, and even have opposite physiological functions. Therefore, obtaining high-purity EPA is of great significance.

[0004] The biggest challenge in preparing high-purity EPA is that EPA often coexists with DHA in natural raw materials or concentrated fish oil. Due to the high similarity in the chemical structures of EPA and DHA, it is difficult to completely separate them using conventional separation techniques, which results in EPA purity failing to reach the ideal level. In addition, some separation methods may introduce new impurities, cause EPA oxidation or cause EPA loss, further increasing the difficulty of preparing high-purity, high-quality EPA products.

[0005] EPA is an important n-3 series polyunsaturated fatty acid that plays an important role in the human body, but its access to it is limited. Although linolenic acid can be converted into EPA in the human body, the conversion amount and speed are at a low level, so it must be obtained from food. At present, the EPA content in marine fish oil is generally less than 20%, and existing research has not found a targeted method to produce EPA. For the acquisition of high-purity EPA, the most effective method is to prepare the liquid phase, but the cost is too high, and the produced EPA is only used in the production of some special drugs, and its application is relatively narrow. The enzymatic method for producing high-concentration EPA fatty acids has the advantages of high efficiency, specificity, mild reaction conditions, few by-products, and high enzyme reuse, and has a very high application value in the field of high value-added structural lipid production. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide an enzymatic method for preparing a fatty acid product with a high content of EPA and a low content of DHA.

[0009] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing a fatty acid product with high EPA content and low DHA content by enzymatic method, characterized in that:

[0010] Mixing the oil containing EPA and DHA, alcohol solvent and water, and performing an enzymatic reaction at 35-65° C. for 4-16 hours, wherein the molar ratio of the oil containing EPA and DHA to the alcohol solvent is 1:(11.5-46), and the oil-water ratio is 1:(1-5) in terms of w / w;

[0011] After the reaction is completed, the lipase and the solvent are removed, and the fatty acid products with EPA>70wt% and DHA<4wt% are obtained by further separation and purification.

[0012] As a preferred embodiment of the preparation method of the present invention, wherein: the alcohol solvent is 1,2-propylene glycol and / or tert-butyl alcohol.

[0013] As a preferred embodiment of the preparation method of the present invention, the molar ratio of the oil containing EPA and DHA to the alcohol solvent is 1:(23-34.5), and the oil-water ratio in w / w is 1:(2-5).

[0014] As a preferred embodiment of the preparation method of the present invention, the reaction temperature is 35°C to 45°C.

[0015] As a preferred embodiment of the preparation method of the present invention, the reaction time is 8 to 16 hours.

[0016] As a preferred embodiment of the preparation method of the present invention, the oil containing EPA and DHA comprises natural and / or synthetic lipids with EPA>15wt%.

[0017] As a preferred embodiment of the preparation method of the present invention, the preparation method of the synthetic lipid comprises: conducting an enzymatic reaction catalyzed by lipase at a ratio of greater than 3:1 between non-glyceride and glycerol at 40 to 80°C for 4 to 12 hours, removing the enzyme and unreacted substrate to obtain a synthetic lipid rich in EPA; wherein the pressure of the reaction system is below 1000 Pa.

[0018] As a preferred embodiment of the preparation method of the present invention, the method for separating the enzyme and the solvent is centrifugation, and the upper layer after centrifugal stratification is the crude product; the centrifugal speed is >3000r / min; and the centrifugal time is 3 to 20min.

[0019] As a preferred embodiment of the preparation method of the present invention, the molecular distillation comprises the following steps: adding the crude product obtained by centrifugal separation into an injector, obtaining a heavy phase and a light phase by short-path distillation under certain pressure and temperature conditions, collecting the light phase, and obtaining a crude fatty acid product containing EPA; the temperature is 100 to 260°C; the pressure is 0.1 to 50 Pa; the feed flow rate is 1 to 20 mL / min; and the scraper speed is 100 to 500 r / min.

[0020] As a preferred embodiment of the preparation method of the present invention, the urea inclusion complex comprises the following steps: the crude fatty acid product containing EPA is mixed with urea in a certain proportion in a 95% ethanol solution, heated and stirred until the entire mixture is a transparent homogeneous solution, and crystallized at a certain temperature. The obtained crystals (urine inclusion phase) and the liquid phase (non-urine inclusion phase) are separated and continue to crystallize at a lower temperature, and the non-urine inclusion phase is finally separated. The non-urine inclusion phase is acidified, water and n-hexane are added, the fatty acids are extracted, the organic phase is separated, and the solvent is removed under reduced pressure to obtain a fatty acid product with a high content of EPA and a low content of DHA.

[0021] As a preferred embodiment of the preparation method of the present invention, the mass ratio of the crude fatty acid product containing EPA to urea is 1:1 to 1:4; the first crystallization temperature is 0°C to 8°C; the second crystallization temperature is -50°C to -10°C; the pH after acidification is below 5; and the volume ratio of water and n-hexane added is 1:2 to 2:1.

[0022] As a preferred embodiment of the preparation method of the present invention, the lipase is produced by Candida antarctica, Rhizopus oryzae, Aspergillus niger, Candida rugosa, Burkaholderia cepacia and Pseudomonas.

[0023] As a preferred embodiment of the preparation method of the present invention, the lipase comprises one or more of CAL-A, ET 2.0, Novozyme 435, Lipozyme 435, Lipase TL 100L, Lipase PS "Amano", Lipase TL IM and Lipase RM IM, preferably ET 2.0 and Lipase TL 100L.

[0024] Beneficial effects of the present invention:

[0025] 1. The enzymatic reaction system of the present invention utilizes the difference in the difficulty of combining 1,2-propylene glycol / tert-butyl alcohol with different fatty acids to improve the selectivity of the enzyme, and at the same time regulates the balance of the enzymatic hydrolysis reaction of the system to increase the difference in the content of EPA and DHA in the fatty acids;

[0026] 2. The present invention controls the water content of the reaction system to ensure a certain degree of hydrolysis reaction, and jointly regulates the balance of the hydrolysis reaction with 1,2-propylene glycol / tert-butyl alcohol to ensure the product yield.

[0027] 3. The present invention adopts an enzymatic method to prepare fatty acid products with high EPA content and low DHA content, and the reaction conditions are mild, efficient, highly specific, and environmentally friendly;

[0028] 4. The present invention uses EPA-rich oil as raw material to obtain fatty acid products with high EPA content and low DHA content, EPA>70wt%, DHA<4wt%;

[0029] 5. The lipase used in the present invention has a high degree of reusability, which can significantly reduce production costs and meet the requirements of large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a gas chromatography (GC) chromatogram of the fish oil raw material used in Example 1 of the present invention. The data in the figure are the fatty acid composition and content of the fish oil raw material.

[0031] Figure 2 This is a GC chromatogram of the fatty acid product (after urea inclusion complex) prepared in Example 1 of the present invention. The data in the figure are the composition and content of the non-urea inclusion phase fatty acid product obtained after urea inclusion complex.

[0032] Figure 3 The figure is a liquid chromatogram (HPLC-RID) of the crude EPA fatty acid prepared by the enzymatic reaction of the present invention, wherein a is the lipid composition of the crude product of the enzymatic reaction obtained by using ET 2.0 as a catalyst in Example 2, b is the lipid composition of the crude product of the enzymatic reaction obtained under the conditions of Comparative Example 1, and c is the lipid composition of the crude product of the enzymatic reaction obtained under the conditions of Comparative Example 2.

[0033] Figure 4 It is a GC chromatogram of the crude product of EPA fatty acid prepared by molecular distillation of the enzymatic reaction of the present invention, wherein a is the composition and content of the crude fatty acid product after molecular distillation of the lipid obtained by using ET 2.0 as a catalyst in Example 2, and b is the composition and content of the crude fatty acid product after molecular distillation of the lipid obtained under the conditions of Comparative Example 1. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. The present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0037] The raw materials and reagents used in the present invention are as follows: the oil-rich substance is refined fish oil, which is purchased from Noah Shengnuo Biotechnology Co., Ltd., and the product model is OMAX 1812; 1,2-propylene glycol, ethanol, methanol, isopropanol, tert-butanol and other organic reagents are all purchased from Sinopharm Chemical Reagent Co., Ltd.; ET 2.0, TL 100L and CAL-A lipase are all purchased from Wuxi Youpu Biotechnology Co., Ltd.

[0038] Embodiment 1:

[0039] Add 30g fish oil, 60g water and 50g 1,2-propylene glycol to a 250mL conical flask, then add ET 2.0 lipase, the enzyme addition amount is 6.67% (based on the mass fraction of fish oil), mix well, and react for 8h at 45℃ with magnetic stirring at 300r / min. After the reaction is completed, centrifuge at 5000r / min for 5min to take the supernatant and remove the lipase and solvent. The obtained oil phase is molecularly distilled (temperature 190℃, pressure 7Pa, feed flow rate 10mL / min; scraper speed 300r / min) to obtain a crude fatty acid product. The crude product is subjected to urea inclusion (15g crude fatty acid product, 25g urea, 100mL 95% ethanol, after mixing well, inclusion at 4℃ for 8h, separation, supernatant inclusion at -30℃ for 12h) to obtain a high-content EPA and low-content DHA fatty acid product. The lipid composition is detected by HPLC-RID, and the free fatty acid content is calculated. The composition and content of fatty acids were detected by GC.

[0040] The free fatty acid contents in different lipids are shown in Table 1; the EPA and DHA contents in the free fatty acids in different lipids are shown in Table 2.

[0041] Figure 1 The fatty acid composition and content of the fish oil raw material in Example 1; Figure 2 The composition and content of fatty acids in the non-urea-encapsulated fatty acid product obtained after urea encapsulation in Example 1.

[0042] Table 1

[0043]

[0044] Table 2

[0045]

[0046]

[0047] As shown in Table 1, the free fatty acid content in the non-urea inclusion phase product after urea inclusion is as high as 99.69%. As shown in Table 2, the contents of EPA and DHA in the non-urea inclusion phase product after urea inclusion are 73.03% and 3.28%, respectively.

[0048] Embodiment 2:

[0049] Add 3g fish oil, 6g water and a certain amount of alcohol (the molar ratio of alcohol to oil is the same, both 23:1) to a 25mL conical flask, then add ET 2.0 lipase, the enzyme addition amount is 6.67% (based on the mass fraction of fish oil), mix well, and react for 8h at 45℃ with magnetic stirring at 300r / min. After the reaction is completed, centrifuge at 5000r / min for 5min to take the supernatant, remove the lipase and solvent, detect the lipid composition in the reaction product by HPLC-RID, and calculate the free fatty acid content. Glycerides and fatty acids are separated by molecular distillation, and the crude fatty acid product is detected by gas phase GC to detect the composition and content of fatty acids.

[0050] The effect of the type of alcohol in the enzymatic reaction on the free fatty acid content in the crude lipid product (without molecular distillation and urea inclusion treatment) is shown in Table 3; in different alcohol systems, the EPA and DHA contents in the crude fatty acid products after molecular distillation of the lipids obtained by the enzymatic reaction are shown in Table 4. As can be seen from Table 3, the fatty acid content is low in the methanol and ethanol systems. Since its lipid products are mainly composed of methyl esters and ethyl esters, they do not meet the requirements of the patented invention for producing fatty acid products. The data in Table 4 show that the relative difference between EPA and DHA in the crude fatty acid products after molecular distillation of the 1,2-propylene glycol and tert-butanol experimental groups is more significant, with EPA content being 37 times and 15 times that of DHA, respectively, and the EPA content in the crude fatty acid product of the tert-butanol group is as high as 30.68%, which is suitable for further purification of EPA in subsequent processes.

[0051] Figure 3 a is the lipid composition of the crude product of the enzymatic reaction obtained in Example 2 using ET 2.0 as a catalyst.

[0052] Table 3

[0053] Types of alcohol Methanol Ethanol 1,2-Propanediol Isopropyl alcohol Tert-Butanol Free fatty acid content 3.37％ 8.01％ 33.17％ 56.41％ 67.42％

[0054] Table 4

[0055] Types of alcohol Methanol Ethanol 1,2-Propanediol Isopropyl alcohol Tert-Butanol EPA Content 24.26％ 17.27％ 22.24％ 33.71％ 30.68％ DHA content 5.95％ 2.15％ 0.60％ 6.39％ 2.02％

[0056] Embodiment 3:

[0057] Add 3g fish oil, 5g 1,2-propylene glycol and 6g water to a 25mL conical flask, then add lipase, the enzyme addition amount is 6.67% (based on the mass fraction of fish oil), mix well, and react for 8h at 45℃ with magnetic stirring at 300r / min. After the reaction is completed, centrifuge at 5000r / min for 5min to take the supernatant, remove the lipase and solvent, detect the lipid composition in the reaction product by liquid HPLC-RID, and calculate the free fatty acid content in the lipid. Glycerides and fatty acids are separated by molecular distillation, and the crude fatty acid product is detected by gas phase GC to detect the composition and content of fatty acids.

[0058] The effect of the type of enzyme in the enzymatic reaction on the free fatty acid content in the crude lipid product is shown in Table 5; the EPA and DHA contents in the crude fatty acid product after molecular distillation of the lipids obtained by the enzymatic reaction under the catalysis of different lipases are shown in Table 6. The data in Table 6 show that the relative differences of EPA and DHA in the crude fatty acid products after molecular distillation of the ET 2.0 and TL 100L experimental groups are very significant, and their EPA content is 37 times and 50 times that of DHA, respectively.

[0059] Table 5

[0060] Types of enzymes ET2.0 CAL-A TL100L Free fatty acid content 33.17％ 32.01％ 30.13％

[0061] Table 6

[0062] Types of enzymes ET2.0 CAL-A TL100L EPA Content 22.24％ 4.72％ 23.59％ DHA content 0.60％ 0.26％ 0.47％

[0063] Embodiment 4:

[0064] In a 25mL conical flask, add 3g of fish oil, 1,2-propylene glycol and 6g of water according to different oil-alcohol molar ratios, and then add ET 2.0, the enzyme addition amount is 6.67% (based on the mass fraction of fish oil), and after mixing evenly, react at 45°C with magnetic stirring at 300r / min for 8h. After the reaction is completed, centrifuge at 5000r / min for 5min to take the supernatant, remove lipase and solvent, detect the lipid composition in the reaction product by liquid HPLC-RID, and calculate the free fatty acid content. Glycerides and fatty acids are separated by molecular distillation, and the crude fatty acid product is used to detect the composition and content of fatty acids by gas GC.

[0065] The effect of the oleyl alcohol ratio in the enzymatic reaction on the free fatty acid content in the crude lipid product is shown in Table 7; the EPA and DHA contents in the crude fatty acid product after molecular distillation of the lipids obtained by the enzymatic reaction under different oleyl alcohol ratios are shown in Table 8.

[0066] Table 7

[0067]

[0068]

[0069] Table 8

[0070] Oil-to-alcohol ratio 1:11.5 1:23 1:34.5 1:46 EPA Content 25.60％ 22.24％ 24.59％ 23.32％ DHA content 2.40％ 0.60％ 1.61％ 1.78％

[0071] Embodiment 5:

[0072] In a 25mL conical flask, add 3g fish oil, 5g 1,2-propylene glycol and water according to different oil-water mass ratios, then add ET 2.0, the enzyme addition amount is 6.67% (based on the mass fraction of fish oil), mix well, and react for 8h at 45℃ with magnetic stirring at 300r / min. After the reaction is completed, centrifuge at 5000r / min for 5min to take the supernatant, remove lipase and solvent, detect the lipid composition in the reaction product by liquid HPLC-RID, and calculate the free fatty acid content. Glycerides and fatty acids are separated by molecular distillation, and the crude fatty acid product is detected by gas phase GC to detect the composition and content of fatty acids.

[0073] The effect of the oil-water mass ratio in the enzymatic reaction on the free fatty acid content in the crude lipid product is shown in Table 9; under the conditions of different oil-water mass ratios, the EPA and DHA contents in the crude fatty acid product after molecular distillation of the lipids obtained by the enzymatic reaction are shown in Table 10.

[0074] Table 9

[0075] Oil-water ratio 1:1 1:2 1:3 1:5 Free fatty acid content 33.39％ 33.17％ 43.73％ 46.17％

[0076] Table 10

[0077] Oil-water ratio 1:1 1:2 1:3 1:5 EPA Content 23.86％ 22.24％ 19.53％ 21.53％ DHA content 3.61％ 0.60％ 0.91％ 1.19％

[0078] Embodiment 6:

[0079] Add 3g fish oil, 5g 1,2-propylene glycol and 6g water to a 25mL conical flask, then add ET 2.0 lipase, the enzyme addition amount is 6.67% (based on the mass fraction of fish oil), mix well, and react for 8h at 300r / min with magnetic stirring at different temperatures. After the reaction is completed, centrifuge at 5000r / min for 5min to take the supernatant, remove the lipase and solvent, detect the lipid composition in the reaction product by liquid HPLC-RID, and calculate the free fatty acid content. Glycerides and fatty acids are separated by molecular distillation, and the crude fatty acid product is detected by gas phase GC to detect the composition and content of fatty acids.

[0080] The effect of reaction temperature in the enzymatic reaction on the free fatty acid content in the crude lipid product is shown in Table 11; under different reaction temperature conditions, the EPA and DHA contents in the crude fatty acid product after molecular distillation of the lipids obtained by the enzymatic reaction are shown in Table 12.

[0081] Table 11

[0082] Temperature / ℃ 35 45 55 65 Free fatty acid content 24.72％ 33.17％ 34.06％ 38.44％

[0083] Table 12

[0084] Temperature / ℃ 35 45 55 65 EPA Content 19.12％ 22.24％ 20.07％ 15.16％ DHA content 1.01％ 0.60％ 1.93％ 1.65％

[0085] Embodiment 7:

[0086] Add 3g fish oil, 5g 1,2-propylene glycol and 6g water to a 25mL conical flask, then add ET 2.0 lipase, the enzyme addition amount is 6.67% (based on the mass fraction of fish oil), mix well, and react for different times at 45℃ with magnetic stirring at 300r / min. After the reaction is completed, centrifuge at 5000r / min for 5min to take the supernatant, remove the lipase and solvent, detect the lipid composition in the reaction product by liquid HPLC-RID, and calculate the free fatty acid content. Glycerides and fatty acids are separated by molecular distillation, and the crude fatty acid product is detected by gas phase GC to detect the composition and content of fatty acids.

[0087] The effect of reaction time in the enzymatic reaction on the free fatty acid content in the crude lipid product is shown in Table 13; the EPA and DHA contents in the crude fatty acid product after molecular distillation of lipids obtained at different enzymatic reaction times are shown in Table 14.

[0088] Table 13

[0089] Time / h 4 8 12 16 Free fatty acid content 24.72％ 33.17％ 34.06％ 38.44％

[0090] Table 14

[0091] time 4 8 12 16 EPA Content 20.81％ 22.24％ 23.15％ 23.85％ DHA content 0.61％ 0.60％ 0.72％ 1.58％

[0092] Comparative Example 1:

[0093] No alcohol was added to the enzymatic system, and other conditions were consistent with those in Example 2.

[0094] Figure 3 b is the lipid composition of the crude product of the enzymatic reaction under the conditions of Comparative Example 1; Figure 4 b is the composition and content of the crude fatty acid product after molecular distillation of the lipids obtained under the conditions of Comparative Example 1.

[0095] The free fatty acid content of the obtained crude lipid product was 52.18%, and the contents of EPA and DHA were 20.83% and 2.84%, respectively. The EPA content was only 7.33 times that of the DHA content, which was relatively low and insufficient for application in the production of final fatty acid products.

[0096] Comparative Example 2:

[0097] No water was added to this enzymatic system, and other conditions were consistent with those in Example 2.

[0098] Figure 3 c is the lipid composition of the crude product of the enzymatic reaction under the conditions of Comparative Example 2.

[0099] The free fatty acid content in the obtained crude lipid product was only 0.14%.

[0100] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing a high-content EPA, low-content DHA fatty acid product by enzymatic method, characterized in that: include, Mixing oil containing EPA and DHA, alcohol solvent and water, and performing enzymatic reaction at 35-65° C. for 4-16 h, wherein the molar ratio of oil containing EPA and DHA to alcohol solvent is 1:(11.5-46), and the oil-water ratio is 1:(1-5) in w / w terms; the alcohol solvent is 1,2-propylene glycol and / or tert-butyl alcohol; After the reaction, the lipase and the solvent are removed, and the fatty acid product with EPA>70 wt% and DHA<4 wt% is obtained by separation and purification. The lipase is one or more of ET 2.0 and Lipase TL 100L.

2. The method according to claim 1, characterized in that: The molar ratio of the oil containing EPA and DHA to the alcohol solvent is 1:(23-34.5), and the oil-water ratio is 1:(2-5) in w / w terms.

3. The method according to claim 1, characterized in that: The reaction temperature is 35-45°C.

4. The method according to claim 1, wherein: The reaction time is 8 to 16 h.

5. The method according to claim 1, characterized in that: The oils and fats containing EPA and DHA include natural and / or synthetic lipids with EPA>15 wt%.

6. The method according to claim 5, characterized in that: The preparation method of the synthetic lipid comprises: performing an enzymatic reaction catalyzed by lipase at a ratio of greater than 3:1 under the condition of 40-80° C. for 4-12 hours, and then removing the enzyme and unreacted substrate to obtain a synthetic lipid rich in EPA; Wherein, the pressure of the reaction system is below 1000 Pa.

7. The method according to claim 1, characterized in that: The method for separating the enzyme and the solvent is centrifugation, and the upper layer after centrifugation is the crude product; Wherein, the centrifugal speed is>3000 r / min; and the centrifugal time is 3~20 min.

8. The method according to claim 1, characterized in that: The further separation and purification method consists of two steps, namely molecular distillation and urea inclusion complex.

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