Method for producing high EPA ethyl ester and high DHA glyceride by two-step enzymatic method

Abstract

This invention discloses a two-step enzymatic method for producing high-EPA ethyl esters and high-DHA glycerides. First, fish oil is catalyzed by ethanol hydrolysis using the fatty acid-selective lipase Cal-A to obtain a mixture of non-EPA and DHA ethyl esters and EPA and DHA-rich glycerides. Next, the EPA and DHA-rich glycerides are separated by extraction using a hexane / ethanol biphase system. Finally, the EPA and DHA-rich glycerides are further catalyzed by the non-position-specific lipase WKQ to convert EPA in the glycerides into ethyl esters, while retaining DHA in the form of glycerides. This two-step enzymatic ethanol hydrolysis of fish oil allows for the large-scale production of 70-80% EPA ethyl ester products (DHA content less than 5%) and 60-80% DHA glycerides (EPA content less than 5%). Compared to a one-step enzymatic catalysis method, the two-step enzymatic method employed in this invention can effectively enrich and separate EPA and DHA, improving the application value of EPA and DHA in fish oil.

Description

Technical Field

[0001] The invention belongs to the field of oil modification, and in particular relates to a method for producing high-EPA ethyl ester and high-DHA glyceride by a two-step enzymatic method. Background Art

[0002] A large number of clinical experiments have shown that n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are a class of active substances that are beneficial to human health. Studies have shown that EPA helps keep blood vessels open, prevents blood clots, prevents stroke or myocardial infarction, removes fat accumulated in the blood, prevents arteriosclerosis and peripheral vascular obstruction (Zhang HJ, Gao X, Guo XF, et al. Effects of dietary eicosapentaenoic acid and docosahexaenoic acid supplementation on metabolic syndrome: A systematic review and meta-analysis of data from 33 randomized controlled trials[J]. Clinical Nutrition, 2021, 40(7): 4538-4550.); DHA is an indispensable material basis for the formation, development and operation of brain cells. It can promote and coordinate the conduction of neural circuits to maintain the normal operation of brain cells and prevent Alzheimer's disease (Jin J, Jin QZ, Wang XG, et al. High Sn-2docosahexaenoic acid lipids for brain benefits, and their enzymatic syntheses: a review[J]. Engineering, 2020, 6(4): 424-431.). However, humans can only synthesize a limited amount of EPA and DHA (the conversion rate is about 5%). Therefore, nutritional supplements rich in EPA and DHA are favored by consumers.

[0003] At present, EPA and / or DHA glycerides or ethyl esters prepared from fish oil are an important source of n-3 polyunsaturated fatty acids for humans. Rubio-Rodríguez et al. proposed that EPA or DHA in the form of ethyl ester or glyceride is more easily utilized by the human body and exhibits better medical effects (Rubio-Rodríguez N, Beltrán S, Jaime I, et al. Production of omega-3 polyunsaturated fatty acid concentrates: A review[J]. Innovative Food Science & Emerging Technologies, 2010, 11(1): 1-12.). However, ethyl ester or glyceride nutritional supplements developed using fish oil contain both EPA and DHA, two n-3 polyunsaturated fatty acids, at relatively high concentrations. For example, the DHA and EPA contents of BLACKMORES brand fish oil omega3 soft capsules are 12% and 18% respectively; the EPA and DHA ratio of NYO3 brand highly concentrated deep-sea fish oil is 4:3. A large number of studies have shown that in the treatment of specific diseases, the effect of EPA or DHA alone is stronger than the combined use of EPA and DHA. Eating high-concentration EPA and DHA products at the same time will cause the body to interfere with the absorption and metabolism of EPA and DHA, affecting the implementation of the biological functions of EPA and DHA (Deckelbaum RJ and Calder P C. Is it time to separate EPA from DHA when using omega-3 fatty acids to protect heart and brain [J]. Current Opinion in Clinical Nutrition and Metabolic Care. 2022, 23 (2): 65-67.). Based on this, scientific and technological workers have been committed to enriching and separating EPA and DHA in fish oil to obtain two biological products rich in EPA and DHA.

[0004] At present, scientific and technological workers mainly use physical or chemical methods to separate EPA and DHA. Xia Yaogan and others studied the separation of EPA and DHA using supercritical fluid chromatography technology (Xia Yaogan, Wang Yu, Zou Wenyan, et al. Study on the separation of fish oil fatty acids EPA and DHA [J]. Journal of Huaiyin Institute of Technology, 2020, 29(05): 49-52.). Dong Qian studied the enrichment and separation methods of EE-type EPA and DHA. First, the concentration of EE-type DHA and EPA was enriched to 80.49% by urea embedding method, and then the concentration of EPA and DHA was further enriched to 99% by fixed bed chromatography. Finally, EPA and DHA were separated by simulated moving bed chromatography. The concentrations of EPA-EE and DHA-EE can reach 91.6% and 93.6%, respectively (Dong Qing. Study on the separation process of EPA-EE and DHA-EE [D]. Zhejiang University, 2018.). However, the above methods are not conducive to the application of industrial production. Compared with chemical catalysis, the enzyme catalysis method enriches and / or separates EPA and DHA. Its reaction conditions are mild, the enzyme shows substrate selectivity, the degree of oxidation of EPA and DHA is low, and it can be easily scaled up for industrial-scale production. Compared with other enzyme catalysis methods (such as hydrolysis reaction and glycerolysis reaction), the ethanol hydrolysis of fish oil catalyzed by lipase has attracted widespread attention. The reasons are as follows: 1) Ethanol is a green solvent, cheap and renewable; 2) Ethanol can be used as both a substrate and a medium, and the separation and purification method is simple; 3) The products are mainly ethyl esters and glycerides. EPA / DHA is easily absorbed and utilized by the human body in the form of ethyl esters or glycerides (He Y, Wang X, Zhang Y, et al. Enzymatic ethanolysis subjected to SchizochYrium biomass: Sequential processing for DHA enrichment and biodiesel production[J]. Energy Conversion and Management, 2019, 184(MAR.): 159-171.). He et al. found that lipase Cal-A has fatty acid selectivity or weak sn-2 position selectivity (He Y, Li J, Kodali S, et al. The near-idealcatalyic property of Candida antarctica Lipase A to highly concentrate n-3polyunsaturated fatty acids in monoacylglycerols via one-step ethanolysis oftriacylglycerols[J]. Bioresource Technology, 2016:466-478.), while lipases CAL-B, NS, TL, and ET have sn-1,3 position specific selectivity (He Y, Li J, Kodali S, et al. Liquid lipases forenzymatic concentration of n-3 polyunsaturated fatty acids inmonoacylglycerols via ethanolysis: Catalyic specificity and parameterization[J]. Bioresource Technology, 2016, 224: 445-456.). Among the above lipases, CAL-A ethanol alcoholysis catalyzed fish oil and showed the strongest EPA and DHA enrichment ability, that is, the EPA and DHA contents could reach 50.86% and 26.3%, respectively. On the other hand, the experimental results show that these lipases only have the ability to enrich EPA and DHA, but have not yet shown the characteristics of separating EPA and DHA. In the research work of Taiwo et al., it was found that the distribution characteristics of EPA and DHA in fish oil in the glycerol backbone are as follows: DHA is mainly distributed in the sn-2 position of the glycerol backbone, while EPA is evenly distributed in the three sites of the glycerol backbone (Taiwo OA, Jacqui LA, Colin J B. Selective concentration of EPA and DHA using Thermomyces lanuginosus lipase is due to fatty acid selectivity and notregioselectivity[J]. Food Chemistry, 2013,138(1): 615-620.). Therefore, the results of these reported research works can be inferred that if only sn-1,3 position specific lipase is selected to enzymatically catalyze the ethanolysis of fish oil in one step, the sn-2 position saturated fatty acids, monounsaturated fatty acids and EPA cannot be effectively removed. Although the selection of lipase with fatty acid selectivity can effectively increase the n-3 polyunsaturated fatty acids in the product, it cannot effectively separate EPA and DHA. Therefore, it is urgent to find a large-scale production method for efficiently enriching and separating EPA and DHA from fish oil, that is, to develop a method for using lipase to catalyze the alcoholysis of fish oil to first efficiently enrich EPA and DHA in fish oil, and then separate EPA and DHA.

[0005] Chinese patent CN112592939B discloses a method for enriching n-3 polyunsaturated fatty acids by enzyme method. The invention uses fish oil as raw material and uses lipases including those derived from Candida cylindracea One or more lipases and lipases Candida antarctica Lipase A reacts in two ways: alcoholysis and hydrolysis. First, take oil, aqueous solution and Candida cylindracea The lipase is reacted in a reactor for a certain period of time to obtain a mixture. After removing the free fatty acids in the mixture, an oil rich in n-3 polyunsaturated fatty acids can be obtained. Then the obtained oil, alcohol aqueous solution and lipase are mixed Candida antarctica Lipase A reacts in a reactor for a certain period of time to obtain a mixture, which is purified to obtain oils rich in n-3 polyunsaturated fatty acids. Candida cylindracea Lipase and lipase Candida antarctica Lipase A effectively increases the content of n-3 polyunsaturated fatty acids in fish oil. However, this method uses an enzyme-catalyzed hydrolysis reaction, and the byproducts produced during the reaction are free fatty acids, which are complicated to separate and purify. At the same time, the lipases selected in this invention are all fatty acid selective enzymes, which cannot effectively separate EPA and DHA, limiting the concentration of EPA and DHA in oils and fats.

[0006] Chinese patent CN111647633B discloses a method for enriching EPA and DHA in deep-sea fish oil. The invention dissolves sn-1,3 position selective lipase in Tris-HCl buffer, then adds acyl migration promoter silica particles, and prepares a dispersion containing lipase and silica by magnetic stirring. Deep-sea fish oil is added to the prepared dispersion system, nitrogen is filled and sealed during the reaction, and magnetic stirring is continuously performed. After the reaction is completed, potassium hydroxide solution and n-hexane are added to the reaction system, which is fully shaken and centrifuged, the upper solution is transferred and collected, and the n-hexane is removed by nitrogen blowing. The remaining liquid after removing the n-hexane is a lipid rich in EPA and DHA. The invention uses sn-1,3 position selective lipase, which cannot effectively remove saturated and monounsaturated fatty acids at the sn-2 position. In addition, this method has complex process and cumbersome procedures, which is not conducive to large-scale industrial production.

[0007] Chinese patent CN112521270B discloses a method for separating DHA and EPA by high vacuum continuous distillation. The invention preheats the ethyl ester fish oil and adds it to a degassing tower to remove low-boiling substances. The product after removing the low-boiling substances then enters the biodiesel separation tower, and biodiesel is separated from the top of the tower. The remaining material then enters the DHA and EPA separation tower, EPA is separated from the top of the tower, and the remaining material passes through a two-stage scraper evaporator in turn to obtain a DHA component and a reboiler component, respectively. This invention can achieve a better separation of EPA and DHA, but the separation temperature of this method is around 180-220°C, which can easily lead to oxidation of DHA and EPA; and this method requires the fish oil to be prepared into an ethyl ester type first, and the reaction cost is relatively high.

[0008] Based on the above conclusions, it is particularly important to develop a large-scale production method that is green, energy-saving, simple to operate, low-cost, and can simultaneously enrich and separate EPA and DHA. Summary of the invention

[0009] The present invention provides a method for large-scale production of high-EPA ethyl ester and high-DHA glyceride by two-step enzymatic ethanolysis of fish oil. The method can obtain high-EPA ethyl ester products and high-DHA glyceride products by large-scale co-production through the two-step enzymatic catalysis method.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] A method for large-scale production of high-EPA ethyl ester and high-DHA glyceride of fish oil by two-step enzymatic ethanolysis catalysis comprises the following steps:

[0012] 1) The fish oil is catalyzed by ethanol hydrolysis of the fatty acid selective lipase Cal-A to obtain a mixture of non-EPA and DHA ethyl esters and EPA and DHA rich glycerides

[0013] Take fish oil, add ethanol, Cal-A enzyme and water to carry out enzyme ethanolysis catalytic reaction, in which the mass ratio of ethanol to fish oil in the reaction system is 3:1-6:1, the enzyme loading is 5-20%, the water content of the reaction system is 5-10%, the temperature is 25-35°C, the reaction time is 48-60h, and the reaction obtains a mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA;

[0014] 2) Extraction and separation of non-EPA and DHA ethyl esters and EPA and DHA-rich glycerides using n-hexane / ethanol two-phase system

[0015] The mixture of ethyl ester and glyceride obtained in step 1) is added into a n-hexane / ethanol system for extraction, and the extraction reaction conditions are: the mass ratio of n-hexane to ethanol is 0.5:1-1:1, the ethanol concentration is 50-70%, and the number of extractions is 2-5 times;

[0016] After extraction, the non-EPA and DHA ethyl esters are distributed in the upper n-hexane phase, and the EPA and DHA-rich glycerides are distributed in the lower ethanol phase. The ethanol phase is evaporated to remove the organic solvent to obtain EPA and DHA-rich glycerides. After EPA and DHA-rich glycerides are methylated, the fatty acid composition and content are analyzed by gas chromatography. The EPA and DHA contents in the EPA and DHA-rich glycerides are 40-50% and 20-30%, respectively.

[0017] 3) Lipase WKQ further catalyzes the conversion of EPA- and DHA-rich glycerides into ethyl esters, while DHA is retained in the form of glycerides, as follows:

[0018] Take EPA- and DHA-rich glycerides, add lipase WKQ, ethanol and water, and carry out a second ethanol alcoholysis reaction. The reaction conditions are: the mass ratio of ethanol to EPA- and DHA-rich glycerides is 0.5:1-2:1, the enzyme loading is 10-30%, the water content of the system is 5-15%, the temperature is 25-35°C, and the reaction time is 24-48h;

[0019] The mixture obtained after the reaction is extracted and separated into high-EPA ethyl ester and high-DHA glyceride through a n-hexane / ethanol two-phase system, the upper n-hexane phase is evaporated to remove the organic solvent to obtain a high-EPA ethyl ester product, and the fatty acid composition and content are analyzed by gas chromatography. The EPA content of the high-EPA ethyl ester product can reach 70-80%, and the DHA content is less than 5%; the lower ethanol phase is evaporated to remove the organic solvent to obtain a high-DHA glyceride product, the high-DHA glyceride product is methylated, and the fatty acid composition and content are analyzed by gas chromatography. The DHA content of the high-DHA glyceride product is 60-80%, and the EPA content is less than 5%.

[0020] The present invention adopts the above technical scheme, firstly, ethanol hydrolysis of fish oil is catalyzed by lipase Cal-A to obtain a mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA, then the ethyl esters other than EPA and DHA and the glycerides rich in EPA and DHA are extracted and separated by a n-hexane / ethanol two-phase system, and finally the glycerides rich in EPA and DHA are further ethanolyzed and catalyzed by a non-positionally specific lipase WKQ to convert EPA in the glycerides into ethyl esters, and the DHA is retained in the form of glycerides.

[0021] The present invention has the following advantages: after the fish oil is catalyzed by two-step enzymatic alcoholysis, EPA ethyl ester with a content of 70-80% (DHA content is less than 5%) and DHA glyceride with a content of 60-80% (EPA content is less than 5%) are obtained. Compared with the one-step enzymatic method, the two-step enzymatic method adopted by the present invention can effectively enrich and separate EPA and DHA in fish oil, thereby improving the application value of EPA and DHA in fish oil. DETAILED DESCRIPTION

[0022] The lipase Cal-A (Novozymes, Denmark) and lipase WKQ (Vekic Biotech, China) described in the present invention are commercially available products.

[0023] Example 1

[0024] A method for large-scale production of high-EPA ethyl ester and high-DHA glyceride from fish oil using a two-step enzymatic process comprises the following steps:

[0025] 1) The fish oil is catalyzed by Cal-A enzyme ethanol hydrolysis to obtain a mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA.

[0026] Take 1 ton of fish oil (EPA 20%, DHA 8%), add ethanol, Cal-A enzyme and water for ethanol hydrolysis reaction, place in a 10 t stirred reactor, the alcohol-oil mass ratio of the reaction system is 4.5:1, the enzyme loading is 15%, the water content of the reaction system is 7.5%, the temperature is 30°C, the reaction time is 55h, and the reaction obtains a mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA;

[0027] 2) Extraction and separation of non-EPA and DHA ethyl esters and EPA and DHA-rich glycerol using n-hexane / ethanol two-phase system.

[0028] The mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA obtained in step 1) is added into a n-hexane / ethanol system for extraction, and the extraction reaction conditions are: a mass ratio of n-hexane to ethanol of 0.75:1, an ethanol concentration of 60%, and extraction times of 3 times;

[0029] After extraction, the ethyl esters other than EPA and DHA are distributed in the upper n-hexane phase, and the glycerides rich in EPA and DHA are distributed in the lower ethanol phase. The ethanol phase is evaporated to remove the organic solvent to obtain glycerides rich in EPA and DHA. After methyl esterification of the glycerides rich in EPA and DHA, the fatty acid composition and content are analyzed by gas chromatography. The EPA and DHA contents of the glycerides are 50% and 30%, respectively.

[0030] After steps 1) and 2), 300 kg of glycerides rich in EPA and DHA can be obtained;

[0031] 3) Catalyze glycerides rich in EPA and DHA, convert EPA in glycerides into ethyl esters, and retain DHA in the form of glycerides, as follows:

[0032] Take the EPA- and DHA-rich glyceride obtained in step 2), add lipase WKQ, ethanol and water to carry out a second ethanolysis reaction, and the reaction conditions are as follows: the mass ratio of ethanol to EPA- and DHA-rich glyceride is 2:1, the lipase WKQ enzyme loading is 30%, the water content of the system is 10%, the temperature is 35°C, and the reaction time is 48h;

[0033] The mixture obtained after the reaction was extracted and separated into high EPA ethyl ester and high DHA glyceride through a n-hexane / ethanol two-phase system. After the upper n-hexane phase was evaporated to remove the organic solvent, 120 kg of high EPA ethyl ester type product was obtained. The fatty acid composition and content were analyzed by gas chromatography. The EPA content of the high EPA ethyl ester type product was 80%, and the DHA content was 3%. After the lower ethanol phase was evaporated to remove the organic solvent, 180 kg of high DHA glyceride type product was obtained. After the high DHA glyceride was methylated, the fatty acid composition and content were analyzed by gas chromatography. The DHA content of the high DHA glyceride type product was 80%, and the EPA content was 4%.

[0034] Example 2

[0035] A method for large-scale production of high-EPA ethyl ester and high-DHA glyceride of fish oil by two-step enzymatic ethanolysis catalysis comprises the following steps:

[0036] 1) The fish oil is catalyzed by Cal-A enzyme ethanol hydrolysis to obtain a mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA.

[0037] Take 1 ton of fish oil (EPA 20%, DHA 8%), add ethanol, Cal-A enzyme and water for ethanol hydrolysis reaction, place in a 10 ton stirred reactor, the alcohol-oil mass ratio of the reaction system is 3:1, the enzyme loading is 5%, the water content of the reaction system is 5%, the temperature is 25°C, the reaction time is 48 hours, and the reaction obtains a mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA;

[0038] 2) Extraction and separation of non-EPA and DHA ethyl esters and EPA and DHA-rich glycerides using n-hexane / ethanol two-phase system

[0039] The mixture of EPA- and DHA-free ethyl esters and EPA- and DHA-rich glycerides obtained in step 1) is added to a n-hexane / ethanol system for extraction. The extraction reaction conditions are: a mass ratio of n-hexane to ethanol of 0.5:1, an ethanol concentration of 50%, and two extractions.

[0040] After extraction, the non-EPA and DHA ethyl esters are distributed in the upper n-hexane phase, and the EPA and DHA-rich glycerides are distributed in the lower ethanol phase. The ethanol phase is evaporated to remove the organic solvent to obtain EPA and DHA-rich glycerides. After methyl esterification of the EPA and DHA-rich glycerides, the fatty acid composition and content are analyzed by gas chromatography. The EPA and DHA contents of the glycerides are 40% and 20%, respectively.

[0041] After steps 1) and 2), 280 kg of glycerides rich in EPA and DHA can be obtained;

[0042] 3) Catalyze glycerides rich in EPA and DHA, convert EPA in glycerides into ethyl esters, and retain DHA in the form of glycerides, as follows:

[0043] Take the EPA- and DHA-rich glyceride obtained in step 2), add lipase WKQ, ethanol and water to carry out a second ethanol hydrolysis reaction, and the enzymatic hydrolysis reaction conditions are as follows: the mass ratio of ethanol to EPA- and DHA-rich glyceride is 0.5:1, the lipase WKQ enzyme loading is 20%, the water content of the system is 15%, the temperature is 25°C, and the reaction time is 24h;

[0044] The mixture obtained after the reaction was extracted and separated into high-EPA ethyl ester and high-DHA glyceride through a n-hexane / ethanol two-phase system. After the upper n-hexane phase was evaporated to remove the organic solvent, 105 kg of high-EPA ethyl ester type product was obtained. The fatty acid composition and content were analyzed by gas chromatography. The EPA content of the high-EPA ethyl ester type product was 70%, and the DHA content was 4%. After the lower ethanol phase was evaporated to remove the organic solvent, 175 kg of high-DHA glyceride type product was obtained. After the high-DHA glyceride was methylated, the fatty acid composition and content were analyzed by gas chromatography. The DHA content of the high-DHA glyceride type product was 60%, and the EPA content was 2%.

[0045] Example 3

[0046] The method for large-scale production of high-EPA ethyl ester and high-DHA glyceride of fish oil by two-step enzymatic ethanolysis comprises the following steps:

[0047] 1) The fish oil is catalyzed by Cal-A enzyme ethanol hydrolysis to obtain a mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA.

[0048] Take 0.8t of fish oil (EPA20%, DHA8%), add ethanol, Cal-A enzyme and water for ethanol hydrolysis reaction, place in a 10t stirred reactor, the alcohol-oil mass ratio of the reaction system is 6:1, the enzyme loading is 20%, the water content of the reaction system is 10%, the temperature is 35°C, the reaction time is 48h, and the reaction obtains a mixture of ethyl esters other than EPA and DHA and glycerides rich in EPA and DHA;

[0049] 2) Extraction and separation of non-EPA and DHA ethyl esters and EPA and DHA-rich glycerides using n-hexane / ethanol two-phase system

[0050] The non-EPA and DHA ethyl esters and the mixture of EPA and DHA-rich glycerides obtained in step 1) are added into a n-hexane / ethanol system for extraction. The extraction reaction conditions are: a mass ratio of n-hexane to ethanol of 1:1, an ethanol concentration of 70%, and 5 extraction times;

[0051] After extraction, ethyl esters other than EPA and DHA are distributed in the upper n-hexane phase, and glycerides rich in EPA and DHA are distributed in the lower ethanol phase. The ethanol phase is evaporated to remove the organic solvent, thereby obtaining glycerides rich in EPA and DHA. After methyl esterification of the glycerides rich in EPA and DHA, the fatty acid composition and content are analyzed by gas chromatography, and the EPA and DHA contents of the glycerides are 45% and 25%, respectively. After steps 1) and 2), 202 kg of glycerides rich in EPA and DHA can be finally obtained.

[0052] 3) Catalyze glycerides rich in EPA and DHA, convert EPA in glycerides into ethyl esters, and retain DHA in the form of glycerides, as follows:

[0053] Take the EPA- and DHA-rich glyceride obtained in step 2), add lipase WKQ, ethanol and water to carry out a second ethanolysis reaction, and the reaction conditions are: the mass ratio of ethanol to EPA- and DHA-rich glyceride is 2:1, the lipase WKQ enzyme loading is 10%, the water content of the system is 5%, the temperature is 30°C, and the reaction time is 36h;

[0054] The mixture obtained after the reaction was extracted and separated into high-EPA ethyl ester and high-DHA glyceride through a n-hexane / ethanol two-phase system. After the upper n-hexane phase was evaporated to remove the organic solvent, 93 kg of high-EPA ethyl ester type product was obtained. The fatty acid composition and content were analyzed by gas chromatography. The EPA content of the high-EPA ethyl ester type product was 75%, and the DHA content was 2%. After the lower ethanol phase was evaporated to remove the organic solvent, 109 kg of high-DHA glyceride type product was obtained. After the high-DHA glyceride was methylated, the fatty acid composition and content were analyzed by gas chromatography. The DHA content of the high-DHA glyceride product was 70%, and the EPA content was 4%.

[0055] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for large-scale production of high-EPA ethyl ester and high-DHA glyceride of fish oil by two-step enzymatic ethanolysis, characterized in that: The following steps are involved: 1) Fish oil is catalyzed by ethanol hydrolysis with fatty acid-selective lipase to obtain a mixture of ethyl esters and glycerides Take fish oil, add ethanol, Cal-A enzyme and water to carry out ethanol alcoholysis reaction of the enzyme to obtain a mixture of ethyl ester and glyceride; wherein the mass ratio of ethanol to fish oil is 3:1-6:1, the enzyme loading is 5-20%, the water content of the reaction system is 5-10%, the temperature is 25-35°C, and the reaction time is 48-60h; 2) Extraction and separation of ethyl ester and glyceride by n-hexane / ethanol two-phase system The mixture of ethyl ester and glyceride obtained in step 1) is added to a n-hexane / ethanol system for extraction, wherein the ethyl esters other than EPA and DHA are distributed in the upper n-hexane phase, and the glycerides rich in EPA and DHA are distributed in the lower ethanol phase. The ethanol phase is removed to remove the organic solvent, thereby obtaining glycerides rich in EPA and DHA. 3) Lipase WKQ ethanol alcoholysis catalyzes EPA- and DHA-rich glycerides to produce high-EPA ethyl esters and high-DHA glycerides Taking glycerides rich in EPA and DHA, adding lipase WKQ, ethanol and water, and carrying out a second ethanolysis reaction, extracting the mixture obtained after the reaction through a n-hexane / ethanol two-phase system to separate ethyl esters and glycerides, taking the upper n-hexane phase and evaporating to remove the organic solvent to obtain high-EPA ethyl esters; taking the lower ethanol phase and evaporating to remove the organic solvent to obtain high-DHA glycerides; The lipase WKQ was purchased from Vicogen Biotechnology Company.

2. The method of claim 1, wherein the method comprises: The extraction reaction conditions of step 2) are: the mass ratio of n-hexane to ethanol is 0.5:1-1:1, the concentration of ethanol is 50-70%, and the number of extractions is 2-5 times.

3. The method of claim 1, wherein the method comprises: In step 3), the reaction conditions for the second alcoholysis using lipase WKQ are as follows: the mass ratio of ethanol to EPA- and DHA-rich glycerides is 0.5:1-2:1, the enzyme loading is 10-30%, the water content of the system is 5-15%, the temperature is 25-35°C, and the reaction time is 24-48h.