Glycerin rich in fish oil omega-3-PUFA and preparation method thereof

Abstract

The invention discloses glyceride rich in fish oil omega-3-PUFA and a preparation method thereof.The preparation method comprises the steps that 1, refined fish oil, ethyl alcohol and glycerin are subjected to transesterification under the catalytic action of a basic catalyst, and part of ethanol hydrolysis oil is obtained; (2) carrying out crystallization fractionation on a part of the ethanol hydrolysis oil to obtain a PUFA part glyceride enriched product; (3) preheating a PUFA part of glyceride enrichment, molecular distillation grade omega-3-PUFA ethyl ester and optional glycerol to an enzymatic transesterification reaction temperature, and carrying out vacuum homogeneous mixing; then adding lipase, and carrying out ester exchange reaction; after the reaction is finished, residual fatty acid ethyl ester is removed from an obtained reaction product through molecular distillation, and glyceride rich in fish oil omega-3-PUFA is obtained. The method can solve the technical problems of low conversion efficiency and large lipase consumption in the process of producing the oil ester rich in fish oil omega-3-PUFA by taking molecular distillation-grade fish oil omega-3-PUFA ethyl ester as a main raw material.

Description

Technical Field

[0001] The invention belongs to the technical field of deep processing of oils and fats, and more specifically, relates to a glyceride rich in fish oil ω-3-PUFA and a preparation method thereof. Background Art

[0002] Docosahexaenoic acid (hereinafter referred to as DHA) and eicosapentaenoic acid (hereinafter referred to as EPA) are ω-3 long-chain polyunsaturated fatty acids (ω-3-PUFA) with special physiological value, which have many physiological functions such as promoting the development of infant nervous system, preventing and treating cardiovascular diseases, inhibiting the formation of tumors, improving fat cell glucose and lipid metabolism, and improving body immunity. Deep-sea fish oil is one of its main sources. In industry, deep-sea fish oil is usually converted into fatty acid ethyl esters by completing ethanol hydrolysis, and then separated and purified by molecular distillation, urea inclusion, crystallization and other operations to obtain high-purity molecular distillation grade ω-3-PUFA ethyl esters. However, the market prefers ω-3-PUFA glycerides with good stability, high bioavailability and low safety risks. At present, ω-3-PUFA-rich glycerides are mainly prepared by using molecular distillation grade ω-3-PUFA ethyl esters as raw materials and ester exchange reaction with glycerol using immobilized lipase as catalyst.

[0003] Due to the large difference in polarity between molecular distillation grade ω-3-PUFA ethyl ester and glycerol, the direct reaction of the two faces severe mass transfer limitations. Therefore, it is usually necessary to increase the stirring and mixing intensity of the reaction system, increase the dosage of immobilized lipase, significantly extend the reaction time or increase the reaction temperature to promote the reaction, which causes serious loss of lipase preparations and leads to high product costs. Adding hydrocarbon organic solvents to the above molecular distillation grade ω-3-PUFA ethyl ester-glycerol hydrolysis reaction is an effective means to break through mass transfer limitations and improve reaction efficiency, but most organic solvents have safety and environmental limitations, and also add an additional burden on solvent recovery, so their industrial application is greatly limited.

[0004] It has been found that due to the good miscibility of glycerides such as triglycerides, diesters or monoglycerides with fatty acid ethyl esters, higher reaction efficiency can be obtained at a lower enzyme dosage in the enzymatic transesterification reaction, and the enzyme preparation has a longer cycle life because it is not easy to be inactivated, which is an effective way to reduce the cost of enzyme preparations. However, most of the fatty acids contained in the vast majority of commercial triglycerides, diesters or monoglycerides are not ω-3-PUFA. If such glycerides are used to synthesize glycerides with molecular distillation grade ω-3-PUFA ethyl esters, the ω-3-PUFA content in the product will inevitably be severely reduced, and ω-3-PUFA-rich glycerides cannot be obtained.

[0005] It can be seen that how to break through the mass transfer barrier of the core reaction of converting ω-3-PUFA ethyl ester to glyceride without significantly reducing the content of fish oil PUFA in glyceride products, so as to minimize the dosage and loss of immobilized lipase, is a key technical problem that needs to be solved. Summary of the invention

[0006] The purpose of the present invention is to provide a glyceride rich in fish oil ω-3-PUFA and a preparation method thereof. The preparation method of the present invention can solve the technical problems of low conversion efficiency and high lipase consumption in the process of producing glyceride rich in fish oil ω-3-PUFA using molecular distillation grade fish oil ω-3-PUFA ethyl ester as the main raw material, and can also improve the unit yield of converting ω-3-PUFA ethyl ester into ω-3-PUFA glyceride.

[0007] In order to achieve the above object, one aspect of the present invention provides a method for preparing glyceride rich in fish oil ω-3-PUFA, the preparation method comprising:

[0008] (1) subjecting refined fish oil, ethanol and glycerol to an ester exchange reaction under the catalytic action of an alkaline catalyst to obtain a partial ethanolysis oil;

[0009] (2) fractionating the partial ethanolysis oil by crystallization to obtain a PUFA partial glyceride enriched product;

[0010] (3) Preheating the PUFA partial glyceride enrichment, molecular distillation grade ω-3-PUFA ethyl ester and optional glycerol to the enzymatic ester exchange reaction temperature and performing vacuum homogenization mixing at the enzymatic ester exchange reaction temperature; then adding lipase to perform ester exchange reaction; after the reaction is completed, the reaction product is subjected to molecular distillation to remove residual fatty acid ethyl ester to obtain glyceride rich in fish oil ω-3-PUFA.

[0011] In the present invention, in step (3), the optional glycerol refers to the glycerol added after step (2), that is, the optional added glycerol.

[0012] According to the present invention, preferably, in step (1), the molar ratio of the ethanol to the triglyceride in the refined fish oil is (3-3.5):1;

[0013] The molar ratio of glycerol to ethanol is (1-3):1.

[0014] In the present invention, in the partial ethanolysis step, the molar ratio of ethanol to triglycerides in refined fish oil is 3-3.5:1, and the molar ratio of glycerol to ethanol is 1-3:1. Specifically, the technical goal of partial ethanolysis is to increase the conversion rate of triglycerides as much as possible, so that multiple fatty acids located on the same glycerol skeleton are released to ensure the purity of PUFA partial glycerides; at the same time, the proportion of partial glycerides converted into glycerol and ethyl esters should be reduced as much as possible to ensure the yield of PUFA partial glycerides. According to the reaction stoichiometric relationship, when the molar ratio of ethanol to triglycerides reaches 2:1 or more, theoretically all triglycerides can be converted into monoglycerides and fatty acid ethyl esters. But in fact, since monoglycerides are very easy to further alcoholyze into glycerol and fatty acid ethyl esters, only a very small amount of partial glycerides will be retained in the final reaction product, and a large amount of triglycerides will still not participate in the reaction. However, the inventors have found that when glycerol is added to the reaction system and the molar ratio of glycerol to ethanol is preferably 1-3:1, the reaction balance control effect can be better exerted, the ethanol conversion rate can be stabilized between 60-70%, and a higher yield of some glycerides in the product can be obtained; at the same time, due to the sufficient supply of ethanol, the conversion rate of triglycerides can still reach more than 80%.

[0015] According to the present invention, preferably, in step (1), the temperature of the transesterification reaction is 40-55° C. and the time is 10-12 hours;

[0016] The alkaline catalyst is sodium methoxide; based on the mass of the refined fish oil, the amount of the alkaline catalyst used is 0.5-0.8wt%.

[0017] In the present invention, in the partial ethanolysis step, a combination of a low reaction temperature of 40-55°C and a long reaction time of 10-12 hours is preferably used. In general glyceride alcohol reactions, a temperature close to the boiling point of alcohol (75-78°C for ethanol) is usually selected as the reaction condition to achieve a full alcoholysis effect more quickly (usually 1-2 hours). In the partial ethanolysis described in the method of the present invention, a lower reaction temperature is used to preferentially convert the liquid glyceride rich in PUFA, providing favorable conditions for the enrichment of PUFA monoglycerides in the subsequent steps. Specifically, glycerides containing more saturated fatty acids or monounsaturated fatty acids in fish oil form supramolecular aggregates due to their high freezing point or viscosity during low-temperature reactions, and the probability of contact with free ethanol molecules is reduced, resulting in a lower alcoholysis reaction rate, while glycerides containing more PUFA are easier to fully contact with ethanol-glycerol due to their good fluidity and undergo alcoholysis reactions. The difference in the reactivity of the two can accumulate more PUFA partial glycerides in the product. At the same time, due to the low reaction temperature, the reaction time needs to be increased to 10-12 hours to obtain a more satisfactory partial glyceride yield.

[0018] According to the present invention, preferably, step (2) comprises: uniformly mixing the partial ethanolysis oil and the filter aid, and then performing a first crystallization fractionation to obtain a partial ethanolysis liquid oil; finally, performing a second crystallization fractionation on the partial ethanolysis liquid oil to obtain a solid phase PUFA partial glyceride enriched product;

[0019] The first crystallization fractionation preferably comprises: crystallizing the mixture of the partial ethanolysis oil and the filter aid at -30 to -32°C to obtain a partial ethanolysis liquid oil;

[0020] The second crystallization fractionation preferably comprises: first cooling the partial ethanolysis oil to -58 to -62°C to freeze, and then heating and maintaining at -48 to -52°C until the liquid phase is drained to obtain a solid phase PUFA partial glyceride enriched product.

[0021] According to the present invention, preferably, in step (2), in step (2), the first crystallization fractionation comprises: precooling the mixture of the partial ethanolysis oil and the filter aid to -30 to -32°C at a rate of 2°C per hour, maintaining it at -30 to -32°C for more than 12 hours, and then separating by filter pressing to obtain the partial ethanolysis liquid oil;

[0022] In the second crystallization fractionation, the temperature is lowered to -58 to -62°C at a rate of 5°C per hour; and the temperature is raised to -48 to -52°C at a rate of 2°C per hour.

[0023] In the present invention, it is preferred to use two crystallization and fractionation operations to obtain a higher purity PUFA partial glyceride enrichment. The first is at -30°C, most of the non-PUFA (including all saturated fatty acids and monounsaturated fatty acids in fish oil) ethyl esters and glycerides crystallize due to their high freezing points, while PUFA ethyl esters and some glycerides remain in liquid form, and solid-liquid separation can be completed by filter pressing. In this process, the ethanol-glycerol mixed solution remaining in the aforementioned partial ethanolysis reaction also acts as a solvent, improving the crystallization heat transfer conditions and solid-liquid separation effect. The second crystallization and fractionation is at -50°C, using the characteristic that the freezing point of PUFA partial glycerides is 20-30°C higher than that of PUFA ethyl esters, so that the PUFA partial glycerides are crystallized and separated from the liquid PUFA ethyl esters, thereby further improving the enrichment effect of PUFA partial glycerides. It should be pointed out that the product obtained after the first crystallization and fractionation contains a large amount of PUFA partial glycerides, which can be directly used in the subsequent enzymatic transesterification reaction, but it still contains a large amount of non-ω-3-PUFA ethyl esters. To address this problem, the inventors took advantage of the fact that PUFA ethyl esters and PUFA partial glycerides have a relatively high freezing point difference and set up a secondary crystallization and extraction step, which further increased the content of PUFA partial glycerides, which is beneficial to improving the efficiency of subsequent enzymatic ester exchange reactions and the content of ω-3-PUFA in the glyceride product.

[0024] In the present invention, preferably, based on the solid-liquid ratio relationship unique to the partial ethanolysis product, different crystallization fractionation methods are used in the two crystallization fractionation. For example, specifically, in the first crystallization fractionation, the partial ethanolysis product is rapidly cooled to -30°C at a cooling rate of 5°C per hour and the crystals are grown for 12 hours. At this time, due to the presence of the ethanol-glycerol mixed solution, the solid phase ratio of the crystallization is only 30-40%, which is conducive to the solid-liquid separation through the filter press operation, and the obtained liquid phase is mainly PUFA ethyl ester, PUFA partial glyceride, ethanol and glycerol; in the second crystallization fractionation operation, the partial ethanolysis liquid oil is first cooled to -60°C for freezing (under this condition, most of the PUFA partial glyceride has been crystallized), and then gradually heated at a rate of 2°C per hour to sweat, and kept warm after reaching -50°C. In this process, PUFA ethyl ester, ethanol and glycerol are gradually drained, and the frozen solid phase obtained by draining is a PUFA partial glyceride enrichment.

[0025] According to the present invention, preferably, in step (3), the lipase is an immobilized lipase;

[0026] The temperature of the transesterification reaction is 55-65° C., the pressure is -0.1 MPa, and the reaction time is 3-5 hours.

[0027] According to the present invention, preferably, in step (3), when the content of diglyceride in the glyceride rich in fish oil ω-3-PUFA is greater than 40wt%:

[0028] The mass ratio of the molecular distillation grade ω-3-PUFA ethyl ester to the PUFA partial glyceride enrichment is (1.5-2):1, the dosage ratio of the lipase to the molecular distillation grade ω-3-PUFA ethyl ester is (10-20)U:1g; and the added amount of glycerol is 0.

[0029] In the present invention, high-purity ω-3-PUFA ethyl ester is used as an acyl donor, and the PUFA partial glyceride enrichment is used as an acyl acceptor. After the two are mixed homogenously under vacuum at a mass ratio of 1.5-2:1, they are contacted with immobilized lipase and undergo an ester exchange reaction. Since the acyl donor and the acceptor in the reaction system are highly compatible, the reaction equilibrium can be reached within 4 hours at a low enzyme amount of 10-20 U / g, and the product includes ω-3-PUFA diglyceride as the main component and less triglyceride.

[0030] According to the present invention, preferably, in step (3), when the content of triglycerides in the glycerides rich in fish oil ω-3-PUFA is greater than 80wt%, and the amount of glycerol added is 0:

[0031] The mass ratio of the molecular distillation grade ω-3-PUFA ethyl ester to the PUFA partial glyceride enrichment is (3-3.5):1; the dosage ratio of the lipase to the molecular distillation grade ω-3-PUFA ethyl ester is (10-30)U:1g.

[0032] According to the present invention, preferably, in step (3), when the content of triglycerides in the glycerides rich in fish oil ω-3-PUFA is greater than 80 wt %, and the amount of glycerol added is not 0:

[0033] The mass ratio of the molecular distillation grade ω-3-PUFA ethyl ester to the PUFA partial glyceride enriched product is (5-7):1; the dosage ratio of the lipase to the molecular distillation grade ω-3-PUFA ethyl ester is (20-40)U:1g; based on the mass dosage of the molecular distillation grade ω-3-PUFA ethyl ester, the added amount of the glycerol is 4-6wt%.

[0034] In the present invention, when the amount of glycerol added is 0, in the enzymatic transesterification reaction, the mass of molecular distillation grade ω-3-PUFA ethyl ester and the PUFA partial glyceride enrichment is increased to 3-3.5:1, and the amount of immobilized lipase is increased to 10-30U / g molecular distillation grade ω-3-PUFA ethyl ester, and a product whose main component is ω-3-PUFA triglyceride can be obtained due to sufficient acyl donors. It should be pointed out that the small amount of glycerol remaining in the mixed crystallization fractionation step in the PUFA partial glyceride enrichment also participates in the transesterification reaction as an acyl acceptor and further improves the conversion rate of molecular distillation grade ω-3-PUFA ethyl ester.

[0035] In the present invention, when the amount of glycerol added is not 0, in the enzymatic transesterification reaction, the mass ratio of molecular distillation grade ω-3-PUFA ethyl ester to the PUFA partial glyceride enrichment is increased to 6:1 or above, and the amount of immobilized lipase is increased to 20-40U / g molecular distillation grade ω-3-PUFA ethyl ester, and a product whose main component is ω-3-PUFA triglyceride can be obtained due to sufficient acyl donors and supplementation of glycerol. It should be pointed out that under the above reaction conditions, the mass ratio of the PUFA partial glyceride enrichment in the entire reaction system is reduced to less than 15%, and the mixed non-ω-3-PUFA has little effect on the fatty acid composition of the final triglyceride product, so the DHA and EPA content in the obtained triglyceride type ω-3-PUFA product is very close to that of molecular distillation grade PUFA ethyl ester. When the DHA and EPA content in the molecular distillation grade ω-3-PUFA ethyl ester raw material used is as high as 60% or above, and glycerides with a considerable content of ω-3-PUFA are prepared, this method is more suitable.

[0036] In the present invention, the method of the present invention includes three key steps: first, the triglycerides in the refined fish oil are converted into partial glycerides (including monoglycerides and diglycerides) through a partial ethanolysis reaction; then, non-PUFA ethyl esters, monoglycerides and diglycerides with higher freezing points are removed through crystallization fractionation to obtain a PUFA partial glyceride enriched product; finally, the PUFA partial glyceride enriched product is used as an acyl acceptor to undergo an ester exchange reaction with high-purity ω-3-PUFA ethyl ester (molecular distillation grade ω-3-PUFA ethyl ester) under the catalysis of lipase to obtain an ω-3-PUFA glyceride product. Different from the current mainstream technical scheme that uses glycerol as the acyl acceptor, the method of the present invention uses PUFA partial glyceride as the acyl acceptor, which has excellent compatibility with ω-3-PUFA ethyl ester, can greatly improve the reaction efficiency and reduce lipase consumption; in addition, the PUFA partial glyceride obtained by crystallization and fractionation itself has a high ω-3-PUFA content, and therefore will not significantly reduce the ω-3-PUFA content in the glyceride product; furthermore, the PUFA partial glyceride used in the method of the present invention is directly extracted from secondary raw materials such as refined fish oil, which is not only low-cost, but also can improve the unit yield of glyceride products synthesized from high-purity ω-3-PUFA ethyl ester.

[0037] The method of the present invention adds glycerol as a reaction balance controller in the partial ethanolysis reaction to increase the yield of some glycerides. Specifically, the alcoholysis of triglycerides with ethanol involves three consecutive reactions: (1) the first step is the alcoholysis of triglycerides to form diglycerides, which has the lowest reaction constant; (2) the second step is the alcoholysis of diglycerides to form monoglycerides, which has the second highest reaction constant; (3) the third step is the alcoholysis of monoglycerides to form glycerol, which has the highest reaction constant. Therefore, in a general glyceride alcoholysis reaction system, monoglycerides and diglycerides are easily converted into glycerol and fatty acid ethyl esters, and it is difficult to achieve a high yield of monoglycerides and diglycerides, which deviates from the technical goal of the method of the present invention. However, it has been found that replacing pure ethanol with an ethanol-glycerol mixture in the partial ethanolysis reaction can simultaneously achieve a high conversion rate of triglycerides and a high retention rate of monoglycerides and diglycerides, thereby obtaining a higher yield of some glycerides.

[0038] The method of the present invention uses crystallization fractionation to separate and purify PUFA partial glycerides from partial ethanol hydrolysis products. Since the freezing points of PUFA ethyl esters, monoglycerides and diglycerides are much lower than the freezing points of saturated fatty acids and monounsaturated fatty acid ethyl esters and partial glycerides, it is theoretically possible to separate the two by crystallization fractionation. However, in actual operation, the ethyl esters or partial glycerides of saturated and monounsaturated fatty acids in fish oil tend to form a dense network structure during crystallization, which can wrap the liquid PUFA ethyl esters and partial glycerides therein, making it difficult to completely separate the solid and liquid phases. In the method described in the present invention, the presence of unreacted ethanol-glycerol mixed liquid in the partial ethanol hydrolysis products plays a role in reducing the viscosity of the system, optimizing the heat transfer efficiency and avoiding the formation of a solid ester network during the crystallization process, which can greatly improve the efficiency of the crystallization fractionation operation, and effectively improve the purity and yield of the PUFA partial glyceride enrichment.

[0039] The method of the present invention uses the PUFA partial glyceride enrichment as an acyl acceptor and molecular distillation grade ω-3-PUFA ethyl ester to undergo an ester exchange reaction under the catalysis of lipase to generate an ω-3-PUFA glyceride product. One of the products of the ester exchange reaction is ethanol, which can be continuously vaporized and removed during the reaction. Since the PUFA partial glyceride enrichment and the molecular distillation grade ω-3-PUFA ethyl ester have excellent compatibility, efficient conversion can be achieved without strong mixing during the reaction and without the need for a large amount of immobilized enzyme input. In addition, the reaction system also avoids the competition of high-concentration glycerol for water on the surface of the lipase, and can reduce the risk of lipase inactivation from both physical and chemical factors, which is conducive to the improvement of the enzyme cycle life.

[0040] Before the enzymatic ester exchange, the PUFA partial glyceride enrichment and the high-purity ω-3-PUFA ethyl ester are preheated to the reaction temperature and subjected to vacuum homogenization treatment to form a very uniform mixed solution and remove residual ethanol, which is beneficial to further improve the reaction efficiency; after the enzymatic ester exchange reaction, the obtained product is subjected to molecular distillation to recover the high-purity ω-3-PUFA ethyl ester that has not participated in the reaction and is recycled again, while obtaining pure ω-3-PUFA glyceride.

[0041] Another aspect of the present invention provides glyceride rich in fish oil ω-3-PUFA prepared by the above preparation method.

[0042] The technical solution of the present invention has the following beneficial effects:

[0043] The preparation method of the present invention can solve the technical problems of low conversion efficiency and high lipase consumption in the process of producing glycerides rich in fish oil ω-3-PUFA using molecular distillation grade fish oil ω-3-PUFA ethyl ester as the main raw material, and can also improve the unit yield of converting ω-3-PUFA ethyl ester into ω-3-PUFA glycerides.

[0044] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0046] The present invention is further described below by examples:

[0047] Example 1

[0048] Refined sardine oil (EPA content 11wt%, DHA content 13.8wt%), 17.5wt% of ethanol by mass of refined sardine oil (equivalent to a molar ratio of 3.5:1 between ethanol and triglycerides in refined sardine oil), 70wt% of glycerol by mass of refined sardine oil (equivalent to a molar ratio of 2:1 between glycerol and ethanol) and 0.5wt% of sodium methoxide by mass of refined sardine oil are mixed, the temperature is raised to 40°C and the mixture is reacted with sealing stirring for 12 hours to obtain a partial ethanolysis oil; the contents of monoglyceride and diglyceride in the obtained partial ethanolysis oil are 12wt% and 23wt%, respectively.

[0049] 1 wt% diatomaceous earth (based on the weight of the partial ethanolysis oil) was mixed into the partial ethanolysis oil and stirred evenly, precooled to -30°C at a rate of 2°C per hour and crystallized at -30°C for 24 hours, then filtered to remove part of the ethanolysis solid esters, and at the same time, part of the ethanolysis liquid oil was collected. The obtained partial ethanolysis liquid oil was cooled to -60°C at a rate of 5°C per hour and frozen, then slowly heated to -50°C at a rate of 2°C per hour and kept for 6 hours until the liquid phase was drained, and the solid phase PUFA partial glyceride enriched product was collected. The PUFA partial glyceride enrichment and 2030 type ethyl ester (molecular distillation product, EPA content 20wt%, DHA content 30wt%) (PUFA partial glyceride enrichment and 2030 type ethyl ester mass ratio of 1:2) were preheated to 60°C, and after vacuum homogenization at 60°C, immobilized lipase was added at a ratio of 10U lipase per gram of 2030 type ethyl ester, and the reaction was shaken at 60°C and -0.1MPa for 4 hours, and the conversion rate of 2030 type ethyl ester reached 62%. After filtering and recovering the immobilized lipase, the fatty acid ethyl ester was recovered by molecular distillation, and the triglyceride content of the obtained product (glyceride rich in fish oil ω-3-PUFA) was 36wt%, the diglyceride content was 62wt%, and the rest was residual monoglyceride; the EPA content in the fatty acid was 20wt%, and the DHA content was 28wt%.

[0050] Comparative Example 1

[0051] Refined sardine oil (EPA content 11wt%, DHA content 13.8wt%), 17.5wt% of ethanol (equivalent to an alcohol-oil molar ratio of 3.5:1) in an amount of 17.5wt% of the mass of refined sardine oil, and 0.5wt% of sodium methoxide in an amount of 0.5wt% of the mass of refined sardine oil were mixed, and the mixture was heated to 40°C and reacted with sealing stirring for 12 hours to obtain a partial ethanolysis oil; the contents of monoglyceride and diglyceride in the obtained partial ethanolysis oil were 0.5wt% and 1.6wt%, respectively.

[0052] Comparative Example 2

[0053] Refined sardine oil (EPA content 11wt%, DHA content 13.8wt%), 10wt% of ethanol (equivalent to an alcohol-oil molar ratio of 2:1) in an amount of refined sardine oil, and 0.5wt% of sodium methoxide in an amount of refined sardine oil were mixed, heated to 40°C and reacted with sealing stirring for 12 hours to obtain a partial ethanolysis oil; the contents of monoglyceride and diglyceride in the obtained partial ethanolysis oil were 7wt% and 15wt%, respectively.

[0054] 1 wt % diatomaceous earth (based on the weight of the partial ethanolysis oil) was mixed into the partial ethanolysis oil and stirred evenly, precooled to -30°C at a rate of 2°C per hour and grown at -30°C for 24 hours to form a plastic continuous solid phase, making it difficult to separate the partial ethanolysis oil.

[0055] By comparing the above embodiments and comparative examples, it can be seen that: as can be seen from Example 1, adding a sufficient amount of glycerol in the fish oil ethanolysis reaction can play a role in controlling the reaction balance, so that a large amount of monoglycerides and diglycerides are accumulated in the reaction product; and in the subsequent crystallization and fractionation operation, the solid phase ratio is reduced to less than 40% due to the presence of a sufficient amount of glycerol-ethanol solution in the system, so that part of the ethanolysis solid esters and liquid oil are completely separated, providing good conditions for the subsequent enzymatic transesterification reaction. As can be seen from Comparative Example 1, if sufficient glycerol is not added, the ethanolysis reaction will tend to be complete, and sufficient partial glycerides cannot be obtained, and sufficient acyl acceptors cannot be provided for the subsequent enzymatic transesterification. As can be seen from Comparative Example 2, it is also difficult to obtain sufficient partial glyceride yields by reducing the amount of ethanol, because a large amount of triglycerides do not participate in the reaction; and in the crystallization and fractionation link, a plastic continuous solid phase is formed due to the excessively high solid ester content, which wraps up part of the ethanolysis liquid oil and is difficult to separate.

[0056] Example 2

[0057] Refined anchovy oil (EPA content 17wt%, DHA content 8.5wt%, saturated and monoenoic acid 70wt%), 15wt% of ethanol (equivalent to a molar ratio of ethanol to triglycerides in refined anchovy oil of 3:1) in an amount of 15wt% of refined anchovy oil, 90wt% of glycerol (equivalent to a molar ratio of glycerol to ethanol of 3:1) in an amount of 90wt% of refined anchovy oil and 0.8wt% of sodium methoxide in an amount of 0.8wt% of refined anchovy oil were mixed, heated to 55°C and reacted in a sealed container with stirring for 10 hours to obtain a partial ethanolysis oil; the contents of monoglyceride and diglyceride in the obtained partial ethanolysis oil were 10wt% and 22wt%, respectively.

[0058] 2 wt% diatomaceous earth (based on the weight of the partial ethanolysis oil) was mixed into the partial ethanolysis oil and stirred evenly, precooled to -30°C at a rate of 2°C per hour and crystallized at -30°C for 24 hours, then filtered to remove part of the ethanolysis solid esters, and part of the ethanolysis liquid oil was collected. The obtained partial ethanolysis liquid oil was cooled to -60°C at a rate of 5°C per hour and frozen, then slowly heated to -50°C at a rate of 2°C per hour and kept for 6 hours until the liquid phase was drained, and the solid phase PUFA partial glyceride enriched product was collected. The PUFA partial glyceride enrichment and 4010 type ethyl ester (molecular distillation product, EPA content 42wt%, DHA content 11wt%) (PUFA partial glyceride enrichment and 4010 type ethyl ester mass ratio of 1:3.5) were preheated to 60°C, and after vacuum homogenization at 60°C, immobilized lipase was added at a ratio of 30U lipase per gram of 4010 type ethyl ester, and the reaction was shaken at 60°C and -0.1MPa for 4 hours, and the conversion rate of 4010 type ethyl ester reached 46%. After filtering and recovering the immobilized lipase, the fatty acid ethyl ester was recovered by molecular distillation, and the triglyceride content of the obtained product (glyceride rich in fish oil ω-3-PUFA) was 89wt%, the diglyceride content was 6wt%, and the rest was residual monoglyceride; the EPA content in the fatty acid was 41wt%, and the DHA content was 10wt%.

[0059] Comparative Example 3

[0060] The same raw materials and conditions as in Example 2 were used to prepare some ethanolysis oil.

[0061] 2 wt% diatomaceous earth (based on the weight of the partial ethanolysis oil) was mixed into the partial ethanolysis oil and stirred evenly, precooled to -30°C at a rate of 2°C per hour and crystallized at -30°C for 24 hours, then filtered to remove part of the ethanolysis solid ester, and collect part of the ethanolysis liquid oil. The obtained partial ethanolysis liquid oil and 4010 type ethyl ester (molecular distillation product, EPA content 42wt%, DHA content 11wt%) (the mass ratio of partial ethanolysis liquid oil to 4010 type ethyl ester is 1:3.5) were preheated to 60°C, and after vacuum homogenization at 60°C, immobilized lipase was added at a ratio of 30U lipase per gram of 4010 type ethyl ester, and the reaction was shaken at 60°C and -0.1MPa for 4 hours, and the conversion rate of 4010 type ethyl ester reached 25%. After filtering and recovering the immobilized lipase, the triglyceride content in the obtained product was 13wt%, and the diglyceride content was 19wt%. After molecular distillation, the EPA content in the obtained glyceride is 38wt%, and the DHA content is 9wt%.

[0062] From the comparison between the above examples and comparative examples, it can be seen that: from Example 2, it can be seen that the first step of crystallization and fractionation operation described in the present invention can remove most of the saturated and monounsaturated fatty acid esters in the refined fish oil, so that the ω-3-PUFA content in the final glyceride product is basically equivalent to 4010 ethyl ester; while the second step of crystallization and fractionation operation removes most of the PUFA ethyl ester and glycerol, so that some glycerides are enriched, and the efficiency of the enzymatic transesterification reaction is guaranteed. Comparative Example 3 only uses one round of crystallization and fractionation, and the ω-3-PUFA content in the final glyceride product decreases slightly, while the conversion rate and triglyceride yield both decrease significantly.

[0063] Example 3

[0064] Refined herring oil (EPA content 7.5wt%, DHA content 6.8wt%), 15wt% of ethanol by mass of refined herring oil (equivalent to a molar ratio of 3:1 between ethanol and triglycerides in refined herring oil), 30wt% of glycerol by mass of refined herring oil (equivalent to a molar ratio of 1:1 between glycerol and ethanol) and 0.6wt% of sodium methoxide by mass of refined herring oil were mixed, heated to 50°C and reacted with sealing stirring for 12 hours to obtain a partial ethanolysis oil; the contents of monoglyceride and diglyceride in the obtained partial ethanolysis oil were 16wt% and 35wt%, respectively.

[0065] 1.5 wt% diatomaceous earth (based on the weight of the partial ethanolysis oil) was mixed into the partial ethanolysis oil and stirred evenly, precooled to -30°C at a rate of 2°C per hour and crystallized at -30°C for 24 hours, then filtered to remove part of the ethanolysis solid esters, and part of the ethanolysis liquid oil was collected. The obtained partial ethanolysis liquid oil was cooled to -60°C at a rate of 5°C per hour and frozen, then slowly heated to -50°C at a rate of 2°C per hour and kept for 8 hours until the liquid phase was drained, and the solid phase PUFA partial glyceride enriched product was collected. The PUFA partial glyceride enrichment, 3020 type ethyl ester (molecular distillation product, EPA content 33wt%, DHA content 21wt%) (PUFA partial glyceride enrichment and 3020 type ethyl ester mass ratio of 1:6) and glycerol (PUFA partial glyceride enrichment and glycerol mass ratio of 1:0.3) were preheated to 60°C, and after vacuum homogenization at 60°C, immobilized lipase was added at a ratio of 40U lipase per gram of 3020 type ethyl ester, and the reaction was shaken at 60°C and -0.1MPa for 4 hours, and the conversion rate of 3020 type ethyl ester reached 53%. After filtering and recovering the immobilized lipase, the fatty acid ethyl ester was recovered by molecular distillation, and the triglyceride content of the obtained product (glyceride rich in fish oil ω-3-PUFA) was 97wt%, the diglyceride content was 2wt%, and the rest was residual monoglyceride; the EPA content in the fatty acid was 30wt%, and the DHA content was 18wt%. The recovered immobilized lipase was recycled for subsequent batches of reactions under the same conditions. After 6 rounds of reaction, the enzyme activity decreased by 21%. Statistics on product unit consumption showed that 0.72 kg of 3020 ethyl ester was consumed to prepare 1 kg of ω-3-PUFA-rich glyceride product.

[0066] Comparative Example 4

[0067] After glycerol and 3020 ethyl ester (molecular distillation product, EPA content 33wt%, DHA content 21wt%) (mass ratio of glycerol to 3020 ethyl ester is 1:6) were vacuum homogenized at 60°C, and immobilized lipase was added at a ratio of 40U lipase per gram of 3020 ethyl ester, and the reaction was shaken at 60°C and -0.1MPa for 4 hours, and the ethyl ester conversion rate was 12%. After filtering and recovering the immobilized lipase, the fatty acid ethyl ester was recovered by molecular distillation, and the triglyceride content of the obtained product was 37wt%, the diglyceride content was 49wt%, and the monoglyceride content was 13wt%. The EPA and DHA contents in the fatty acids were 33wt% and 21wt%, respectively. The recovered immobilized lipase was circulated for subsequent batches to react under the same conditions, and the enzyme activity decreased by 69% after 6 rounds of reaction.

[0068] Comparative Example 5

[0069] After glycerol and 3020 ethyl ester (molecular distillation product, EPA content 33wt%, DHA content 21wt%) (mass ratio of glycerol to 3020 ethyl ester is 1:6) were vacuum homogenized at 60°C, and immobilized lipase was added at a ratio of 200U lipase per gram of 3020 ethyl ester, and the reaction was shaken at 60°C and -0.1MPa for 4 hours, and the ethyl ester conversion rate was 45%. After filtering and recovering the immobilized lipase, the fatty acid ethyl ester was recovered by molecular distillation, and the triglyceride content of the obtained product was 92wt%, the diglyceride content was 6wt%, and the rest was residual monoglyceride. The EPA and DHA contents in the fatty acids were 33wt% and 21wt%, respectively.

[0070] Comparative Example 6

[0071] The product obtained in comparative example 4, 3020 ethyl ester (molecular distillation product, EPA content 33wt%, DHA content 21wt%) (the mass ratio of the product obtained in comparative example 4 to 3020 ethyl ester is 1:6) and glycerol (the mass ratio of the product obtained in comparative example 4 to the comparison is 1:0.3) were vacuum homogenized at 60°C, and immobilized lipase was added at a ratio of 40U lipase per gram of 3020 ethyl ester, and the reaction was shaken at 60°C and -0.1MPa for 4 hours, and the ethyl ester conversion rate was 42%. After filtering and recovering the immobilized lipase, the fatty acid ethyl ester was recovered by molecular distillation, and the triglyceride content in the obtained product was 92wt%, and the diglyceride content was 5wt%. The EPA and DHA contents in the fatty acids were 33wt% and 21wt%, respectively. Statistics on the unit consumption of the product show that 0.93 kg of 3020 ethyl ester is consumed to prepare 1 kg of ω-3-PUFA-rich glyceride product.

[0072] From the comparison between the above embodiments and comparative examples, it can be seen that: from comparative example 4, when glycerol is used as the acyl acceptor in the enzymatic transesterification reaction, both the reaction efficiency and the enzyme cycle life are relatively low; and from example 3, it can be seen that after adding the PUFA partial glyceride enrichment to glycerol, the reaction efficiency and the enzyme cycle life are greatly increased, which can greatly reduce the consumption of lipase. In comparative example 5, glycerol is still used as the acyl acceptor, and the amount of immobilized lipase is greatly increased, so that a reaction conversion rate close to that of example 3 can be obtained, but the cost of the enzyme preparation is significantly increased. In comparative example 6, the product obtained in comparative example 4 is put into the enzymatic transesterification reaction of glycerol and 3020 type ethyl ester, which also improves the reaction efficiency. The reason is that it also contains a large amount of partial glyceride, which plays a role in breaking through the mass transfer barrier and improving the reaction efficiency. It can be seen that the use of comparative example 4, comparative example 5 alone or the combination of comparative examples 4 and 6 can synthesize glyceride products rich in ω-3-PUFA, but whether it is the reaction time, enzyme preparation consumption, or the unit consumption of 3020 type ethyl ester is much higher than that of example 3.

[0073] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for preparing glycerides rich in fish oil ω-3-PUFA, characterized in that: The preparation method comprises: (1) subjecting refined fish oil, ethanol and glycerol to an ester exchange reaction under the catalytic action of an alkaline catalyst to obtain a partial ethanolysis oil; (2) fractionating the partial ethanolysis oil by crystallization to obtain a PUFA partial glyceride enriched product; (3) Preheating the PUFA partial glyceride enrichment, molecular distillation grade ω-3-PUFA ethyl ester and optional glycerol to the enzymatic ester exchange reaction temperature and performing vacuum homogenization mixing at the enzymatic ester exchange reaction temperature; then adding lipase to perform ester exchange reaction; after the reaction is completed, the reaction product is subjected to molecular distillation to remove residual fatty acid ethyl ester to obtain glyceride rich in fish oil ω-3-PUFA.

2. The preparation method according to claim 1, wherein In step (1), the molar ratio of the ethanol to the triglyceride in the refined fish oil is (3-3.5):1; The molar ratio of glycerol to ethanol is (1-3):

1.

3. The preparation method according to claim 1, wherein In step (1), the temperature of the transesterification reaction is 40-55° C. and the time is 10-12 hours; The alkaline catalyst is sodium methoxide; based on the mass of the refined fish oil, the amount of the alkaline catalyst used is 0.5-0.8wt%.

4. The preparation method according to claim 1, wherein Step (2) comprises: uniformly mixing the partial ethanolysis oil and the filter aid, and then performing a first crystallization fractionation to obtain a partial ethanolysis liquid oil; and finally, performing a second crystallization fractionation on the partial ethanolysis liquid oil to obtain a solid phase PUFA partial glyceride enriched product; The first crystallization fractionation preferably comprises: crystallizing the mixture of the partial ethanolysis oil and the filter aid at -30 to -32°C to obtain a partial ethanolysis liquid oil; The second crystallization fractionation preferably comprises: first cooling the partial ethanolysis oil to -58 to -62°C to freeze, and then heating and maintaining at -48 to -52°C until the liquid phase is drained to obtain a solid phase PUFA partial glyceride enriched product.

5. The preparation method according to claim 4, wherein In step (2), the first crystallization fractionation comprises: precooling the mixture of the partial ethanolysis oil and the filter aid to -30 to -32°C at a rate of 2°C per hour, maintaining the mixture at -30 to -32°C for more than 12 hours, and then separating the partial ethanolysis liquid oil by filter pressing; In the second crystallization fractionation, the temperature is lowered to -58 to -62°C at a rate of 5°C per hour; and the temperature is raised to -48 to -52°C at a rate of 2°C per hour.

6. The preparation method according to claim 1, wherein In step (3), the lipase is an immobilized lipase; The temperature of the transesterification reaction is 55-65° C., the pressure is -0.1 MPa, and the reaction time is 3-5 hours.

7. The preparation method according to claim 1, wherein In step (3), when the content of diglyceride in the glyceride rich in fish oil ω-3-PUFA is greater than 40wt%: The mass ratio of the molecular distillation grade ω-3-PUFA ethyl ester to the PUFA partial glyceride enrichment is (1.5-2):1, the dosage ratio of the lipase and the molecular distillation grade ω-3-PUFA ethyl ester is (10-20)U:1g; and the added amount of glycerol is 0.

8. The preparation method according to claim 1, wherein In step (3), when the content of triglyceride in the glyceride rich in fish oil ω-3-PUFA is greater than 80wt%, and the amount of glycerol added is 0: The mass ratio of the molecular distillation grade ω-3-PUFA ethyl ester to the PUFA partial glyceride enrichment is (3-3.5):1; the dosage ratio of the lipase to the molecular distillation grade ω-3-PUFA ethyl ester is (10-30)U:1g.

9. The preparation method according to claim 1, wherein In step (3), when the content of triglyceride in the glyceride rich in fish oil ω-3-PUFA is greater than 80wt%, and the amount of glycerol added is not 0: The mass ratio of the molecular distillation grade ω-3-PUFA ethyl ester to the PUFA partial glyceride enriched product is (5-7):1; the dosage ratio of the lipase to the molecular distillation grade ω-3-PUFA ethyl ester is (20-40)U:1g; based on the mass dosage of the molecular distillation grade ω-3-PUFA ethyl ester, the added amount of the glycerol is 4-6wt%.

10. Glyceride rich in fish oil ω-3-PUFA prepared by the preparation method according to any one of claims 1 to 9.