A method for preparing n-3 polyunsaturated fatty acid glycerides by enzymatic catalysis

Abstract

The present invention discloses an enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides. Liquid CAL-A enzyme is used as a biocatalyst to enrich n-3 PUFA in glycerides. Results show that during the enzymatic alcoholysis reaction, without the addition of new enzyme, the liquid CAL-A enzyme can only be reused 3 to 4 times, and its enzyme activity drops to less than 80% of the initial enzyme activity. However, by supplementing a certain amount of fresh CAL-A liquid enzyme, the number of reuses of the CAL-A enzyme can be greatly increased, ranging from 2 to 3 times. Each increase in the number of reuses of the lipase significantly reduces the cost of enzymatic preparation of n-3 PUFA glycerides. Simultaneously, the present invention effectively reduces wastewater discharge and solvent usage. The method of the present invention for enriching n-3 PUFA glycerides has good effect, low cost, and is suitable for commercial production.

Description

Technical Field

[0001] The invention belongs to the field of deep processing of oils and fats, and particularly relates to an enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides. Background Art

[0002] n-3 polyunsaturated fatty acids (n-3 PUFAs) possess a variety of physiological activities and functions, such as anti-inflammatory, lipid-lowering, immune-enhancing, and memory-enhancing properties. The physiological activity of n-3 PUFAs depends on their purity and form. Currently, commercially available n-3 PUFA products primarily exist in the form of ethyl esters, free fatty acids, and glycerides. Ethyl ester n-3 PUFAs possess excellent oxidative stability, but their bioavailability is low, primarily due to the inability of pancreatic lipase in the human body to effectively hydrolyze the ester bond of ethyl esters. Currently, commercially available n-3 PUFA products primarily exist in the form of ethyl esters, as existing separation and purification methods can yield high-purity ethyl ester n-3 PUFAs. Similarly, existing separation techniques such as urea encapsulation, crystallization, or molecular distillation can also yield high-purity free fatty acid n-3 PUFAs. However, free n-3 PUFAs are highly susceptible to oxidation, resulting in only a small fraction of free fatty acid n-3 PUFAs being commercially available. Compared with the other two forms, glyceride-type n-3 PUFA has better oxidative stability and higher bioavailability, and is the naturally occurring form of n-3 PUFA. However, the disadvantage is that the content of n-3 PUFA in natural fish oil or algae oil is low, and the low n-3 PUFA content also affects its bioavailability.

[0003] Therefore, more and more scholars have begun to study the preparation and enrichment of glyceride-type n-3PUFA. Generally speaking, traditional separation and purification methods cannot obtain high-concentration glyceride-type n-3PUFA. Enzymatic enrichment is a common method for preparing n-3PUFA glycerides. Lipase can selectively hydrolyze or alcoholyze saturated fatty acids and monounsaturated fatty acids in fish oil or algae oil, while n-3PUFA such as DHA and EPA are difficult to hydrolyze or alcoholyze, and can be enriched in glycerides.

[0004] At present, the main problem with the enzymatic preparation and enrichment of n-3 PUFA is the high cost of the enzyme. Enzyme immobilization can increase the number of times lipase can be reused and reduce the cost of lipase use. However, the enzyme immobilization process is cumbersome, the immobilized carrier is easy to fall off, and it is also relatively expensive. In addition, the immobilization process is easy to change the conformation of the lipase and may affect the specificity of the lipase. These shortcomings also limit the application of immobilized lipase in industry.

[0005] Therefore, there is an urgent need in the art to develop a method for increasing the number of times liquid lipase is reused to solve the problems of low number of times liquid lipase is reused and low catalytic activity of the recovered lipase. Summary of the Invention

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not 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 object of the present invention is to overcome the deficiencies in the prior art and provide an enzymatic preparation method for n-3 polyunsaturated fatty acid glycerides.

[0009] In order to solve the above technical problems, the present invention provides the following technical solution: an enzymatic preparation method of n-3 polyunsaturated fatty acid glyceride, comprising:

[0010] n-3 PUFA-rich oil and an alcohol aqueous solution are mixed in a reactor, liquid lipase is added, and after alcoholysis reaction, the alcohol-water phase containing the liquid lipase is recovered by layering to obtain a recovered aqueous phase containing the liquid lipase. The oil phase is collected and free fatty acids and fatty acid esters in the crude product are removed to obtain a glyceride product rich in n-3 PUFA.

[0011] Re-taking the n-3 PUFA-rich oil and fat, adding it to the recovered aqueous phase containing the liquid lipase, performing an alcoholysis reaction again, recovering the alcohol-water phase containing the liquid lipase by layering, collecting the oil phase and removing free fatty acids and fatty acid esters in the crude product to obtain a glyceride product rich in n-3 PUFA;

[0012] The aqueous phase containing liquid lipase is repeatedly recovered and added to n-3 PUFA-rich oil for alcoholysis reaction to produce n-3 polyunsaturated fatty acid glycerides. If the enzyme activity drops below 80% of the initial enzyme activity, fresh liquid lipase and alcohol aqueous solution are added.

[0013] As a preferred embodiment of the enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides of the present invention, the liquid lipase is derived from Candida antarctica lipase A CAL-A.

[0014] As a preferred embodiment of the enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides of the present invention, the oil rich in n-3 PUFA is algae oil or fish oil.

[0015] As a preferred embodiment of the enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides of the present invention, the fish oil includes 1812 fish oil, Omax fish oil and tuna oil.

[0016] As a preferred embodiment of the enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides of the present invention, the alcohol is one or both of methanol and ethanol.

[0017] As a preferred embodiment of the enzymatic preparation method of n-3 polyunsaturated fatty acid glyceride of the present invention, the alcoholysis reaction is carried out at a temperature of 25 to 45° C. and a reaction time of 10 to 24 hours.

[0018] As a preferred embodiment of the enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides described in the present invention, the method comprises: mixing n-3 PUFA-rich oil and an alcohol aqueous solution in a reactor, adding liquid lipase, wherein the amount of lipase added is more than 300 LU per gram of oil, the mass fraction of alcohol in the alcohol aqueous solution is 30% to 60%, and the molar ratio of alcohol to oil is 5:1 to 70:1.

[0019] As a preferred embodiment of the enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides of the present invention, the molar ratio of the alcohol to the oil is 10:1 to 70:1.

[0020] As a preferred embodiment of the enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides according to the present invention, if the enzyme activity drops below 80% of the initial enzyme activity, fresh liquid lipase and an aqueous solution of alcohol are supplemented, wherein the amount of fresh liquid lipase supplemented is 80LU to 200LU of fresh liquid lipase per gram of oil, and the amount of fresh alcohol solvent supplemented is 0.8 to 10 mol of alcohol per mole of oil.

[0021] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an enzymatic preparation method for n-3 polyunsaturated fatty acid glycerides for use in the field of oil and fat processing.

[0022] Beneficial effects of the present invention:

[0023] (1) The present invention provides an enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides, wherein fresh liquid lipase CAL-A is added to the first alcoholysis reaction, and the liquid lipase, water and alcohol used in the next enzymatic alcoholysis reaction are the aqueous phase collected from the previous reaction (or a small amount of fresh lipase and alcohol are newly added). This lipase addition strategy not only ensures that the recycled lipase has a good enrichment effect on n-3 PUFA in glycerides, but also increases the number of reuses of the CAL-A enzyme, reduces the amount of lipase used and the cost of preparing n-3 PUFA glycerides, and is economical and efficient.

[0024] (2) The water used in the multiple enzymatic alcoholysis reactions of the present invention mostly comes from the recovered aqueous phase in the previous enzymatic alcoholysis, rather than discarding the water separated after the previous alcoholysis, thereby reducing the generation of wastewater. At the same time, the alcohol also comes from the alcohol recovered in the previous reaction, which also greatly reduces the consumption of solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0026] Figure 1 This is a gas chromatogram of the glyceride fatty acid composition of 1812 fish oil before alcoholysis in Example 1 of the present invention;

[0027] Figure 2 This is a gas chromatogram of the glyceride fatty acid composition of 1812 fish oil after enzymatic alcoholysis in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] 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 the embodiments of the specification.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0031] (1) Determination of n-3 PUFA content and yield

[0032] Refer to the method in Yang Zhuangzhuang's master's thesis (Yang Zhuangzhuang. Research on two-step enzymatic hydrolysis of fish oil to enrich n-3 polyunsaturated fatty acid glycerides. Master's thesis of Jiangnan University, 2021).

[0033] (2) Molecular distillation conditions

[0034] The oil phase after alcoholysis consists of fatty acid methyl esters and free fatty acids, which need to be separated from the glycerides. The molecular distillation separation conditions are as follows: system pressure below 1-2 Pa, feed temperature 35°C, evaporation temperature 160°C, condensation temperature 25°C, scraper speed 150 r / min, and feed rate 1.5 mL / min. Molecular distillation produces a heavy phase primarily composed of glycerides (including monoglycerides, diglycerides, and triglycerides) and a light phase primarily composed of fatty acid methyl esters and free fatty acids.

[0035] The liquid lipase Candida antarctica lipase A (specific enzyme activity of 6.0 LU / mg) in the present invention is from Darwin Enzyme Company, trade name AD L.

[0036] The oils used in the present invention are all commercially available, and the algae oil rich in n-3 PUFA is fermented from Schizochytrium algae. The DHA content in the oil is 43.26%, and the total n-3 PUFA content is 44.81%.

[0037] The total n-3 PUFA content of 1812 fish oil is 38.13% (EPA content 18% to 20%, DHA content 12% to 14%), and the total n-3 PUFA content of tuna oil is 34.30%. Other reagents are commercially available unless otherwise specified.

[0038] Example 1

[0039] (1) 3.0 g of 1812 fish oil (n-3 PUFA content 38.13%) and 1200 U of fresh liquid CAL-A lipase (enzyme added at 400 LU / g oil) were added to an enzyme reactor. A 50% methanol aqueous solution was then added at a methanol:oil molar ratio of 65:1. The reaction was carried out at 30°C for 13 h. During the reaction, the stirring speed was set at 300 rpm, and a small amount of evaporated methanol was refluxed into the reaction system using a condenser. After the reaction was completed, the oil phase and the aqueous phase containing the CAL-A enzyme were centrifuged. After removing free acids and methyl esters from the oil phase, n-3 PUFA-rich glycerides were obtained.

[0040] (2) 3.0 g of 1812 fish oil was weighed again, and the recovered aqueous phase (containing water, methanol, CAL-A liquid enzyme, etc.) was added to the mixture. A second enzymatic alcoholysis reaction was performed under the same reaction conditions as in (1). After the reaction was completed, the aqueous phase containing lipase from the previous enzymatic alcoholysis was recovered again and used as a catalyst and reaction system to perform alcoholysis reactions under the same conditions to study the number of times the lipase could be reused.

[0041] When the activity of the recovered liquid lipase fell below 80% of the initial activity, 360 LU of fresh CAL-A liquid enzyme (enzyme addition rate: 120 LU / g oil) was added as a catalyst, and a methanol-water solution was added to the reactor at a methanol-to-oil molar ratio of 1:1, with a methanol-water solution having a methanol mass fraction of 80%. The reaction was continued under the conditions of (1) for a period of time (i.e., 30°C, 13 h, 300 rpm). The results are shown in the following table.

[0042] Table 1 Reuse times of lipase

[0043]

[0044] As shown in the table above, the n-3 PUFA content in natural fish oil is approximately 38.13%. During the first reaction, 400 LU / g of liquid lipase CAL-A was added. After a period of enzymatic hydrolysis, the total n-3 PUFA content in the glycerides increased from 38.13% to 69.22%, of which EPA accounted for approximately 39% and DHA for approximately 26%. Liquid CAL-A slightly enriched EPA more than DHA.

[0045] After three repetitions of the reaction, the CAL-A enzyme activity remained above 80%. However, by the fourth repetition, the activity had dropped below 80%. Generally speaking, when the activity of a lipase drops below 80% of its initial activity, it is unsuitable for reuse and must be discarded. Therefore, the CAL-A liquid enzyme was reused three times in this alcoholysis reaction system.

[0046] After three repetitions, we added 120 LU / g of fresh enzyme solution and an 80% methanol-water solution to the recovered aqueous phase containing lipase, alcohol, and water for a fourth re-use. As shown in the table, adding fresh enzyme and methanol-water to the recovered enzyme solution after three reactions, using this catalyst and reaction system, increased the n-3 PUFA content in fish oil from an initial 38.13% to 68.39%. When the recovered lipase solution was used as a catalyst for n-3 PUFA enrichment in the fifth and sixth re-uses, over 80% of the initial enzyme activity was retained.

[0047] Therefore, this lipase addition strategy can increase the number of reuses of liquid CAL-A enzyme from 3 times to 6 times, while the enzyme addition amount only increases by 120LU / g oil. At the same time, the methanol aqueous solution recovered after the previous reaction is reused in the n-3PUFA enrichment process, reducing wastewater discharge and solvent consumption. Therefore, this n-3PUFA glyceride enrichment method is economical and efficient.

[0048] Example 2

[0049] (1) 3.0 g of Schizochytrium truncatum oil (n-3 PUFA content 44.81%) and 1800 U of fresh liquid CAL-A lipase (enzyme added at 600 LU / g oil) were added to an enzyme reactor. A 35% methanol aqueous solution was then added at a methanol:oil molar ratio of 20:1. The reaction was carried out at 35°C for 18 h. During the reaction, the stirring speed was set at 300 rpm, and a small amount of volatilized methanol was recovered by a condenser and returned to the reaction system. After the reaction, the oil phase and the aqueous phase containing the CAL-A enzyme were centrifuged. After removing free acids and methyl esters from the oil phase, n-3 PUFA-rich glycerides were obtained.

[0050] (2) 3.0 g of algae oil was weighed again, and the recovered aqueous phase (containing water, methanol, CAL-A liquid enzyme, etc.) was added to the mixture. A second enzymatic alcoholysis reaction was performed under the same reaction conditions as in (1). After the reaction was completed, the aqueous phase containing lipase from the previous enzymatic alcoholysis was recovered and used as a catalyst and reaction system to conduct a reaction under the same conditions to study the number of times the lipase could be reused.

[0051] When the activity of the liquid lipase in the recovered aqueous phase fell below 80% of the initial activity, 540 U of fresh CAL-A liquid enzyme (enzyme addition amount was 180 LU / g oil) was added to the aqueous phase, and a methanol-water solution was added to the reactor at a methanol-to-oil molar ratio of 1:5, with a methanol mass fraction of 75%. The reaction was continued under the conditions of (1) for a period of time (i.e., 35°C, 18 h, 300 rpm). The results are shown in the following table.

[0052] Table 2 Number of times lipase is reused

[0053]

[0054] As shown in Table 2, liquid CAL-A enzyme can be reused four times without adding new enzyme, retaining 80% of its initial activity. After the fifth reaction, its activity drops below 80%. Therefore, after four enzymatic reactions, adding 180 LU / g of fresh lipase and a 75% by mass methanol aqueous solution to the aqueous phase containing free enzyme can allow for a further four reuses, for a total of eight. This significantly increases the number of lipase reuses, reduces enzyme usage costs, and reduces wastewater discharge and waste solvent recovery costs.

[0055] Example 3

[0056] (1) 3.0 g of tuna oil (n-3 PUFA content 34.3%) and 1800 U of fresh liquid CAL-A lipase (enzyme addition amount is 600 LU / g oil) were added to an enzyme reactor. Subsequently, a 45% methanol aqueous solution was added at a methanol:oil molar ratio of 80:1. The reaction was carried out at 40°C for 20 h. During the reaction, the stirring speed was set at 300 rpm, and a small amount of volatilized methanol was recovered by a condenser and returned to the reaction system. After the reaction was completed, the oil phase and the aqueous phase containing CAL-A enzyme were centrifuged and free acids and methyl esters were removed from the oil phase to obtain n-3 PUFA-rich glycerides.

[0057] (2) 3.0 g of tuna oil was weighed again, and the recovered aqueous phase (containing water, methanol, CAL-A liquid enzyme, etc.) was added to the mixture. A second enzymatic alcoholysis reaction was carried out under the same reaction conditions as in (1). After the reaction was completed, the aqueous phase containing lipase from the previous enzymatic alcoholysis was recovered and reacted under the same reaction conditions to study the number of times the lipase could be reused.

[0058] When the activity of the liquid lipase in the recovered aqueous phase fell below 80% of the initial activity, 450 U of fresh CAL-A liquid enzyme (enzyme addition amount was 150 LU / g oil) was added to the aqueous phase, and a methanol-water solution was added to the reactor at a methanol-to-oil molar ratio of 1:2, with a methanol mass fraction of 85%. The reaction was continued under the conditions of (1) for a period of time (i.e., 40°C, 20 h, 300 rpm). The results are shown in the following table.

[0059] Table 3 Reuse times of lipase

[0060]

[0061] As shown in Table 3, the liquid CAL-A enzyme can only be reused three times without adding new enzyme, and its enzyme activity can retain 80% of the initial enzyme activity. After the fourth reaction, its enzyme activity drops below 80%.

[0062] Therefore, after three enzymatic reactions, adding 150 LU / g of fresh lipase and an 85% methanol-water solution to the aqueous phase containing the free enzyme allowed the reaction to be reused two more times, for a total of five times. This significantly increases the number of lipase reuses, reduces enzyme costs, reduces wastewater discharge, and reduces waste solvent recovery costs. Furthermore, we found that different n-3 PUFA oils and fats used as substrates resulted in different enrichment effects. Tuna oil, when used as a substrate, enriched n-3 PUFA glycerides less efficiently than algae oil and 1812 fish oil.

[0063] Comparative Example 1

[0064] The reaction conditions and process were similar to those of Example 1, except that 15% or 80% aqueous methanol was added in the initial reaction instead of 50% aqueous methanol. Four alcoholysis reactions were performed. No fresh liquid CAL-A enzyme or aqueous alcohol solution was added in the first four alcoholysis reactions. The results are shown in the following table.

[0065] Table 4 Reuse times of lipase

[0066]

[0067] As shown in Table 4, when the concentration of added methanol is not appropriate, the content of n-3 PUFA in the enriched glycerides decreases. At the same time, in the experimental group in which 80% aqueous alcohol solution was added to the initial reaction system, the number of reuses of liquid CAL-A enzyme decreased from 3 to 2.

[0068] Comparative Example 2

[0069] The reaction conditions and process were similar to those of Example 1, except that the liquid CAL-A enzyme was reused three times, and only 120 LU / g oil of fresh lipase was added to the reactor. No 80% methanol aqueous solution with a 1:1 methanol to oil molar ratio was added to the reactor. The results are shown in the table below.

[0070] Table 5 Reuse times of lipase

[0071]

[0072] As shown in Table 5, when only 120 LU / g of CAL-A enzyme was newly added to the aqueous phase solution after the lipase was reused three times without supplementing with 80% methanol aqueous solution, the n-3 PUFA content in the glyceride was lower than that in Example 1. Moreover, the number of reuses of the aqueous phase with the newly added enzyme was reduced from 3 to 2, i.e., the lipase activity after the fourth and fifth reactions retained more than 80%. After the sixth reaction, the n-3 PUFA content in the glyceride was less than 55.38% (i.e., 69.22% × 80%).

[0073] The present invention uses liquid CAL-A enzyme as a biocatalyst to enrich n-3 PUFA in glycerides. Results show that during the enzymatic alcoholysis reaction, without fresh enzyme replenishment, the liquid CAL-A enzyme can only be reused 3 to 4 times, with its enzyme activity dropping to less than 80% of its initial activity. However, replenishing a certain amount of fresh CAL-A liquid enzyme can significantly increase the number of reuses of the CAL-A enzyme by 2 to 3 times. Each increase in the number of lipase reuses significantly reduces the cost of enzymatic preparation of n-3 PUFA glycerides. The present invention also effectively reduces wastewater discharge and solvent usage. The present method for enriching n-3 PUFA glycerides is effective, low-cost, and suitable for commercial production.

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. 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 may 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 claims of the present invention.

Claims

1. An enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides, characterized in that: include, An n-3 PUFA-rich oil and an alcohol aqueous solution are mixed in a reactor, liquid lipase is added, and after alcoholysis reaction, the alcohol-water phase containing the liquid lipase is recovered by layering to obtain a recovered aqueous phase containing the liquid lipase. The oil phase is collected and free fatty acids and fatty acid esters in the crude product are removed to obtain an n-3 PUFA-rich glyceride product, wherein the amount of lipase added is 300 LU or more per gram of oil, the mass fraction of alcohol in the alcohol aqueous solution is 30% to 60%, and the molar ratio of alcohol to oil is 5:1 to 70:1; Re-taking the n-3 PUFA-rich oil and fat, adding it to the recovered aqueous phase containing the liquid lipase, performing an alcoholysis reaction again, recovering the alcohol-water phase containing the liquid lipase by layering, collecting the oil phase and removing free fatty acids and fatty acid esters in the crude product to obtain a glyceride product rich in n-3 PUFA; The aqueous phase containing liquid lipase is repeatedly recovered and added to n-3 PUFA-rich oil for alcoholysis reaction to produce n-3 polyunsaturated fatty acid glycerides. If the enzyme activity drops below 80% of the initial enzyme activity, fresh liquid lipase and alcohol aqueous solution are supplemented. The amount of fresh liquid lipase supplemented is 80LU to 200LU of fresh liquid lipase per gram of oil, and the amount of fresh alcohol solvent supplemented is 0.8 to 10 mol of alcohol per mole of oil.

2. The enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides according to claim 1, characterized in that: The liquid lipase is derived from Candida antarcticalipase A CAL-A from Candida antarctica.

3. The enzymatic preparation method of n-3 polyunsaturated fatty acid glyceride according to claim 1 or 2, characterized in that: The oil rich in n-3 PUFA is algae oil or fish oil.

4. The enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides according to claim 3, characterized in that: The fish oils include 1812 fish oil, Omax fish oil and tuna oil.

5. The enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides according to any one of claims 1, 2 or 4, characterized in that: The alcohol is one or both of methanol and ethanol.

6. The enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides according to claim 5, characterized in that: The alcoholysis reaction has a reaction temperature of 25 to 45° C. and a reaction time of 10 to 24 hours.

7. The enzymatic preparation method of n-3 polyunsaturated fatty acid glycerides according to claim 1, characterized in that: The molar ratio of the alcohol to the oil is 10:1 to 70:

1.

8. Use of the method according to any one of claims 1 to 7 in the field of oil and fat processing.

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