Method for enriching n-3 polyunsaturated fatty glyceride by enzyme method

Abstract

The invention belongs to the technical field of grease modification and lipid deep processing, and particularly relates to a method for enriching n-3 polyunsaturated fatty glyceride by an enzymic method. According to the method, alkyl-modified hollow mesoporous silica spheres are used as an immobilized enzyme carrier and an emulsifier, grease rich in n-3 polyunsaturated fatty acid is used as a raw material in a stable Pickering emulsion system, saturated fatty acid and monounsaturated fatty acid are selectively hydrolyzed by lipase, and polyunsaturated fatty glyceride is enriched. The method has the characteristics of fast reaction, high yield, wide substrate application range and reusability of the immobilized enzyme, and the content of polyunsaturated fatty acid in the polyunsaturated fatty glyceride reaches 58.05-90.53%.

Description

Technical Field

[0001] The invention belongs to the technical field of oil modification and lipid deep processing, and specifically relates to a method for enriching n-3 polyunsaturated fatty acid glyceride by enzymatic method. Background Art

[0002] n-3 polyunsaturated fatty acids (n-3PUFA) are essential fatty acids for the human body and play an important role in human health. They can not only reduce the risk of cardiovascular disease, diabetes, hypertension and other inflammatory diseases, but also prevent obesity, depression and promote the development of the brain and visual nervous system of infants and young children. Studies have shown that different forms of n-3PUFA have different bioavailability characteristics. The bioavailability of ethyl ester n-3PUFA is poor, the bioavailability of free fatty acids is moderate, and the bioavailability of glyceride n-3PUFA is the highest. At the same time, glyceride n-3PUFA has good flavor and taste, so the demand in the food, health care products and medical pharmaceutical industries is also increasing. Green, environmentally friendly, cost-effective and efficient n-3PUFA enrichment methods have also received more attention.

[0003] The lipase method has been favored by researchers due to its advantages such as strong specificity, safety and environmental protection. The enzyme method mostly uses a free enzyme oil-water two-phase system to enrich n-3PUFA glycerides. The method is simple and environmentally friendly, but it has problems such as low efficiency, the free enzyme cannot be reused, and it is easy to cause product pollution due to residues. Therefore, the field urgently needs to find a new efficient and safe enzyme catalysis system for enriching glyceride-type n-3PUFA. Summary of the invention

[0004] In view of this, in order to solve the problems existing in the prior art, the present invention provides a method for enriching n-3 PUFA glycerides based on enzymatic hydrolysis at the emulsion interface.

[0005] In order to achieve the above objects, the first object of the present invention is to provide a method for enzymatically enriching n-3 polyunsaturated fatty acid glycerides.

[0006] The invention discloses an enzymatic method for enriching n-3 polyunsaturated fatty acid glycerides, wherein the saturated fatty acids and monounsaturated fatty acids in n-3PUFA oils are selectively hydrolyzed by an immobilized enzyme, and the n-3 polyunsaturated fatty acid glycerides are obtained after deacidification of the organic phase.

[0007] It is worth noting that the existing selective hydrolysis process for enriching n-3PUFA glycerides usually adopts a free enzyme oil-water two-phase system, which has the problems of long reaction time, low conversion efficiency, difficulty in product separation, inability to reuse free enzymes, and easy residues causing product pollution. In view of the above problems, the present invention provides a method for enriching n-3PUFA glycerides based on enzymatic hydrolysis at the emulsion interface. The present invention regulates the hydrophilicity and hydrophobicity of the carrier surface, and the immobilized enzyme particles obtained not only provide a suitable catalytic microenvironment for lipase, but also improve the catalytic activity and stability of the enzyme; it can also stabilize the oil-in-water type Pickering emulsion, greatly improve the interface area of ​​the oil-water two phases and the catalytic efficiency of the enzyme, accelerate the reaction rate, and the 2-hour reaction rate of enriching n-3PUFA glycerides can be increased by 9.17% to 23.09%. In addition, the immobilized lipase can be reused, which significantly reduces the production cost.

[0008] Furthermore, the carrier of the immobilized enzyme is a hollow mesoporous silica sphere with surface alkyl hydrophobically modified, and the loaded lipase is one or more of AY "Amano" 400SD, AY "Amano" SD, AY "Amano" AYS, and AY "Amano" DF-15.

[0009] Furthermore, the preparation method of the immobilized enzyme includes: preparing hollow mesoporous silica spheres HMSS by a sol-gel method, and preparing a hydrophobic carrier HMSS-C by hydrophobic modification. n ; Then, the lipase was immobilized on the hydrophobic carrier by physical adsorption to obtain the immobilized enzyme Lipase@HMSS-C n , where n is the carbon chain length of the silane reagent used for hydrophobic modification.

[0010] Furthermore, the silane reagent used for the hydrophobic modification includes one or more of methyltrichlorosilane, ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, and octadecyltrimethoxysilane, and the added amount of the silane reagent is 5-100 mmol / g carrier.

[0011] More specifically, the preparation method of the immobilized enzyme comprises:

[0012] S1. Preparation of carrier by sol-gel method: 1,2-bis(triethoxysilane)ethane (BTSE), tetraethyl orthosilicate (TEOS) and hexadecyltrimethylammonium bromide (CTAB) were dispersed in water, stirred to react to obtain solid particles, and calcined to form hollow mesoporous silica spheres (HMSS); then, triethylamine was used as a catalyst in a toluene environment, and one or more silane reagents selected from methyltrichlorosilane, ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane and octadecyltrimethoxysilane were used to modify the surface hydrophobicity of HMSS, and the surface alkyl-modified hollow mesoporous silica spheres HMSS-C were obtained after filtration, collection and drying. n ;

[0013] S2, adding free lipase to a phosphate buffer solution with a pH of 5.0-9.0 to prepare an enzyme solution with a concentration of 10-50 mg / mL; then adding the HMSS-C obtained in step S1 n Add to the enzyme solution, the carrier mass and enzyme solution volume ratio is 1:100-3.5:100g / mL, the immobilization time is 0.5-2h, the incubation temperature is 20-40℃, centrifugal filtration, freeze-drying to obtain the immobilized enzyme Lipase@HMSS-C n .

[0014] It is worth noting that the present invention fixes free lipase on silica spheres that are allowed to be added as food additives, thereby improving the thermal stability and reusability of the enzyme, reducing production costs, and being green and economical. In addition, the immobilized enzyme particles simultaneously serve as enzyme carriers and emulsifiers to construct a stable oil-in-water type Pickering emulsion interface catalytic system, significantly increasing the oil-water two-phase interface area, and greatly improving the catalytic efficiency compared to the traditional reaction system. The whole process does not require the addition of organic solvents, the raw material concentration is high, and it is suitable for industrial production.

[0015] Furthermore, the n-3 PUFA oil includes fish oil, algae oil or vegetable oil containing polyunsaturated fatty acids.

[0016] Furthermore, the n-3PUFA oil includes fish oil, DHA algae oil, ARA algae oil, linseed oil, perilla oil or safflower seed oil with different polyunsaturated fatty acid contents. Different raw materials can be selected according to needs to enrich n-3PUFA glycerides with different contents to meet the needs of different application scenarios.

[0017] Furthermore, the specific steps of the method for enzymatic enrichment of n-3 polyunsaturated fatty acid glycerides described in the present invention include: mixing n-3PUFA oil with phosphate buffer solution, adding immobilized enzyme and emulsifier, homogenizing to form Pickering emulsion, and conducting selective hydrolysis reaction at a constant temperature. After the reaction is completed, centrifugal demulsification and separation are performed, the upper organic phase is retained, and n-3 polyunsaturated fatty acid glycerides are obtained by deacidification treatment, and the lower immobilized enzyme is separated and collected by precipitation filtration.

[0018] Furthermore, the pH range of the phosphate buffer solution is 5.0-9.0, the amount of immobilized enzyme added is 0.1%-2% of the substrate mass, and the emulsifier is HMSS-C n , and the amount of emulsifier added is 0.5%-2.5% of the substrate mass, the oil-water ratio is 3:7-7:3, the reaction temperature is 25-65°C, and the reaction time is 0.5-6h.

[0019] Furthermore, the deacidification treatment is to use 0.5M potassium hydroxide-ethanol solution for acid-base neutralization to remove free fatty acids, separate the organic phase and dry it to obtain n-3 polyunsaturated fatty acid glycerides.

[0020] Furthermore, the immobilized enzyme particles collected by precipitation filtration separation are washed with n-hexane, dried, and recycled.

[0021] The immobilized enzyme-stabilized Pickering emulsion interfacial catalytic system for enriching n-3 PUFA glyceride disclosed in the present invention has the advantages of high catalytic efficiency, good substrate adaptability, stability and reusability. After 11 cycles of use, the increment of n-3 PUFA in the hydrolyzate is still 49.50% of the relative increment.

[0022] Compared with the prior art, the present invention uses alkyl-modified hollow mesoporous silica balls as immobilized enzyme carriers and emulsifiers, and uses fish oil, algae oil or vegetable oil and other oils rich in polyunsaturated fatty acids as raw materials in a stable Pickering emulsion system, and selectively hydrolyzes saturated fatty acids and monounsaturated fatty acids by lipase to enrich polyunsaturated fatty acid glycerides. The method has the characteristics of fast reaction, high yield, wide substrate application range, and reusable immobilized enzymes. The polyunsaturated fatty acid content in the polyunsaturated fatty acid glycerides reaches 58.05%-90.53%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0024] Figure 1 These are SEM (a) and TEM (b) images of the support material HMSS obtained in Example 1 of the present invention.

[0025] Figure 2 HMSS and HMSS-C of the present invention 18 , AY 400SD and AY 400SD@HMSS-C 18 FTIR spectrum (a) and thermogravimetric analysis (b).

[0026] Figure 3 For this invention, AY 400SD@HMSS-C 18 Confocal laser scanning microscopy images of the stable Pickering emulsion (Nile Red labeled the immobilized enzyme, FITC labeled the aqueous phase).

[0027] Figure 4 These are the results of repeated use of the Pickering interface enzymatic hydrolysis to enrich n-3 PUFA glycerides in Example 1 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0030] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0031] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0032] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0033] The fatty acid composition of the n-3 PUFA glyceride of the present invention is mainly detected by gas chromatography.

[0034] Gas chromatography conditions: equipped with a hydrogen flame ionization detector (FID) and a capillary column (DB-FastFAME, 30m×0.250mm×0.25μm). The injection volume was 1μL, the carrier gas (nitrogen) flow rate was set to 2.0mL / min, and the split ratio was 1:10. The column oven temperature program was 165℃ for 3.6min, then increased to 230℃ at a rate of 20℃ / min and maintained for 6min. The temperatures of the detector and injection port were 260℃ and 280℃, respectively. The fatty acid composition was determined based on the retention time of the fatty acid methyl ester standard. The content of each fatty acid was calculated based on the peak area normalization method.

[0035] The preparation method of the immobilized enzyme used in the embodiment of the present invention is:

[0036] (1) Preparation of HMSS: 2.4 g of hexadecyltrimethylammonium bromide (CTAB), 3.75 mL of 1,2-bis(triethoxysilane)ethane (BTSE) and 3.75 mL of tetraethyl orthosilicate (TEOS) were placed in a three-necked flask, and an ethanol / water solution containing ammonia (water, 1125 mL; ethanol, 450 mL; ammonia, 15 mL) was added. The mixture was stirred at 35°C and 300 rpm for 24 h. After the reaction was completed, the carrier particles were collected by filtration and washed with anhydrous ethanol. The obtained white particles were dried at 60°C overnight. Ultrasonic dispersion was performed in ultrapure water, and HMSS was collected by filtration after incubation in an oven at 70°C for 20 h. The solid powder HMSS was calcined in a tube furnace at 550°C for 6 h to obtain solid powder HMSS. The particle morphology was observed by TEM and SEM ( Figure 1 ).

[0037] (2) Modification of HMSS: 1.0 g HMSS, 15 μL triethylamine, and 10 mmol octadecyltrimethoxysilane were added to a reaction kettle containing 10 mL toluene. The mixture was reacted at 130 °C for 20 h, filtered and washed with ethanol, and dried in an oven at 60 °C to obtain HMSS-C 18 spare.

[0038] (3) Immobilization of free enzyme: Accurately weigh 1.5 g of AY "Amano" 400SD and dissolve it in 50 mL of phosphate buffer solution (50 mM, pH = 7). Mix the carrier and enzyme solution at a solid-liquid ratio of 10 mg / mL, ultrasonically disperse for 5 min, evacuate for 5 min, incubate on a shaker at 30°C for 40 min, and freeze-dry to obtain the homemade immobilized enzyme.

[0039] (4) Optimization of Pickering emulsion conditions: Weigh the oil phase and water phase with a mass ratio of 3:7-7:3 and place them in a reaction bottle, add 1%-2.5% of the emulsifier AY 400SD@HMSS-C based on the substrate 18 , a stable Pickering emulsion was formed by stirring, and the size and type of the emulsion droplets were observed by optical microscopy and laser confocal microscopy ( Figure 3 ).

[0040] The HMSS and HMSS-C prepared according to the above method 18 , AY 400SD and AY400SD@HMSS-C 18 The infrared spectrum (a) and thermogravimetric analysis (b) of Figure 2 shown.

[0041] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0042] Example 1

[0043] Algae oil (n-3 PUFA content of 70.16%) and phosphate buffer (pH = 7.0, 50mM) were mixed in a mass ratio of 1:1 (1.5g:1.5g), and 2% of the substrate weight of immobilized enzyme and emulsifier (0.5% AY400SD@HMSS-C 18 and 1.5% HMSS-C 18 ), a stable Pickering emulsion is formed by stirring, and the enzymatic hydrolysis reaction is carried out at 35°C for 2 hours. The supernatant is centrifuged to obtain the crude product of n-3 PUFA-rich glyceride; a KOH-ethanol solution is used for deacidification to obtain n-3 PUFA-rich glyceride.

[0044] The results showed that the content of n-3PUFA in the hydrolyzate was 86.77% and the recovery rate of n-3PUFA was 90.1%.

[0045] Reuse of immobilized enzyme AY 400SD@HMSS-C 18 After 11 times, the relative enrichment increment of n-3 PUFA in the hydrolyzate glyceride was 49.50%.

[0046] Example 2

[0047] As in Example 1, algae oil (n-3 PUFA content of 77.71%) and phosphate buffer (pH = 7.0, 50 mM) were mixed at a mass ratio of (2.0 g: 1.0 g), and the amount of immobilized enzyme and emulsifier added was 1.0% of the total mass of the substrate (0.5% AY400SD@HMSS-C 18 and 0.5% HMSS-C 18 ), and the rest of the operations were the same. The content of n-3 PUFA in the obtained glyceride was 89.02%, and the recovery rate of n-3 PUFA was 90.24%.

[0048] The fatty acid composition of n-3 PUFA glycerides in the hydrolyzate of Example 1-2 is shown in Table 1.

[0049] Table 1 Fatty acid composition of n-3 PUFA glycerides in the hydrolyzate of Example 1-2

[0050]

[0051] Example 3

[0052] As in Example 1, fish oil (n-3 PUFA content of 34.96%) and phosphate buffer (pH = 7.0, 50 mM) were mixed in a mass ratio of (2.1 g: 0.9 g), and the amount of immobilized enzyme and emulsifier added was 3.0% of the total mass of the substrate (0.5% AY400SD@HMSS-C 18 and 2.5% HMSS-C 18 ), the enzymatic hydrolysis reaction was carried out at 45°C for 0.5h, the content of n-3PUFA in the obtained glyceride was 58.05%, and the recovery rate of n-3PUFA was 91.24%.

[0053] Example 4

[0054] As in Example 1, fish oil (n-3 PUFA content of 39.61%) and phosphate buffer (pH = 7.0, 50 mM) were mixed in a mass ratio of (1.8 g: 1.2 g), and the immobilized enzyme and emulsifier AY 400SD@HMSS-C were added. 18 The addition amount was 2.0% of the total mass of the substrate (0.5% AY 400SD@HMSS and 1.5% HMSS-C 18 ), the enzymatic hydrolysis reaction was carried out at 40°C for 3.0 h, the content of n-3 PUFA in the obtained glyceride was 60.57%, and the recovery rate of n-3 PUFA was 92.96%.

[0055] Example 5

[0056] As in Example 1, fish oil (n-3 PUFA content of 71.84%) and phosphate buffer (pH = 7.0, 50 mM) were mixed in a mass ratio of (1.2 g:1.8 g), and the immobilized enzyme and emulsifier AY 400SD@HMSS-C were added. 18 The addition amount was 2.0% of the total mass of the substrate (0.5% AY 400SD@HMSS-C 18 and 1.5% HMSS-C 18 ), the enzymatic hydrolysis reaction was carried out at 25°C for 6.0 h, the content of n-3 PUFA in the obtained glyceride was 81.85%, and the recovery rate of n-3 PUFA was 90.58%.

[0057] Example 6

[0058] As in Example 1, fish oil (n-3 PUFA content of 81.36%) and phosphate buffer (pH = 7.0, 50 mM) were mixed in a mass ratio of (0.9 g: 2.1 g), and the immobilized enzyme and emulsifier AY 400SD@HMSS-C were added. 18 The addition amount was 2.0% of the total mass of the substrate (0.5% AY 400SD@HMSS-C 18 and 1.5% HMSS-C 18 ), the enzymatic hydrolysis reaction was carried out at 65°C for 4.0 h, the content of n-3 PUFA in the obtained glyceride was 90.53%, and the recovery rate of n-3 PUFA was 91%.

[0059] The fatty acid composition of n-3 PUFA glycerides in the hydrolyzate of Example 3-6 is shown in Table 2.

[0060] Table 2 Fatty acid composition of n-3 PUFA glycerides in hydrolyzates of Examples 3-6

[0061]

[0062] Example 7

[0063] Preparation of homemade immobilized enzyme:

[0064] Accurately weigh 2.0 g of AY "Amano" DF-15, dissolve it in 50 mL of phosphate buffer solution (50 mM, pH = 8.0), mix the carrier and enzyme solution at a solid-liquid ratio of 10 mg / mL, ultrasonically disperse for 5 min, vacuumize for 5 min, incubate on a shaker at 20 ° C for 2.0 h, centrifuge, and freeze-dry to obtain the homemade immobilized enzyme DF-15@HMSS-C. 18 .

[0065] As in Example 1, perilla oil (n-3 PUFA content of 68.95%) and phosphate buffer (pH = 7.0, 50 mM) were mixed in a mass ratio of (1.5 g: 1.5 g), and the immobilized enzyme and emulsifier DF-15@HMSS-C were added. 18 The addition amount was 2.0% of the total mass of the substrate (1.0% DF-15@HMSS-C 18 , and 1.0% HMSS-C 18 ), the enzymatic hydrolysis reaction was carried out at 45°C for 3.0 h, the content of n-3 PUFA in the obtained glyceride was 83.25%, and the recovery rate of n-3 PUFA was 90.8%.

[0066] Example 8

[0067] As in Example 1, linseed oil (n-3 PUFA content of 52.97%) and phosphate buffer (pH = 7.0, 50 mM) were mixed in a mass ratio of (1.5 g: 1.5 g), and the immobilized enzyme and emulsifier DF-15@HMSS-C were added. 18 The addition amount was 2.0% of the total mass of the substrate (1.0% DF-15@HMSS-C 18 and 1.0% HMSS-C 18 ), the enzymatic hydrolysis reaction was carried out at 45°C for 2.5h, the content of n-3 PUFA in the obtained glyceride was 67.39%, and the recovery rate of n-3 PUFA was 90.2%.

[0068] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the properties and application performance of the method for enzymatic enrichment of polyunsaturated fatty acids in n-3PUFA oils disclosed in the present invention are further illustrated by the following comparative examples, but they should not be construed as limitations on the present invention. The properties of the method obtained by other determination experiments conducted by technicians in this field based on the above-mentioned invention content and the applications based on the above-mentioned properties are also deemed to fall within the protection scope of the present invention.

[0069] Comparative Example 1

[0070] As in Example 1, algae oil (n-3 PUFA content of 70.16%) and phosphate buffer (pH = 7.0, 50 mM) were mixed in a mass ratio of 1:1 (1.5 g: 1.5 g), and 0.5% of the substrate mass of free lipase AY 400SD was added to the system. The rest of the operations were the same. It was determined that the content of n-3 PUFA in the hydrolyzate was 80.22%, and the recovery rate of n-3 PUFA was 93.75%.

[0071] After 4 repetitions, the relative enrichment increment of n-3 PUFA in the hydrolysate glycerides was only 3.75%.

[0072] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for enzymatically enriching n-3 polyunsaturated fatty acid glycerides, characterized in that: The saturated fatty acids and monounsaturated fatty acids in n-3PUFA oils were selectively hydrolyzed by immobilized enzymes, and n-3 polyunsaturated fatty acid glycerides were obtained after deacidification of the organic phase.

2. The method according to claim 1, characterized in that The carrier of the immobilized enzyme is a hollow mesoporous silica sphere with surface alkyl hydrophobically modified, and the loaded lipase is one or more of AY "Amano" 400SD, AY "Amano" SD, AY "Amano" AYS, and AY "Amano" DF-15.

3. The method according to claim 2, characterized in that The preparation method of the immobilized enzyme comprises: preparing hollow mesoporous silica spheres HMSS by sol-gel method, and preparing a hydrophobic carrier HMSS-C by hydrophobic modification. n ; Then, the lipase was immobilized on the hydrophobic carrier by physical adsorption to obtain the immobilized enzyme Lipase@HMSS-C n , where n is the carbon chain length of the silane reagent used for hydrophobic modification.

4. The method according to claim 3, characterized in that The silane reagent used for the hydrophobic modification includes one or more of methyltrichlorosilane, ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, and octadecyltrimethoxysilane, and the added amount of the silane reagent is 5-100 mmol / g carrier.

5. The method according to claim 1, characterized in that The n-3 PUFA oils include fish oil, algae oil or vegetable oil containing polyunsaturated fatty acids.

6. The method according to claim 5, characterized in that The n-3 PUFA oil includes fish oil, DHA algae oil, ARA algae oil, flaxseed oil, perilla oil or safflower seed oil.

7. The method according to any one of claims 1 to 6, characterized in that: The specific steps include: mixing n-3PUFA oil with phosphate buffer solution, adding immobilized enzyme and emulsifier, homogenizing to form Pickering emulsion, performing selective hydrolysis reaction at constant temperature, and after the reaction, centrifuging to break the emulsion and separate, retaining the upper organic phase, and obtaining n-3 polyunsaturated fatty acid glyceride through deacidification treatment, and collecting the lower immobilized enzyme through precipitation filtration separation.

8. The method according to claim 7, characterized in that The pH range of the phosphate buffer solution is 5.0-9.0, the amount of immobilized enzyme added is 0.1%-2% of the substrate weight, and the emulsifier is HMSS-C n , and the amount of emulsifier added is 0.5%-2.5% of the substrate mass, the oil-water ratio is 3:7-7:3, the reaction temperature is 25-65°C, and the reaction time is 0.5-6h.

9. The method according to claim 7, characterized in that: The deacidification treatment is to use 0.5M potassium hydroxide-ethanol solution for acid-base neutralization to remove free fatty acids, separate the organic phase and dry it to obtain n-3 polyunsaturated fatty acid glycerides.

10. The method according to claim 7, characterized in that The immobilized enzyme particles collected by precipitation filtration separation are washed and dried with n-hexane and recycled.

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