A method for preparing a high content triglyceride type fish oil

Abstract

The application relates to the technical field of fish oil refining, and discloses a preparation method of high-content triglyceride type fish oil. Choline chloride and glycerol are used to prepare low-water-activity choline chloride-glycerol natural deep eutectic solvent (NADES) as a glycerol carrier and a reaction microenvironment regulation medium; the low-water-activity choline chloride-glycerol natural deep eutectic solvent is mixed with fish oil ethyl ester and immobilized lipase, re-esterification is carried out under the conditions of inert gas protection and reduced pressure, ethanol is removed in situ, phase separation, selective hydrolysis of partial glyceride lipase and molecular distillation are carried out, and high-content triglyceride type fish oil is obtained. The method can improve the conversion degree of fish oil ethyl ester to triglyceride, reduce the residues of fatty acid ethyl ester and diglyceride, and realize the recycling of NADES.

Description

Technical Field

[0001] This invention relates to the field of fish oil refining technology, specifically to a method for preparing high-triglyceride-type fish oil. Background Technology

[0002] EPA, DHA, and other ω-3 polyunsaturated fatty acids are important active ingredients in fish oil. Fish oil products rich in EPA and DHA typically include ethyl ester, free fatty acid, and triglyceride forms. Among these, triglyceride-type fish oil is closer to its natural form, exhibiting better stability and applicability. Therefore, converting ethyl esters of fish oil rich in EPA and DHA into triglyceride-type fish oil is an important direction in the deep processing of fish oil.

[0003] CN102994580A discloses a method for preparing high-purity triglyceride-type PUFA. The method uses lipase Novozym435 as a catalyst to react the acyl donor of PUFA with glycerol, yielding a mixture of triglyceride-type PUFA and glyceryl-type PUFA. The glyceryl-type PUFA is then hydrolyzed or alcoholyzed with a hydroxyl donor under conditions not exceeding 5°C using glyceryl-type lipase SMG1. Finally, the mixture is purified to obtain high-purity triglyceride-type PUFA. The key feature of this technology is the use of a specific glyceryl-type lipase to remove the glyceryl-type component at low temperatures. However, the reesterification stage still mainly relies on the conventional enzymatic reaction between the PUFA acyl donor and glycerol. The method does not disclose the use of a choline chloride-glycerol natural eutectic solvent to regulate the glycerol supply microenvironment, control water activity, or achieve solvent recycling through polar / non-polar phase separation.

[0004] CN107823137A discloses a method for preparing refined fish oil for injection. This method uses edible-grade fish oil as raw material and employs lipase hydrolysis, glyceryl ester lipase hydrolysis, alkali refining and deacidification, and column chromatography to remove impurities to prepare triglyceride-type refined fish oil for injection. This method emphasizes maintaining the natural fish oil triglyceride configuration and reducing impurities and oxidation indicators. It does not disclose the use of fish oil ethyl ester as a starting material for re-esterification, nor does it disclose the use of a low-water-activity choline chloride-glycerol natural eutectic solvent to assist in the re-esterification of fish oil ethyl ester, in-situ deethanolination, or the recycling of the natural eutectic solvent.

[0005] In the enzymatic reesterification of fish oil ethyl esters and glycerol, glycerol has poor compatibility with the oil phase, and the reaction microenvironment and system moisture content can affect the reesterification efficiency and side reactions. If the residual fatty acid ethyl esters and diglycerides are high during the reesterification stage, the subsequent hydrolysis of glycerides and molecular distillation will increase the load, potentially leading to adverse effects on acid value, oxidation indicators, and product yield. Therefore, there is still a need for a method to prepare high-triglyceride-content fish oil that can increase the conversion of fish oil ethyl esters to triglycerides during the reesterification stage, reduce the subsequent refining load, and is suitable for industrial reuse. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing high-triglyceride fish oil. This method uses a low-water-activity choline chloride-glycerol natural eutectic solvent as a glycerol carrier and a reaction microenvironment control medium. Combined with in-situ deethanolination during reesterification, the conversion of fish oil ethyl esters to triglycerides is improved, and the residues of fatty acid ethyl esters and diglycerides are reduced, thereby reducing the load on subsequent selective hydrolysis and molecular distillation.

[0007] To achieve the above objectives, the present invention provides a method for preparing high-triglyceride-type fish oil, comprising the following steps: 1) Mix choline chloride with glycerol and heat until a clear and transparent liquid is formed. After dehydration, a low water activity choline chloride-glycerol natural deep eutectic solvent is obtained. 2) Mix fish oil ethyl ester, the low water activity choline chloride-glycerol natural eutectic solvent obtained in step 1), and immobilized lipase, and carry out a re-esterification reaction under inert gas protection and reduced pressure, and remove the generated ethanol in situ during the reaction; after the reaction, separate the immobilized lipase, and allow the reaction solution to stand and separate into layers, and separate the upper layer of re-esterified fish oil and the lower layer of natural eutectic solvent phase. 3) Add water and metaglycerol lipase to the heavily esterified fish oil obtained in step 2) to carry out selective hydrolysis reaction, and separate the upper oil phase after the reaction; 4) Perform molecular distillation on the upper oil phase obtained in step 3) and collect the heavy phase product to obtain high-triglyceride fish oil.

[0008] Preferably, in step 1), the molar ratio of choline chloride to glycerol is 1:1.5-3.0, more preferably 1:2. This ratio is beneficial for forming a stable choline chloride-glycerol natural deep eutectic solvent and provides a glycerol donor for the re-esterification of fish oil ethyl esters.

[0009] Preferably, in step 1), choline chloride and glycerol are stirred at 60-90°C for 30-120 min to form a clear and transparent choline chloride-glycerol natural deep eutectic solvent; the dehydration treatment is carried out at 50-80°C and a vacuum degree of -0.080MPa to -0.098MPa for 1-5 h.

[0010] Preferably, the water content of the low water activity choline chloride-glycerol natural deep eutectic solvent after dehydration treatment in step 1) is 0.1-0.5 wt%, and the water activity is 0.05-0.18. By controlling the low water activity, the hydrolysis side reaction in the re-esterification stage can be reduced, and the effective supply of glycerol in the lipase reaction system can be improved.

[0011] Preferably, the immobilized lipase in step 2) is Novozym 435 immobilized lipase; the amount of the immobilized lipase added is 1-5 wt% based on the mass of fish oil ethyl ester. The amount of the low water activity choline chloride-glycerol natural deep eutectic solvent used is 10-30 wt% of the mass of fish oil ethyl ester. The immobilized lipase can catalyze the reesterification reaction of fish oil ethyl ester and glycerol under relatively mild conditions, and is also easy to filter and recover.

[0012] Preferably, in step 2), the temperature of the re-esterification reaction is 45-70℃, the time is 24-60h, the stirring speed is 100-400r / min, and the vacuum degree is -0.080MPa to -0.098MPa.

[0013] Preferably, in step 2), the ethanol generated during the reaction is collected in a cold trap under reduced pressure. In-situ removal of ethanol can promote the re-esterification reaction towards the formation of triglycerides and reduce the residual fatty acid ethyl esters.

[0014] Preferably, after the reaction in step 2) is completed, the oil phase and the natural eutectic solvent phase are allowed to stand and separate into layers based on their polarity differences, resulting in an upper layer of re-esterified fish oil and a lower layer of natural eutectic solvent. The lower layer of natural eutectic solvent can be treated by removing residual ethanol and water, and then adding glycerol to restore the molar ratio of choline chloride to glycerol to 1:1.5-3.0, thus obtaining a regenerated natural eutectic solvent, which can be reused in the next batch of re-esterification reactions.

[0015] Preferably, in step 3), the metaglycerol lipase is Lipase G50; based on the mass of the re-esterified fish oil, the amount of water added is 5-15 wt%, and the amount of metaglycerol lipase added is 0.1-1.0 wt%. The selective hydrolysis reaction is carried out at a temperature of 35-60℃ for 8-24 hours, with a stirring speed of 100-300 r / min. This metaglycerol lipase can selectively reduce the metaglycerol components such as diglycerides in the re-esterified fish oil, making it easier to obtain fish oil products with high triglyceride content through subsequent molecular distillation.

[0016] Preferably, in step 4), the evaporation temperature of molecular distillation is 120-170℃, the condensation temperature is 20-50℃, the system pressure is 1-10Pa, and the scraping speed is 100-400r / min.

[0017] Preferably, the obtained high-triglyceride fish oil contains not less than 98.0% triglycerides, not more than 0.6% fatty acid ethyl esters, not more than 1.2% diglycerides, and no monoglycerides are detected.

[0018] The beneficial effects of this invention include: This invention uses a low water activity choline chloride-glycerol natural deep eutectic solvent as a glycerol carrier and reaction microenvironment control medium to improve the supply status of glycerol in the fish oil ethyl ester system and increase the proportion of triglycerides generated during the re-esterification stage. In this invention, ethanol is continuously removed from the depressurized cold trap during the reesterification process to achieve in-situ deethanolization, which helps to shift the reaction equilibrium toward triglycerides and reduce the residual fatty acid ethyl esters. This invention recovers NADES by separating and recovering the oil phase / natural deep eutectic solvent phase. The process does not require a large amount of organic solvent and is suitable for recycling and industrial scale-up. This invention combines selective hydrolysis with glyceryl lipase and molecular distillation to obtain triglyceride-type fish oil with high triglyceride content, low residual fatty acid ethyl esters and diglycerides, and low acid value and oxidation index. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to the embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Unless otherwise stated, all percentages are by mass.

[0020] The main raw materials used in the examples are as follows: Ethyl fish oil is a raw material rich in EPA and DHA, and is specified as a concentrate of ω-3 polyunsaturated fatty acid ethyl esters. Based on the mass of the ethyl fish oil used, the test results are as follows: EPA content 36.2%, DHA content 24.8%, total EPA and DHA content 61.0%, total fatty acid ethyl ester content 96.5%, acid value 0.7 mg KOH / g, peroxide value 2.4 meq O2 / kg, and anisidine value 8.1. Before use, the ethyl fish oil should be sealed and stored under nitrogen protection, away from light, and protected from oxidation caused by high temperatures.

[0021] The glycerin is food-grade or pharmaceutical-grade.

[0022] Choline chloride, also known as choline chloride, is a food-grade or pharmaceutical-grade choline chloride.

[0023] The immobilized lipase is Novozym 435 immobilized lipase, derived from Candida antarcticis lipase B, and immobilized on macroporous acrylic resin. It appears as granular immobilized enzyme, with an esterification / transesterification activity of 5000 U / g. Store in a sealed container before use to prevent moisture absorption.

[0024] The metaglycerol lipase is Lipase G50, a powdered enzyme preparation with a labeled enzyme activity of 50,000 U / g. This enzyme is used for the selective hydrolysis of diglycerides and other metaglycerol components in fish oil. Store in a sealed container before use to prevent moisture absorption.

[0025] Example 1

[0026] A method for preparing high-triglyceride fish oil includes the following steps: 1) Preparation of a low water activity choline chloride-glycerol natural deep eutectic solvent: Weigh 20.5 g of choline chloride and 27.0 g of glycerol into a beaker, place in an 80°C water bath and stir for 60 min until a clear and transparent liquid is formed, thus obtaining a choline chloride-glycerol natural deep eutectic solvent, wherein the molar ratio of choline chloride to glycerol is 1:2; Place the obtained natural deep eutectic solvent under 70°C and a vacuum of -0.090 MPa for 3 h for dehydration treatment, cool to room temperature and seal for storage, thus obtaining a low water activity choline chloride-glycerol natural deep eutectic solvent, denoted as E1-NADES; According to the method described in Test Example 1, the water content of E1-NADES is 0.28 wt%, and the water activity is 0.12; 2) Low water activity choline chloride-glycerol natural eutectic solvent-assisted reesterification reaction of fish oil ethyl ester: 300g of fish oil ethyl ester, the low water activity choline chloride-glycerol natural eutectic solvent obtained in step 1), and 9.0g of immobilized lipase Novozym 435 were added to a 500mL three-necked flask; the three-necked flask was connected to a mechanical stirrer, a vacuum system, and a cold trap collection device; after nitrogen purging three times, the reaction system was heated to 60℃, the stirring speed was controlled at 250r / min, and the vacuum degree was maintained at -0.085MPa to -0.095MPa, so that the ethanol generated during the reaction was continuously collected and removed by the cold trap; after the reaction was 36h, the heating was stopped, the immobilized lipase was recovered by filtration, the resulting reaction solution was allowed to stand for 30min to separate the upper oil phase and the lower natural eutectic solvent phase, the upper oil phase was collected to obtain reesterified fish oil, denoted as E1-1; 3) Selective hydrolysis with metaglycerol lipase: Take the heavily esterified fish oil obtained in step 2), add 8% of its mass of pure water and 0.4% of metaglycerol lipase Lipase G50, and hydrolyze it at 50℃ and 180 r / min for 16 h; after the reaction is completed, centrifuge at 4000 r / min for 10 min to separate and remove the lower aqueous phase and enzyme phase, and collect the upper oil phase, which is recorded as E1-2; 4) Molecular distillation purification: The upper oil phase obtained in step 3) is subjected to molecular distillation. The molecular distillation conditions are: evaporation temperature 145℃, condensation temperature 35℃, system pressure 5Pa, scraper rotation speed 250r / min, and feed rate 1.5mL / min. The heavy phase product is collected to obtain high-triglyceride fish oil, denoted as E1-3. 5) Recycling of natural deep eutectic solvent: The lower layer of natural deep eutectic solvent obtained in step 2) is subjected to 70°C and vacuum of -0.090MPa for 2 hours to remove residual ethanol and water. Then, glycerol is added to restore the molar ratio of choline chloride to glycerol to 1:2, and the regenerated natural deep eutectic solvent, denoted as E1-NADES-R, is obtained. It can be used for the next batch of fish oil ethyl ester reesterification reaction.

[0027] Comparative Example 1 A method for preparing triglyceride-type fish oil includes the following steps: 1) Ethyl fish oil re-esterification reaction: 300g ethyl fish oil, 27.0g glycerol, and 9.0g immobilized lipase Novozym 435 were added to a 500mL three-necked flask. The three-necked flask was connected to a mechanical stirrer, a vacuum system, and a cold trap collection device. After nitrogen purging three times, the reaction system was heated to 60℃, the stirring speed was controlled at 250r / min, and the vacuum degree was maintained at -0.085MPa to -0.095MPa, so that the ethanol generated during the reaction was continuously collected and removed by the cold trap. After the reaction was carried out for 36h, the heating was stopped, the immobilized lipase was recovered by filtration, and the re-esterified fish oil was obtained, denoted as D1-1. 2) Selective hydrolysis with metaglycerol lipase: Take the heavily esterified fish oil obtained in step 1), add 8% of its mass of pure water and 0.4% of metaglycerol lipase Lipase G50, and hydrolyze it at 50℃ and 180 r / min for 16 h; after the reaction is completed, centrifuge at 4000 r / min for 10 min to separate and remove the lower aqueous phase and enzyme phase, and collect the upper oil phase, which is recorded as D1-2; 3) Molecular distillation purification: The upper oil phase obtained in step 2) is subjected to molecular distillation. The molecular distillation conditions are: evaporation temperature 145℃, condensation temperature 35℃, system pressure 5Pa, scraper rotation speed 250r / min, and feed rate 1.5mL / min. The heavy phase product is collected to obtain triglyceride-type fish oil, denoted as D1-3.

[0028] Comparative Example 2 A method for preparing triglyceride-type fish oil includes the following steps: 1) Preparation of undehydrated choline chloride-glycerol natural deep eutectic solvent: Weigh 20.5g of choline chloride and 27.0g of glycerol into a beaker, place in an 80℃ water bath and stir for 60min until a clear and transparent liquid is formed, thus obtaining the choline chloride-glycerol natural deep eutectic solvent, wherein the molar ratio of choline chloride to glycerol is 1:2; the obtained choline chloride-glycerol natural deep eutectic solvent is not subjected to vacuum dehydration treatment and is denoted as D2-NADES; according to the method described in Test Example 1, the water content of D2-NADES is 2.4wt% and the water activity is 0.46; 2) Re-esterification reaction of ethyl fish oil assisted by undehydrated choline chloride-glycerol natural eutectic solvent: 300g of ethyl fish oil, the choline chloride-glycerol natural eutectic solvent obtained in step 1), and 9.0g of immobilized lipase Novozym 435 were added to a 500mL three-necked flask. The three-necked flask was connected to a mechanical stirrer, a vacuum system, and a cold trap collection device. After nitrogen purging three times, the reaction system was heated to 60℃, the stirring speed was controlled at 250r / min, and the vacuum degree was maintained at -0.085MPa to -0.095MPa, so that the ethanol generated during the reaction was continuously collected and removed by the cold trap. After reacting for 36h, heating was stopped, the immobilized lipase was recovered by filtration, and the resulting reaction solution was allowed to stand for 30min to separate the upper oil phase and the lower natural eutectic solvent phase. The upper oil phase was collected to obtain re-esterified fish oil, denoted as D2-1. 3) Selective hydrolysis with metaglycerol lipase: Take the heavily esterified fish oil obtained in step 2), add 8% of its mass of pure water and 0.4% of metaglycerol lipase Lipase G50, and hydrolyze it at 50℃ and 180 r / min for 16 h; after the reaction is completed, centrifuge at 4000 r / min for 10 min to separate and remove the lower aqueous phase and enzyme phase, and collect the upper oil phase, which is recorded as D2-2; 4) Molecular distillation purification: The obtained upper oil phase was subjected to molecular distillation under the following conditions: evaporation temperature 145℃, condensation temperature 35℃, system pressure 5Pa, scraper rotation speed 250r / min, and feed rate 1.5mL / min. The heavy phase product was collected to obtain triglyceride-type fish oil, denoted as D2-3.

[0029] Comparative Example 3 A method for preparing triglyceride-type fish oil includes the following steps: 1) Preparation of a low water activity choline chloride-glycerol natural deep eutectic solvent: Weigh 20.5 g of choline chloride and 27.0 g of glycerol into a beaker, place in an 80°C water bath and stir for 60 min until a clear and transparent liquid is formed, obtaining a choline chloride-glycerol natural deep eutectic solvent, wherein the molar ratio of choline chloride to glycerol is 1:2; Dehydrate the obtained choline chloride-glycerol natural deep eutectic solvent at 70°C and a vacuum of -0.090 MPa for 3 h, cool to room temperature and seal for storage, obtaining a low water activity choline chloride-glycerol natural deep eutectic solvent; The obtained low water activity choline chloride-glycerol natural deep eutectic solvent is used for subsequent reactions; According to the method described in Test Example 1, the water content of this low water activity choline chloride-glycerol natural deep eutectic solvent is 0.30 wt%, and the water activity is 0.13; 2) Low water activity choline chloride-glycerol natural eutectic solvent-assisted reesterification reaction of fish oil ethyl ester: 300g of fish oil ethyl ester, the low water activity choline chloride-glycerol natural eutectic solvent obtained in step 1), and 9.0g of immobilized lipase Novozym 435 were added to a 500mL three-necked flask; the three-necked flask was connected to a mechanical stirrer and a nitrogen protection device, but not to a cold trap collection device, and no vacuum ethanol removal was performed during the reaction; after nitrogen purging 3 times, the reaction system was heated to 60℃, the stirring speed was controlled at 250r / min, and the reaction was carried out for 36h under normal pressure and nitrogen protection; after the reaction was completed, heating was stopped, the immobilized lipase was filtered and recovered, the resulting reaction solution was allowed to stand for 30min to separate the upper oil phase and the lower natural eutectic solvent phase, the upper oil phase was collected to obtain reesterified fish oil, denoted as D3-1; 3) Selective hydrolysis with metaglycerol lipase: Take the heavily esterified fish oil obtained in step 2), add 8% of its mass of pure water and 0.4% of metaglycerol lipase Lipase G50, and hydrolyze it at 50℃ and 180 r / min for 16 h; after the reaction is completed, centrifuge at 4000 r / min for 10 min to separate and remove the lower aqueous phase and enzyme phase, and collect the upper oil phase, which is recorded as D3-2; 4) Molecular distillation purification: The upper oil phase obtained in step 3) is subjected to molecular distillation. The molecular distillation conditions are: evaporation temperature 145℃, condensation temperature 35℃, system pressure 5Pa, scraper rotation speed 250r / min, and feed rate 1.5mL / min. The heavy phase product is collected to obtain triglyceride-type fish oil, denoted as D3-3.

[0030] Test Example 1 Effects of different re-esterification conditions on the conversion efficiency of fish oil ethyl esters The heavily esterified fish oil samples obtained in Example 1 and Comparative Examples 1-3 were tested. The heavily esterified fish oil sample of Example 1 was E1-1, the heavily esterified fish oil sample of Comparative Example 1 was D1-1, the heavily esterified fish oil sample of Comparative Example 2 was D2-1, and the heavily esterified fish oil sample of Comparative Example 3 was D3-1.

[0031] The moisture content of the natural eutectic solvent was determined according to GB / T 6283-2008 "Determination of Moisture Content in Chemical Products - Karl Fischer Method (General Method)"; the water activity was determined using a water activity meter in accordance with GB 5009.238-2016 "National Food Safety Standard - Determination of Water Activity in Food". The contents of fatty acid ethyl esters (EE), triglycerides (TG), diglycerides (DG), and monoglycerides (MG) in fish oil samples were determined by high performance liquid chromatography-evaporative light scattering detector (ELSD). 100 mg of fish oil sample was weighed and placed in a 10 mL volumetric flask, dissolved in n-hexane, and diluted to volume. The solution was filtered and used as the test solution. A C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase consisted of acetonitrile and isopropanol, changing from acetonitrile / isopropanol = 40 / 60 to acetonitrile / isopropanol = 0 / 100 within 0-40 min. The flow rate was 1.0 mL / min. An evaporative light scattering detector was used. The relative contents of fatty acid ethyl esters, triglycerides, diglycerides, and monoglycerides, as well as the relative contents of FFA and other visible peaks, were calculated using the area normalization method. In Table 1, "FFA and other peaks" refers to free fatty acid peaks and trace unknown peaks other than those of fatty acid ethyl esters, triglycerides, diglycerides, and monoglycerides. Acid value was determined according to GB 5009.229-2025 "National Food Safety Standard - Determination of Acid Value in Food". The test results are shown in Table 1.

[0032] Table 1. Effects of different re-esterification conditions on the conversion efficiency of fish oil ethyl esters Group NADES moisture content / wt% Water activity EE / % TG / % DG / % MG / % FA and other peaks / % <![CDATA[Acid value / mgKOH·g -1 > Example 1 0.28 0.12 2.8 89.6 7.4 0.2 0.0 0.9 Comparative Example 1 - - 6.9 81.7 11.1 0.3 0.0 1.1 Comparative Example 2 2.4 0.46 4.7 84.3 9.5 0.4 1.1 2.8 Comparative Example 3 0.30 0.13 6.1 83.2 10.3 0.4 0.0 1.0 As shown in Table 1, compared with Comparative Example 1, Example 1 used low water activity choline chloride-glycerol NADES as a glycerol carrier and reaction microenvironment control medium. Under the same in-situ deethanolination conditions, the TG content in the re-esterified fish oil increased from 81.7% to 89.6%, the EE content decreased from 6.9% to 2.8%, and the DG content decreased from 11.1% to 7.4%. This indicates that low water activity NADES can improve the supply status of glycerol in the fish oil ethyl ester system and increase the conversion degree of fish oil ethyl ester to triglycerides.

[0033] Compared to Example 1, Comparative Example 2 used untreated choline chloride-glycerol NADES, resulting in a significant increase in NADES water content and water activity. The resulting heavily esterified fish oil showed a decrease in TG content and an increase in acid value, indicating that controlling the low water activity of NADES is beneficial for reducing hydrolysis side reactions and improving the heavy esterification effect. The presence of 1.1% FFA and other peaks in Comparative Example 2, corresponding to its increased acid value, suggests that the higher water content and water activity in the untreated NADES promote some hydrolysis side reactions, further demonstrating the necessity of controlling the low water activity of NADES.

[0034] Compared to Example 1, Comparative Example 3, although using low water activity choline chloride-glycerol NADES, did not employ in-situ deethanolination under reduced pressure and a cold trap. Instead, the reaction was carried out under atmospheric pressure and nitrogen protection. The resulting re-esterified fish oil showed a significantly increased EE content and a decreased TG content. This result demonstrates that in the reaction system of this invention, the in-situ deethanolination operation, involving continuous removal of ethanol from the cold trap under reduced pressure, is beneficial for reducing residual fatty acid ethyl esters and promoting the re-esterification reaction of fish oil ethyl esters with glycerol towards the formation of triglycerides.

[0035] Test Example 2 Quality testing of oil phase and final product after hydrolysis The oil phase samples after selective hydrolysis with metaglycerol lipase in Examples 1 and Comparative Examples 1-3, as well as the final products after molecular distillation, were analyzed. The oil phase sample after hydrolysis in Example 1 was E1-2, and the final product was E1-3; the oil phase sample after hydrolysis in Comparative Example 1 was D1-2, and the final product was D1-3; the oil phase sample after hydrolysis in Comparative Example 2 was D2-2, and the final product was D2-3; the oil phase sample after hydrolysis in Comparative Example 3 was D3-2, and the final product was D3-3.

[0036] The contents of EE, TG, DG and MG were determined using the high performance liquid chromatography-evaporative light scattering detector method described in Test Example 1.

[0037] Acid value was determined according to GB 5009.229-2025 "National Food Safety Standard - Determination of Acid Value in Food"; peroxide value was determined according to GB 5009.227-2023 "National Food Safety Standard - Determination of Peroxide Value in Food"; and anisidine value was determined according to GB / T 24304-2024 "Determination of Anisidine Value in Animal and Vegetable Oils". Product yield was calculated using the following formula: Product yield = Mass of the heavy phase product obtained after molecular distillation / Mass of fish oil ethyl ester feed × 100%.

[0038] The test results are shown in Tables 2 and 3.

[0039] Table 2. Oil phase composition after selective hydrolysis of glycerol lipase Group Sample number EE / % TG / % DG / % MG / % <![CDATA[Acid value / mgKOH·g -1 > Example 1 E1-2 2.3 96.4 0.9 Not detected 8.5 Comparative Example 1 D1-2 5.1 93.5 1.2 Not detected 10.2 Comparative Example 2 D2-2 3.8 94.8 1.1 Not detected 11.4 Comparative Example 3 D3-2 4.6 94.0 1.2 Not detected 9.7 Table 3. Quality test results of the final product after molecular distillation Group Sample number EE / % TG / % DG / % MG / % <![CDATA[Acid value / mgKOH·g -1 > <![CDATA[Peroxide value / meqO2·kg -1 > Anisamine value Product yield / % Example 1 E1-3 0.3 98.8 0.8 Not detected 0.5 1.8 3.6 82.5 Comparative Example 1 D1-3 0.6 98.1 1.1 Not detected 0.6 2.6 5.2 77.8 Comparative Example 2 D2-3 0.5 98.3 1.0 Not detected 0.6 3.4 6.8 76.9 Comparative Example 3 D3-3 0.6 98.0 1.2 Not detected 0.7 2.3 4.9 78.4 As shown in Table 2, the DG content in each group of samples was significantly reduced after selective hydrolysis with Lipase G50. The oil phase of Example 1 had the highest TG content and the lowest EE content after hydrolysis, indicating that the low EE and low DG base formed in Example 1 during the re-esterification stage can reduce the processing load of subsequent selective hydrolysis and molecular distillation.

[0040] As shown in Table 3, the final product obtained in Example 1 had a TG content of 98.8%, an EE content reduced to 0.3%, a DG content of 0.8%, and an acid value of 0.5 mg KOH / g. Simultaneously, the peroxide value and anisidine value were lower than those of the comparative proportions, and the product yield was also higher. These results indicate that using low water activity choline chloride-glycerol NADES combined with in-situ deethanolination can increase the conversion of fish oil ethyl esters to TG during the re-esterification stage, reducing the subsequent refining load, thereby obtaining triglyceride-type fish oil with high TG, low EE, low acid value, and low oxidation indicators.

[0041] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing high-triglyceride-type fish oil, characterized in that, Includes the following steps: 1) Mix choline chloride with glycerol and heat until a clear and transparent liquid is formed. After dehydration, a low water activity choline chloride-glycerol natural deep eutectic solvent is obtained. 2) Mix fish oil ethyl ester, the low water activity choline chloride-glycerol natural eutectic solvent obtained in step 1), and immobilized lipase, and carry out a re-esterification reaction under inert gas protection and reduced pressure, and remove the generated ethanol in situ during the reaction; after the reaction, separate the immobilized lipase, and allow the reaction solution to stand and separate into layers, and separate the upper layer of re-esterified fish oil and the lower layer of natural eutectic solvent phase. 3) Add water and metaglycerol lipase to the heavily esterified fish oil obtained in step 2) to carry out selective hydrolysis reaction, and separate the upper oil phase after the reaction; 4) Perform molecular distillation on the upper oil phase obtained in step 3) and collect the heavy phase product to obtain high-triglyceride fish oil.

2. The preparation method according to claim 1, characterized in that, In step 1), the molar ratio of choline chloride to glycerol is 1:1.5-3.

0.

3. The preparation method according to claim 1, characterized in that, The water content of the low water activity choline chloride-glycerol natural deep eutectic solvent after dehydration treatment in step 1) is 0.1-0.5 wt%, and the water activity is 0.05-0.

18.

4. The preparation method according to claim 1, characterized in that, In step 1), choline chloride and glycerol are stirred at 60-90℃ for 30-120 min to form a natural deep eutectic solvent. The dehydration treatment is carried out at 50-80℃ and a vacuum degree of -0.080MPa to -0.098MPa for 1-5 h.

5. The preparation method according to claim 1, characterized in that, The immobilized lipase mentioned in step 2) is Novozym 435 immobilized lipase; the amount of the immobilized lipase added is 1-5 wt% based on the mass of fish oil ethyl ester; the amount of the low water activity choline chloride-glycerol natural deep eutectic solvent used in step 2) is 10-30 wt% of the mass of fish oil ethyl ester.

6. The preparation method according to claim 1, characterized in that, In step 2), the temperature of the heavy esterification reaction is 45-70℃, the time is 24-60h, the stirring speed is 100-400r / min, and the vacuum degree is -0.080MPa to -0.098MPa.

7. The preparation method according to claim 1, characterized in that, After removing residual ethanol and water from the lower natural eutectic solvent phase obtained in step 2), glycerol is added to restore the molar ratio of choline chloride to glycerol to 1:1.5-3.0, thus obtaining a regenerated natural eutectic solvent that can be reused in the next batch of re-esterification reactions.

8. The preparation method according to claim 1, characterized in that, The metaglycerol lipase mentioned in step 3) is Lipase G50; based on the mass of the heavily esterified fish oil, the amount of water added is 5-15 wt%, and the amount of metaglycerol lipase added is 0.1-1.0 wt%. In step 3), the selective hydrolysis reaction is carried out at a temperature of 35-60℃ for 8-24 hours and at a stirring speed of 100-300 r / min.

9. The preparation method according to claim 1, characterized in that, In step 4), the evaporation temperature of molecular distillation is 120-170℃, the condensation temperature is 20-50℃, the system pressure is 1-10Pa, and the scraping speed is 100-400r / min.

10. The preparation method according to any one of claims 1-9, characterized in that, The obtained high-triglyceride fish oil contained no less than 98.0% triglycerides, no more than 0.6% fatty acid ethyl esters, no more than 1.2% diglycerides, and no detected monoglycerides.