Preparation method of triglyceride rich in polyunsaturated fatty acid

Abstract

The invention provides a preparation method of triglyceride rich in polyunsaturated fatty acid, and belongs to the technical field of biochemical engineering. The method comprises the following steps: mixing a fatty acid mixture, glycerol and lipase, and carrying out primary esterification synthesis reaction under vacuum; the esterification synthesis reaction is carried out in a vacuum environment, so that the reaction can be promoted to be carried out in the direction of generating triglyceride; according to the method, the ratio of the amount of substance of the fatty acid mixture to the glycerol is controlled to be (1-2.5): 1, and high-saturation fatty acid or monounsaturated fatty acid can preferentially react with the glycerol, so that the high-saturation fatty acid or monounsaturated fatty acid is removed, and the content of polyunsaturated fatty acid in the recovered fatty acid mixture is increased. The method comprises the following steps: mixing the recovered fatty acid mixture with glycerol and lipase, controlling the mass ratio of the recovered fatty acid mixture to be (3-6): 1, and carrying out secondary esterification synthesis reaction under vacuum, so that the recovered fatty acid mixture can completely react as much as possible to obtain the triglyceride rich in polyunsaturated fatty acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, and particularly relates to a preparation method of triglyceride rich in polyunsaturated fatty acid. BACKGROUND

[0002] Polyunsaturated fatty acid (PUFA) refers to a straight-chain fatty acid containing two or more double bonds and having a carbon chain length of 18-22 carbon atoms. PUFA is an important material basis for metabolism of the body, especially activities such as brain development of infants, is a constituent of cell membrane, and mainly plays functions of maintaining cell membrane fluidity, promoting cholesterol esterification, reducing cholesterol and triglyceride, reducing blood viscosity, improving blood circulation, and the like. PUFA also has functions of improving human thinking and enhancing memory. However, the human body cannot synthesize PUFA and needs to obtain PUFA through diet. PUFA is various, and mainly includes ω-3 polyunsaturated fatty acid (ω-3 PUFA), ω-6 polyunsaturated fatty acid (ω-6 PUFA), ω-9 polyunsaturated fatty acid (ω-9 PUFA), and the like, such as α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), linoleic acid (LA), conjugated linoleic acid (CLA), γ-linolenic acid (GLA), arachidonic acid (AA), and the like. Among them, ω-3 polyunsaturated fatty acid (ω-3 PUFA) represented by eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is most familiar and accepted by the public.

[0003] At present, PUFA products on the market mainly include free type products, ethyl ester type products and triglyceride type products. Among them, since PUFA in nature mainly exists in the form of triglyceride, the triglyceride type product is more and more preferred by consumers. The triglyceride type product is obtained by reacting free fatty acid or ethyl ester fatty acid under the action of a chemical catalyst or a biological enzyme catalyst. However, since the starting material of the reaction is a mixture of fatty acids, in addition to polyunsaturated fatty acids, a large amount of saturated and monounsaturated fatty acids are also included in the starting material, so that the proportion of PUFA in the prepared triglyceride type PUFA product is low.

[0004] In order to improve the proportion of polyunsaturated fatty acids in the triglyceride product, the commonly used methods at present include the following two kinds: one is to improve the proportion of polyunsaturated fatty acids in the starting material of the reaction; for example, by using the boiling point difference caused by the carbon number and the number of double bonds between different fatty acids, saturated or monounsaturated fatty acids are removed as much as possible by distillation or rectification, but this method requires high separation effect of the distillation and rectification column; and because the distillation or rectification process has to go through high temperature (the temperature is often above 180℃, even up to 220℃), high temperature is not conducive to the stability of polyunsaturated fatty acids containing more double bonds, destroys the molecular structure of polyunsaturated fatty acids, produces more undesirable oligomers, reduces its biological activity, and even produces carcinogenicity; in addition, for fatty acids with the same carbon atom number and little difference in the number of double bonds, such as C20:0, C20:1, C20:2, C20:3, C20:4 and C20:5, it is difficult to separate by distillation or rectification method. Two is to use the difference in the number of double bonds of different fatty acids, and to selectively remove saturated or monounsaturated fatty acids by urea inclusion, silver ion adsorption and other processes, but a large amount of urea or silver ion solution is used in this process, which is not friendly to food application, so the application is limited.

[0005] It can be seen that the existing method for improving the proportion of polyunsaturated fatty acids in the triglyceride product has the problems of poor improvement effect, harsh conditions, and the use of substances that are not friendly to food application, and how to solve the above problems is a technical problem to be solved in the field. SUMMARY

[0006] The purpose of the present application is to provide a preparation method of triglyceride rich in polyunsaturated fatty acids, the method provided by the present application has mild reaction conditions, does not use reagents that are not friendly to food, and can also significantly improve the proportion of polyunsaturated fatty acids in triglyceride.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a preparation method of triglyceride rich in polyunsaturated fatty acids, comprising the following steps: (1) mixing fatty acid mixture, glycerol and lipase, and performing a first esterification synthesis reaction under vacuum, and then sequentially performing a first water washing, a first degassing and a first distillation to obtain a recovered fatty acid mixture; the substance amount ratio of the fatty acid mixture to glycerol is 1-2.5:1; (2) mixing the recovered fatty acid mixture obtained in step (1) with glycerol and lipase, and performing a second esterification synthesis reaction under vacuum, and then sequentially performing a second water washing, a second degassing and a second distillation to obtain a triglyceride rich in polyunsaturated fatty acids; the substance amount ratio of the recovered fatty acid mixture to glycerol is 3-6:1.

[0008] Preferably, the fatty acid mixture in the step (1) comprises ethyl ester type fatty acids and / or free type fatty acids.

[0009] Preferably, the lipase in the steps (1) and (2) independently comprises Novozyme 435 enzyme, Novozyme TL enzyme, Lipozyme RM IM enzyme, Lipozyme TL IM enzyme or EstC24-EstC27.

[0010] Preferably, the lipase in the step (1) accounts for 1-6% of the mass percentage of the fatty acid mixture.

[0011] Preferably, the temperature of the first esterification synthesis reaction in the step (1) and the temperature of the second esterification synthesis reaction in the step (2) independently are 30-60℃.

[0012] Preferably, the time of the first esterification synthesis reaction in the step (1) is 4-24h.

[0013] Preferably, the time of the second esterification synthesis reaction in the step (2) is 24-72h.

[0014] Preferably, the lipase in the step (2) accounts for 3-10% of the mass percentage of the recovered fatty acid mixture.

[0015] Preferably, the temperature of the first distillation in the step (1) and the temperature of the second distillation in the step (2) independently are 120-180℃.

[0016] Preferably, the pressure of the first distillation in the step (1) and the pressure of the second distillation in the step (2) independently are 2-100Pa.

[0017] The application provides a preparation method of triglyceride rich in polyunsaturated fatty acids, comprising the following steps: mixing fatty acid mixture, glycerol and lipase, performing a first esterification synthesis reaction under vacuum, and then sequentially performing a first water washing, a first degassing and a first distillation to obtain recovered fatty acid mixture; the mass ratio of the fatty acid mixture to the glycerol is 1-2.5:1; mixing the recovered fatty acid mixture with the glycerol and the lipase, performing a second esterification synthesis reaction under vacuum, and then sequentially performing a second water washing, a second degassing and a second distillation to obtain the triglyceride rich in polyunsaturated fatty acids; the mass ratio of the recovered fatty acid mixture to the glycerol is 3-6:1. The fatty acid mixture, the glycerol and the lipase are mixed first, and a first esterification synthesis reaction is performed under vacuum; the esterification synthesis reaction between the glycerol and the fatty acid mixture is an ester exchange reaction or an esterification reaction, which is a reversible reaction, and ethanol or water is generated in the reaction process, and the esterification synthesis reaction is performed under vacuum, so that the reaction is promoted to generate triglyceride; the mass ratio of the fatty acid mixture to the glycerol is controlled to be 1-2.5:1, when the lipase reacts with the fatty acid mixture and the glycerol, the fatty acid mixture and the glycerol are preferentially combined with high-saturation fatty acids and monounsaturated fatty acids at 1,3 positions, when the amount of fatty acids is insufficient, more high-saturation fatty acids and monounsaturated fatty acids are preferentially combined at 1,3 positions, and the mass ratio of the fatty acid mixture to the glycerol is controlled to be 1-2.5:1, so that the high-saturation fatty acids and the monounsaturated fatty acids are allowed to react with the glycerol to remove the high-saturation fatty acids and the monounsaturated fatty acids, and the content of polyunsaturated fatty acids in the recovered fatty acid mixture is increased. The recovered fatty acid mixture is mixed with the glycerol and the lipase, a second esterification synthesis reaction is performed under vacuum, the mass ratio of the recovered fatty acid mixture to the glycerol is 3-6:1, and the recovered fatty acid mixture is allowed to react completely to obtain the triglyceride rich in polyunsaturated fatty acids. The results of examples show that the content of polyunsaturated fatty acids in the fatty acid mixture can be increased from 30%-60% to 50%-95% by the method, and the content of polyunsaturated fatty acids in the final product triglyceride can be as high as more than 90%. DETAILED DESCRIPTION

[0018] The application provides a preparation method of triglyceride rich in polyunsaturated fatty acids, comprising the following steps: (1) mixing fatty acid mixture, glycerol and lipase, performing a first esterification synthesis reaction under vacuum, and then sequentially performing a first water washing, a first degassing and a first distillation to obtain recovered fatty acid mixture; the mass ratio of the fatty acid mixture to the glycerol is 1-2.5:1; (2) mixing the recovered fatty acid mixture obtained in step (1) with glycerol and a lipase, performing a second esterification synthesis reaction under vacuum, and then sequentially performing a second water washing, a second degassing, and a second distillation to obtain a glyceride rich in polyunsaturated fatty acids; the molar ratio of the recovered fatty acid mixture to glycerol is 3-6:1.

[0019] The present application mixes a fatty acid mixture, glycerol, and a lipase, performs a first esterification synthesis reaction under vacuum, and then sequentially performs a first water washing, a first degassing, and a first distillation to obtain a recovered fatty acid mixture.

[0020] In the present application, the fatty acid mixture preferably includes ethyl ester type fatty acids and / or free type fatty acids, and more preferably one or more of fish oil, algal oil, linoleic acid, conjugated linoleic acid, linolenic acid, and arachidonic acid. The present application does not have a special limitation on the source of the fatty acid mixture, and a conventional commercially available product can be used.

[0021] In the present application, the lipase preferably includes Novozyme 435 enzyme, Novozyme TL enzyme, Lipozyme RMIM enzyme, Lipozyme TL IM enzyme, or EstC24-EstC27 enzyme. The present application does not have a special limitation on the source of the lipase, and a conventional commercially available product can be used. In the embodiments of the present application, the source of the Novozyme 435 enzyme, Novozyme TL enzyme, Lipozyme RMIM enzyme, and Lipozyme TL IM enzyme can be Novozymes (China) Biotechnology Co., Ltd.; the EstC24-EstC27 is a new type of esterase derived from a metagenomic library constructed by Professor Xie Tian of Hangzhou University and optimized, which has been disclosed in CN102796715A.

[0022] In the present application, the molar ratio of the fatty acid mixture to glycerol is 1-2.5:1. As an embodiment of the present application, the molar ratio of the fatty acid mixture to glycerol can be 1:1, 1.5:1, 2:1, or 2.5:1. In the present application, when the lipase catalyzes the esterification or transesterification reaction of the fatty acid mixture and glycerol, the 1,3 position preferentially binds to high-saturation fatty acids. When the amount of fatty acids is insufficient, more high-saturation fatty acids and monounsaturated fatty acids will preferentially bind to the 1,3 position. In theory, the molar ratio of fatty acids to glycerol required for complete reaction is 3:1. In the present application, the molar ratio of the fatty acid mixture to glycerol is controlled to be 1-2.5:1, the amount of fatty acids is insufficient, and high-saturation fatty acids and monounsaturated fatty acids are allowed to react with glycerol to remove high-saturation fatty acids and monounsaturated fatty acids, thereby increasing the content of polyunsaturated fatty acids in the recovered fatty acid mixture that does not participate in the reaction.

[0023] In the present application, the lipase is preferably 1 to 6% by mass of the fatty acid mixture. As one embodiment of the present application, the lipase can be 1%, 2%, 3%, 4%, 5% or 6% by mass of the fatty acid mixture. The present application limits the amount of lipase added to the above range, which can have a faster reaction rate, and can prevent the problem of high cost caused by excessive use of lipase.

[0024] The present application does not have a special limitation on the method of mixing the fatty acid mixture, glycerol and lipase, and the three can be mixed uniformly.

[0025] In the present application, the temperature of the first esterification synthesis reaction is preferably 30 to 60°C, and the time of the first esterification synthesis reaction is preferably 4 to 24 hours. As one embodiment of the present application, the temperature of the first esterification synthesis reaction can be 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C, and the time of the first esterification synthesis reaction can be 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours or 24 hours. The present application controls the temperature and time of the first esterification synthesis reaction in the above range, which can make the high-saturation fatty acid and monounsaturated fatty acid fully participate in the reaction.

[0026] In the present application, the pressure of the first esterification synthesis reaction is preferably 9 to 11 Pa, and more preferably 10 to 11 Pa. The present application can provide a suitable vacuum degree under the above pressure, which promotes the esterification synthesis reaction to proceed in the direction of generating triglyceride.

[0027] The present application does not have a special limitation on the operation method of the first water washing, and a conventional water washing method can be used. The present application can remove glycerol in the product of the first esterification synthesis reaction by the first water washing. In the examples of the present application, the method of the first water washing can be washing with 70°C water.

[0028] The present application does not have a special limitation on the operation method of the first degassing, and a conventional degassing method can be used. The present application can remove volatile substances in the mixed solution obtained after water washing by the first degassing, which reduces the impurity content.

[0029] In the present application, the temperature of the first distillation is preferably 120 to 180°C. As an embodiment of the present application, the temperature of the first distillation can be 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, or 180°C. In the present application, the pressure of the first distillation is preferably 2 to 100 Pa. As an embodiment of the present application, the pressure of the first distillation can be selected from 2 Pa, 5 Pa, 10 Pa, 20 Pa, 30 Pa, 40 Pa, 50 Pa, 60 Pa, 70 Pa, 80 Pa, 90 Pa, or 100 Pa. The present application performs the first distillation under the above conditions, and can recover the unreacted fatty acid mixture, and the content of polyunsaturated fatty acids in the obtained recovered fatty acid mixture is significantly increased. The method of the distillation is not particularly limited in the present application, and a conventional distillation method can be used. In the examples of the present application, the method of the distillation can be molecular distillation.

[0030] After obtaining the recovered fatty acid mixture, the present application mixes the recovered fatty acid mixture with glycerol and a lipase, performs a second esterification synthesis reaction under vacuum, and then sequentially performs second water washing, second degassing, and second distillation to obtain a polyunsaturated fatty acid-rich triglyceride.

[0031] In the present application, the lipase is the same as the lipase described in the above technical solution, and will not be described again here.

[0032] In the present application, the molar ratio of the recovered fatty acid mixture to glycerol is 3 to 6:1. As an embodiment of the present application, the molar ratio of the recovered fatty acid mixture to glycerol can be 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, or 6:1. In the present application, the molar ratio of the recovered fatty acid mixture to glycerol is in the above range, which can make the recovered fatty acid mixture and glycerol fully react to obtain a polyunsaturated fatty acid-rich triglyceride.

[0033] In the present application, the mass percentage of the lipase in the recovered fatty acid mixture is preferably 3 to 10%. As an embodiment of the present application, the mass percentage of the lipase in the recovered fatty acid mixture can be 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The present application limits the amount of lipase added to the above range, which can not only have a faster reaction rate, but also prevent the problem of high cost caused by excessive use of lipase.

[0034] The present application does not have a particular limitation on the method of mixing the recovered fatty acid mixture, glycerol, and lipase, and the three can be mixed uniformly.

[0035] In the present application, the temperature of the secondary esterification synthesis reaction is preferably 30 to 60°C, and the time of the secondary esterification synthesis reaction is preferably 24 to 72 hours. As an embodiment of the present application, the temperature of the secondary esterification synthesis reaction can be 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C, and the time of the secondary esterification synthesis reaction can be 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, or 72 hours. By controlling the temperature and time of the secondary esterification synthesis reaction within the above ranges, the present application can make the recovered fatty acid mixture sufficiently participate in the reaction.

[0036] In the present application, the pressure of the secondary esterification synthesis reaction is preferably 9 to 1000 Pa, and more preferably 10 to 500 Pa. The present application can provide a suitable vacuum degree under the above pressure, and promote the esterification synthesis reaction to proceed in the direction of generating triglycerides.

[0037] The present application does not have a special limitation on the operation method of the secondary water washing, and a conventional water washing method can be used. The present application can remove glycerol from the product of the secondary esterification synthesis reaction by the secondary water washing. In the embodiments of the present application, the method of the secondary water washing can be washing with 70°C water.

[0038] The present application does not have a special limitation on the operation method of the secondary degassing, and a conventional degassing method can be used. The present application can remove volatile substances from the mixed solution obtained after the water washing by the secondary degassing, and reduce the impurity content.

[0039] In the present application, the temperature of the secondary distillation is preferably 120 to 180°C. As an embodiment of the present application, the temperature of the secondary distillation can be 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, or 180°C. In the present application, the pressure of the secondary distillation is preferably 2 to 100 Pa. As an embodiment of the present application, the pressure of the secondary distillation can be selected from 2 Pa, 5 Pa, 10 Pa, 20 Pa, 30 Pa, 40 Pa, 50 Pa, 60 Pa, 70 Pa, 80 Pa, 90 Pa, or 100 Pa. The present application can recover the unreacted fatty acid mixture and obtain triglycerides rich in polyunsaturated fatty acids with high purity by performing the secondary distillation under the above conditions.

[0040] The present application removes most of the high-saturation fatty acids and monounsaturated fatty acids by combining as much high-saturation fatty acids and monounsaturated fatty acids as possible with glycerol through twice ester synthesis reactions catalyzed by lipase and controlling the conditions of the first ester synthesis reaction, thereby increasing the proportion of polyunsaturated fatty acids in the recovered fatty acid mixture; then the recovered fatty acid mixture containing a higher proportion of polyunsaturated fatty acids obtained is subjected to a second ester synthesis reaction catalyzed by lipase in the presence of glycerol, and finally, the unreacted glycerol and fatty acids are removed, thereby obtaining high-purity triglyceride rich in polyunsaturated fatty acids. The preparation method provided by the present application is simple in operation, mild in conditions, and does not need to use reagents that are not friendly to food, and can prepare triglyceride rich in polyunsaturated fatty acids.

[0041] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0042] The detection method of the triglyceride rich in polyunsaturated fatty acids in the embodiments of the present application is as follows: 100 mg of the sample is dissolved in 10 mL of mobile phase, and the injection amount is 20 μL.

[0043] HPLC with a differential detector, and the chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX RX-SIL 4.6 mm x 250 mm, the particle size is 5 μm; the mobile phase is n-hexane-isopropyl alcohol (15:1); the flow rate is 0.8 mL / min, and the column temperature is 35 ℃. The peak order is as follows: triglyceride, fatty acid ethyl ester, 1,3-diglyceride, 1,2-diglyceride, and monoglyceride. The content of triglyceride is quantified by area normalization.

[0044] The method for detecting the composition of the recovered fatty acid mixture by gas chromatography in the embodiments of the present application is as follows: Methyl esterification treatment: the treatment method of boron trifluoride methanol in GB / T 17376-2008 "Animal and vegetable fats and oils - Preparation of fatty acid methyl esters". Gas chromatography conditions: DB-23 capillary column (60 m x 0.25 mm x 0.25 μm); the temperature program is 150 ℃ for 2 min, then 3 ℃ / min to 200 ℃, then 2 ℃ / min to 220 ℃, hold for 2 min, then 25 ℃ / min to 230 ℃, hold for 2 min; FID detector temperature 280 ℃; injection port temperature 250 ℃; carrier gas is high-purity helium (purity 99.999%), flow rate 2.0 mL / min; hydrogen flow rate 30 mL / min; air flow rate 450 mL / min; injection volume 1.0 μL; split ratio 20:1. Area normalization method for quantitative determination.

[0045] Example 1 A method for preparing a triglyceride rich in polyunsaturated fatty acids, comprising the steps of: (1) mixing a fatty acid mixture (fish oil: EPA 26.4%, DHA 15.8%, total polyunsaturated fatty acid content 60.4%, saturated and monounsaturated fatty acid content 39.5%), glycerol and lipase (Novozyme 435 enzyme), vacuumizing to make the pressure during reaction 10 Pa, performing a first esterification synthesis reaction at 50 ℃ for 16 h, stopping the reaction, then adding hot water at 70 ℃ to perform a first water washing, then performing a first degassing, and then performing a first distillation by molecular distillation at a temperature of 160 ℃ and a pressure of 15 Pa to obtain a recovered fatty acid mixture; wherein the molar ratio of the fatty acid mixture to glycerol is 2.03:1; the mass percentage of the lipase in the fatty acid mixture is 5%; The composition of the recovered fatty acid mixture is detected by a gas chromatograph: EPA 51.3%, DHA 25.0%, total polyunsaturated fatty acid content 95.2%, saturated and monounsaturated fatty acid content 4.4%; (2) mixing the recovered fatty acid mixture obtained in step (1) with glycerol and lipase (Novozyme 435 enzyme), vacuumizing to make the pressure during reaction 12 Pa, performing a second esterification synthesis reaction at 50 ℃ for 36 h, stopping the reaction, then adding hot water at 70 ℃ to perform a second water washing, then performing a second degassing, and then performing a second distillation by molecular distillation at a temperature of 168 ℃ and a pressure of 11 Pa to obtain a triglyceride rich in polyunsaturated fatty acids; The molar ratio of the recovered fatty acid mixture to glycerol is 4:1; the mass percentage of the lipase in the recovered fatty acid mixture is 8%; The composition of the polyunsaturated fatty acid-rich triglyceride is determined by high performance liquid chromatography to be: the content of triglyceride, fatty acid ethyl ester, diglyceride and monoglyceride is 88.7%, 0.9%, 8.5% and 1.9% respectively.

[0046] The composition of the polyunsaturated fatty acid-rich triglyceride is determined by gas chromatography to be: EPA 50.6%, DHA 24.3%, the total content of polyunsaturated fatty acid is 94.8%, and the content of saturated and monounsaturated fatty acid is 5.1%.

[0047] Comparative Example 1 A method for preparing a triglyceride, comprising the steps of: (1) mixing a fatty acid mixture (fish oil: EPA 26.4%, DHA 15.8%, the total content of polyunsaturated fatty acid is 60.4%, and the content of saturated and monounsaturated fatty acid is 39.5%), glycerol and lipase (Novozyme 435 enzyme), vacuumizing to make the pressure during reaction 10 Pa, performing a first esterification synthesis reaction at 50℃ for 48 h, stopping the reaction, adding hot water at 70℃ to perform a first water washing, then performing a first degassing, and then performing a first distillation by molecular distillation at a temperature of 160℃ and a pressure of 15 Pa to obtain a recovered fatty acid mixture; wherein the molar ratio of the fatty acid mixture to glycerol is 3.02:1, and the mass percentage of the lipase in the fatty acid mixture is 5%; The composition of the recovered fatty acid mixture is determined by gas chromatography to be: EPA 32.4%, DHA 18.9%, the total content of polyunsaturated fatty acid is 73.4%, and the content of saturated and monounsaturated fatty acid is reduced to 26.5%; (2) mixing the recovered fatty acid mixture obtained in step (1) with glycerol and lipase (Novozyme 435 enzyme), vacuumizing to make the pressure during reaction 14 Pa, performing a second esterification synthesis reaction at 50℃ for 72 h, stopping the reaction, adding hot water at 70℃ to perform a second water washing, then performing a second degassing, and then performing a second distillation by molecular distillation at a temperature of 166℃ and a pressure of 10 Pa to obtain a triglyceride; The molar ratio of the recovered fatty acid mixture to glycerol is 3:1, and the mass percentage of the lipase in the recovered fatty acid mixture is 8%; The composition of the triglyceride is determined by high performance liquid chromatography to be: the content of triglyceride, fatty acid ethyl ester, diglyceride and monoglyceride is 89.2%, 0.7%, 8.2% and 1.7% respectively.

[0048] The composition of the triglyceride was detected by a gas chromatograph, and the results were as follows: EPA 31.4%, DHA 17.2%, the total content of polyunsaturated fatty acids was 71.8%, and the content of saturated and monounsaturated fatty acids was 28.2%.

[0049] Comparative Example 2 A method for preparing a triglyceride, comprising the following steps: mixing a fatty acid mixture (fish oil: EPA 26.4%, DHA 15.8%, the total content of polyunsaturated fatty acids was 60.4%, and the content of saturated and monounsaturated fatty acids was 39.5%), glycerol and lipase (Novozyme 435 enzyme), vacuumizing to make the pressure in the reaction 9 Pa, performing esterification synthesis reaction at 50 DEG C for 72 h, stopping the reaction, adding hot water at 70 DEG C to perform water washing, then degassing, and then performing distillation by molecular distillation at a temperature of 157 DEG C and a pressure of 13 Pa to obtain the triglyceride; The molar ratio of the fatty acid mixture to glycerol was 3.01:1, and the mass percentage of the lipase in the fatty acid mixture was 5%. The composition of the triglyceride was detected by a gas chromatograph, and the results were as follows: EPA 31.4%, DHA 17.2%, the total content of polyunsaturated fatty acids was 71.8%, and the content of saturated and monounsaturated fatty acids was 28.2%.

[0050] The composition of the triglyceride was detected by a gas chromatograph, and the results were as follows: EPA 31.4%, DHA 17.2%, the total content of polyunsaturated fatty acids was 71.8%, and the content of saturated and monounsaturated fatty acids was 28.2%.

[0051] As can be seen from Example 1 and Comparative Examples 1-2, the total content of polyunsaturated fatty acids in the triglyceride prepared in Comparative Example 1 was 71.8%, the total content of polyunsaturated fatty acids in the triglyceride prepared in Comparative Example 2 was 52.3%, and the total content of polyunsaturated fatty acids in the polyunsaturated fatty acid-rich triglyceride prepared in Example 1 was 94.8%. It can be seen that, compared with Comparative Examples 1-2, the method provided in Example 1 can significantly improve the proportion of polyunsaturated fatty acids in the triglyceride. This is because, when the proportion of the fatty acid mixture to glycerol in the reaction substrate is lower, especially lower than the theoretical required reaction proportion of 3:1, the high saturated fatty acids and monounsaturated fatty acids in the reaction substrate preferentially react with glycerol to generate triglyceride or diglyceride in a short time, and then the unreacted fatty acids are recovered by molecular distillation due to the difference in boiling points of the fatty acids, triglyceride and diglyceride, and then the reaction is performed again with glycerol under the catalysis of the lipase. At this time, in order to improve the proportion of the triglyceride, the proportion of the fatty acid to glycerol in the reaction substrate should be increased, and the reaction time should be prolonged, and finally the proportion of the triglyceride and diglyceride in the product is more than 85%, and the proportion of polyunsaturated fatty acids is also greatly improved.

[0052] Example 2 A method for preparing a polyunsaturated fatty acid-enriched triglyceride, comprising the steps of: (1) mixing a fatty acid mixture (ethylester type algal oil obtained by fermentation: EPA 4.0%, DHA 25.7%, total polyunsaturated fatty acid content 30.8%), glycerol and a lipase (Lipozyme RM IM enzyme), vacuumizing to make the pressure during reaction 1000 Pa, performing a first esterification synthesis reaction at 60°C for 4 h, stopping the reaction, adding hot water at 70°C to perform a first water washing, then performing a first degassing, and then performing a first distillation by molecular distillation at a temperature of 162°C and a pressure of 12 Pa to obtain a recovered fatty acid mixture; wherein the molar ratio of the fatty acid mixture to glycerol is 1:1; and the mass percentage of the lipase in the fatty acid mixture is 1%; The composition of the recovered fatty acid mixture was detected by a gas chromatograph to be: EPA 11.5%, DHA 66.1%, total polyunsaturated fatty acid content 78.9%, and saturated and monounsaturated fatty acid content 20.8%; (2) mixing the recovered fatty acid mixture obtained in step (1) with glycerol and a lipase (Lipozyme RM IM enzyme), vacuumizing to make the pressure during reaction 478 Pa, performing a second esterification synthesis reaction at 50°C for 24 h, stopping the reaction, adding hot water at 70°C to perform a second water washing, then performing a second degassing, and then performing a second distillation by molecular distillation at a temperature of 180°C and a pressure of 99.6 Pa to obtain a polyunsaturated fatty acid-enriched triglyceride; The molar ratio of the recovered fatty acid mixture to glycerol is 6:1; and the mass percentage of the lipase in the recovered fatty acid mixture is 10%; The composition of the polyunsaturated fatty acid-enriched triglyceride was detected by a high performance liquid chromatograph to be: triglyceride, fatty acid ethyl ester, diglyceride, monoglyceride content 79.2%, 4.3%, 10.7% and 5.6%, respectively.

[0053] The composition of the polyunsaturated fatty acid-enriched triglyceride was detected by a gas chromatograph to be: EPA 10.6%, DHA 64.1%, total polyunsaturated fatty acid content 76.8%, and saturated and monounsaturated fatty acid content 23.1%.

[0054] Example 3 A method for preparing a polyunsaturated fatty acid-enriched triglyceride, comprising the steps of: (1) mixed fatty acid mixture (linoleic acid: conjugated linoleic acid: linolenic acid = 1:2:1, polyunsaturated fatty acid accounted for 76.4%, saturated and monounsaturated fatty acid content was 23.5%), glycerol and lipase (Novozyme TL enzyme) were mixed, vacuum was extracted to make the pressure 11 Pa during the reaction, and the first esterification synthesis reaction was carried out at 30℃ for 4h, then 70℃ hot water was added for the first water washing, and then the first degassing was carried out, and then the first distillation was carried out by molecular distillation at a temperature of 121℃ and a pressure of 2.3 Pa, to obtain a recovered fatty acid mixture; The molar ratio of the fatty acid mixture to glycerol was 2.5:1, and the mass percentage of the lipase in the fatty acid mixture was 6%. The composition of the recovered fatty acid mixture was detected by a gas chromatograph, and the total polyunsaturated fatty acid content was 89.7%, and the saturated and monounsaturated fatty acid content was reduced to 10.3%. (2) the recovered fatty acid mixture obtained in step (1) was mixed with glycerol and lipase (Lipozyme RM IM enzyme), vacuum was extracted to make the pressure 12 Pa during the reaction, and the second esterification synthesis reaction was carried out at 50℃ for 72h, then 70℃ hot water was added for the second water washing, and then the second degassing was carried out, and then the second distillation was carried out by molecular distillation at a temperature of 132℃ and a pressure of 48 Pa, to obtain a polyunsaturated fatty acid-rich triglyceride; The molar ratio of the recovered fatty acid mixture to glycerol was 6:1, and the mass percentage of the lipase in the recovered fatty acid mixture was 10%. The composition of the polyunsaturated fatty acid-rich triglyceride was determined by high performance liquid chromatography, and the contents of triglyceride, fatty acid ethyl ester, diglyceride and monoglyceride were 81.3%, 5.1%, 9.4% and 4.2% respectively.

[0055] The composition of the polyunsaturated fatty acid-rich triglyceride was detected by a gas chromatograph, and the total polyunsaturated fatty acid content was 87.8%, and the saturated and monounsaturated fatty acid content was 12.1%.

[0056] Example 4 A method for preparing a polyunsaturated fatty acid-rich triglyceride, comprising the following steps: (1) mixing the fatty acid mixture (free type arachidonic acid raw material (content 60.9%, in which the ratio of polyunsaturated fatty ester is 74.5%, and the ratio of saturated fatty acid and monounsaturated fatty acid is 25.4%), glycerol and lipase (EstC24 enzyme), vacuumizing to make the pressure 10 Pa during the reaction, and then performing a first esterification synthesis reaction at 45℃ for 4 hours, stopping the reaction, then adding hot water at 70℃ to perform a first water washing, and then performing a first degassing, and then performing a first distillation by molecular distillation at a temperature of 170℃ and a pressure of 2.3 Pa, to obtain a recovered fatty acid mixture; In the above, the molar ratio of the fatty acid mixture to glycerol is 2:1; and the mass percentage of the lipase in the fatty acid mixture is 6%; The composition of the recovered fatty acid mixture is detected by a gas chromatograph, and the total polyunsaturated fatty acid content is 87.3%, and the saturated and monounsaturated fatty acid content is reduced to 12.6%. (2) mixing the recovered fatty acid mixture obtained in the step (1) with glycerol and lipase (EstC26 enzyme), vacuumizing to make the pressure 12 Pa during the reaction, and then performing a second esterification synthesis reaction at 50℃ for 36 hours, stopping the reaction, then adding hot water at 70℃ to perform a second water washing, and then performing a second degassing, and then performing a second distillation by molecular distillation at a temperature of 156℃ and a pressure of 18 Pa, to obtain a polyunsaturated fatty acid-rich triglyceride; The molar ratio of the recovered fatty acid mixture to glycerol is 4.5:1; and the mass percentage of the lipase in the recovered fatty acid mixture is 3%; The composition of the polyunsaturated fatty acid-rich triglyceride is determined by a high performance liquid chromatograph, and the contents of triglyceride, fatty acid ethyl ester, diglyceride and monoglyceride are 80.6%, 2.4%, 10.3% and 6.7% respectively.

[0057] The composition of the polyunsaturated fatty acid-rich triglyceride is detected by a gas chromatograph, and the total polyunsaturated fatty acid content is 85.3%, and the saturated and monounsaturated fatty acid content is 14.7%.

[0058] From the above results, it can be seen that the preparation method of the present application can significantly improve the content of polyunsaturated fatty acid in the triglyceride, and the reaction conditions are mild, and no food-unfriendly reagent is used. This is because, in the present application, the conditions of the first esterification synthesis reaction are controlled to make as many high-saturation fatty acids as possible to combine with glycerol, thereby removing most of the high-saturation fatty acids, increasing the proportion of polyunsaturated fatty acid in the recovered fatty acid mixture, and then performing a second esterification synthesis reaction on the recovered fatty acid mixture with a higher proportion of polyunsaturated fatty acid and glycerol under the catalysis of lipase, and finally removing the unreacted glycerol and fatty acid, to obtain a polyunsaturated fatty acid-rich triglyceride.

[0059] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for preparing triglycerides rich in polyunsaturated fatty acids, characterized in that, Includes the following steps: (1) The fatty acid mixture, glycerol and lipase are mixed and subjected to an esterification synthesis reaction under vacuum. Then, the mixture is washed with water, degassed and distilled in sequence to obtain the recovered fatty acid mixture. The molar ratio of the fatty acid mixture to glycerol is 1~2.5:

1. (2) The recovered fatty acid mixture obtained in step (1) is mixed with glycerol and lipase, and a secondary esterification synthesis reaction is carried out under vacuum. Then, the mixture is subjected to secondary water washing, secondary degassing and secondary distillation to obtain triglycerides rich in polyunsaturated fatty acids. The molar ratio of the recovered fatty acid mixture to glycerol is 3~6:

1.

2. The preparation method according to claim 1, characterized in that, The fatty acid mixture in step (1) includes ethyl ester fatty acids and / or free fatty acids.

3. The preparation method according to claim 1, characterized in that, The lipases in steps (1) and (2) independently include Novozyme 435 enzyme, Novozyme TL enzyme, Lipozyme RM IM enzyme, Lipozyme TL IM enzyme, or EstC24-EstC27 enzyme.

4. The preparation method according to claim 1, characterized in that, In step (1), the lipase accounts for 1-6% of the mass percentage of the fatty acid mixture.

5. The preparation method according to claim 1, characterized in that, The temperatures of the primary esterification synthesis reaction in step (1) and the secondary esterification synthesis reaction in step (2) are independently 30~60℃.

6. The preparation method according to claim 1, characterized in that, The time for one esterification synthesis reaction in step (1) is 4~24h.

7. The preparation method according to claim 1 or 5, characterized in that, The time for the secondary esterification synthesis reaction in step (2) is 24~72h.

8. The preparation method according to claim 1, characterized in that, In step (2), the lipase accounts for 3-10% of the mass percentage of the recovered fatty acid mixture.

9. The preparation method according to claim 1, characterized in that, The temperature of the first distillation in step (1) and the temperature of the second distillation in step (2) are independently 120~180℃.

10. The preparation method according to claim 1 or 9, characterized in that, The pressure of the first distillation in step (1) and the pressure of the second distillation in step (2) are independently 2~100 Pa.