High-purity sn-2 eicosapentaenoic acid monoglyceride grease and preparation method thereof

Abstract

The invention discloses grease of high-purity sn-2 eicosapentaenoic acid monoglyceride and a preparation method of the grease. The preparation method comprises the following steps: S1, carrying out alcoholysis reaction on triEPA triglyceride; s2, a product obtained after alcoholysis is subjected to liquid-liquid extraction through a solvent extraction method, a lower-layer substance is separated, sn-2-EPA-MAG is obtained, and an upper-layer substance obtained through separation comprises EPA ethyl ester; and S3, performing ester exchange treatment on an upper-layer substance obtained after liquid-liquid extraction and glyceride to generate triEPA triglyceride, circularly repeating the steps S1 to S3, and collecting sn-2-EPA-MAG obtained after liquid-liquid extraction in the step S2 in each circulation process. A large amount of EPA ethyl ester products are generated in the alcoholysis head product, the yield of 2-MAG is reduced, the EPA ethyl ester products separated after liquid-liquid extraction are reused for exchange with glyceride to generate triEPA triglyceride, the EPA raw material is recycled, and the recycling rate of EPA in sn-2MAG is increased to 70.64% by three times of cyclic enzymolysis.

Description

Technical Field

[0001] The invention belongs to the field of oil technology and nutrition technology, and specifically relates to a high-purity sn-2 eicosapentaenoic acid monoglyceride oil and a preparation method thereof. Background Art

[0002] Triglycerides (= triacylglycerol) (TAG) are glycerides formed by the esterification of three hydroxyl groups of glycerol with three fatty acid molecules. Hypertriglyceridemia is defined as a fasting serum triglyceride level > 200 mg / dL (2.3 mmol / L). Hypertriglyceridemia (HTG) is a common type of dyslipidemia. According to the China Adult Nutrition and Chronic Disease Monitoring Report 2015, the prevalence of HTG in the Chinese population is as high as 15%, and appropriate drugs are needed to reduce the occurrence and development of hypertriglyceridemia in my country.

[0003] Statins are the most popular cardiovascular drugs in the international market. The global sales of atorvastatin alone exceeds 10 billion US dollars. However, it is reported that 98% of patients experience muscle pain after taking statins. In addition, patients who take statins for 2 years or more have a 4-14 times higher risk of polyneuropathy than the control group. Therefore, other drugs need to be used in combination with statins to reduce the dosage of statins and reduce their side effects.

[0004] It is reported that fibrates, niacin and mixed omega-3 fatty acid drugs were forced to withdraw from clinical trials after entering phase I clinical trials due to insignificant effects on lowering triglycerides. Eicosapentaenoic acid (EPA) ethyl ester is the only drug approved by the FDA (U.S. Food and Drug Administration), EMA (European Medicines Agency), PMDA (Japan Medical Device Administration) and my country for use in combination with statins to lower triglycerides through phase III clinical trials. FDA, EMA, PMDA and NMPA (China National Medical Products Administration) have approved 14, 1, 44 and two EPA ethyl ester drugs, respectively. However, the dosage of EPA ethyl ester approved by the above agencies is as high as 4g / d, which brings many side effects, such as nausea, bloating, diarrhea and other digestive discomfort, bleeding, etc., especially for patients who are taking anticoagulants, who are prone to new atrial fibrillation after taking it.

[0005] Monoglyceride (MAG) has three isomers, namely the positional isomer sn-2-MAG and two enantiomers sn-1-MAG and sn-3-MAG. The digestion, absorption and metabolic mechanisms of the positional isomer monoglyceride (sn-2MAG) and EPA in the form of ethyl ester (EE) are different, including: in terms of digestion, because the fatty acids in 2-MAG are attached to the sn-2 bond of the glycerol backbone, the fatty acids in 2-MAG are absorbed without pancreatic lipase digestion, while the rate of hydrolysis of the ester bond in EE is 10 to 50 times lower than that of glycerol esters; in terms of absorption, 2-MAG is an amphiphilic molecular structure, and its high micellar solubility in the intestinal cavity can enhance the absorption of small intestinal epithelial cells; in terms of metabolism, the fatty acids carried by 2-MAG have better tissue binding ability, while only about 1 / 3 of the amount of EE after hydrolysis and re-esterification is bound to the tissue to play a role. The study of Chevalier and Plourde showed that when the same plasma concentration level is achieved, the amount of EPA in the sn-2 form may only require 1 / 3 of the amount in the EE form. Therefore, 2-MAG is expected to replace EPA ethyl ester as a potential drug for reducing EPA dosage.

[0006] High-purity EPA ethyl ester can be obtained by ethyl esterification of fish oil and further refining and purification, with low production cost. Therefore, the form of EPA approved by many institutions is ethyl ester. However, the preparation and separation of sn-2-MAG is difficult, which seriously hinders further marketization. It is more meaningful to find a convenient and efficient method for preparing sn-2-MAG. However, existing studies have focused more on mixed fatty acid mixed MAGs, and have not paid more attention to sn-2EPA MAG. In addition, due to the structural similarity between sn-1-MAG and sn-2-MAG and the easy acyl transfer of sn-2-MAG, it is difficult to separate sn-1 and sn-2 MAGs, and the recovery rate of EPA in the prepared sn-2MAG is low, and the purity of EPA is low.

[0007] A patent (application number 201510435808.7) discloses a method for synthesizing sn-2-monoglyceride by an enzymatic method. However, the sn-2-monoglyceride obtained by this method contains a large amount of ethyl ester by-product and the purity of the target fatty acid is low.

[0008] A patent (application number 201510643768.5) discloses a high-purity α-linolenic acid monoglyceride and a preparation method thereof, but the method involves complex molecular distillation, which is costly, and the purity of α-linolenic acid is low, ≥40%.

[0009] Therefore, finding a convenient and efficient method for preparing sn-2-MAG has great research significance and market prospects. Summary of the invention

[0010] Purpose of the invention: In view of the above-mentioned deficiencies in the prior art, the present invention provides an enzymatic hydrolysis preparation technology for 2-EPA-MAG with high purity and high recovery rate, so as to solve the problem of low bioavailability of EPA ethyl ester drugs for patients with hypertriglyceridemia. In view of the problems of less sn-2EPA MAG products, low purity, low recovery rate of fatty acids in preparation technology and high cost, a method for efficiently preparing high-purity 2-EPA-MAG is developed, so as to provide a theoretical basis for the optimized design of fish oil health foods or medicines for patients with hypertriglyceridemia.

[0011] It should be noted that, in the present invention, various expressions such as 2-EPA-MAG, sn-2EPA MAG and 2-MAG-EPA all refer to sn-2 eicosapentaenoic acid monoglyceride.

[0012] To achieve the above object, the present invention adopts the following technical solution:

[0013] The first object of the present invention is to provide a method for preparing a high-purity sn-2 eicosapentaenoic acid monoglyceride grease, comprising the following steps:

[0014] S1, alcoholysis reaction of tri-EPA triglyceride;

[0015] S2. The product after alcoholysis is subjected to liquid-liquid extraction by solvent extraction, and the lower layer is separated to obtain sn-2-EPA-MAG, and the upper layer obtained by separation contains EPA ethyl ester;

[0016] S3. The upper layer after liquid-liquid extraction is subjected to transesterification with glyceride to generate tri-EPA triglyceride, and steps S1-S3 are repeated in a recycling process. During each cycle, the sn-2-EPA-MAG obtained after liquid-liquid extraction in step S2 is collected.

[0017] Optionally, in one embodiment of the present invention, in step S1, the purity of the tri-EPA triglyceride is not less than 80%.

[0018] Optionally, in one embodiment of the present invention, in step S1, the alcoholysis reaction method includes: adding ethanol and lipase to triEPA-triglyceride in an enzyme reactor, stirring at 30°C-40°C and 100-800r / min for 2-12h, removing the lipase in the system by suction filtration, and removing the solvent in the reaction system by rotary evaporation at 40-65°C.

[0019] Further optionally, in one embodiment of the present invention, the molar ratio of ethanol to tri-EPA-triglyceride is 1:15-1:120.

[0020] Further optionally, in one embodiment of the present invention, the molar ratio of ethanol to tri-EPA-triglyceride is 1:30-1:120. Still further optionally, in one embodiment of the present invention, the molar ratio of ethanol to tri-EPA-triglyceride is 1:60-1:120. More preferably, in one embodiment of the present invention, the molar ratio of ethanol to tri-EPA-triglyceride is 1:60.

[0021] Further optionally, in one embodiment of the present invention, in step S1, the mass proportion of lipase in the substrate tri-EPA triglyceride is 6-14% (w / w). Further optionally, in one embodiment of the present invention, in step S1, the mass proportion of lipase in the substrate tri-EPA triglyceride is 10-12% (w / w).

[0022] Further optionally, in one embodiment of the present invention, in step S1, the lipase is at least one of Lipozyme 435 lipase, CL "Amano" IM, Lipozyme TL IM and NS40086. Further optionally, in one embodiment of the present invention, in step S1, the lipase is at least one of Lipozyme 435 lipase, CL "Amano" IM and Lipozyme TL IM. Further optionally, in one embodiment of the present invention, in step S1, the lipase is at least one of Lipozyme 435 lipase and CL "Amano" IM. More preferably, in one embodiment of the present invention, the lipase is Lipozyme 435 lipase.

[0023] Further optionally, in one embodiment of the present invention, in step S1, the reaction is carried out at 35°C-40°C with stirring at 300-600 r / min for 3-8 hours. Preferably, in one embodiment of the present invention, in step S1, the reaction is carried out at 35°C with stirring at 300-600 r / min for 4-5 hours.

[0024] Further optionally, in one embodiment of the present invention, in step S1, the solvent in the reaction system is removed by rotary evaporation at 45-60°C.

[0025] Optionally, in one embodiment of the present invention, in step S2, the liquid-liquid extraction method includes: extracting and separating using n-hexane and 85% ethanol-water solution.

[0026] Further optionally, in one embodiment of the present invention, the ratio of n-hexane, 85% ethanol-water solution and alcoholysis product is independently 1:10 w / w, and the alcoholysis product refers to the product obtained by removing the solvent from the alcoholysis product.

[0027] Optionally, in one embodiment of the present invention, in step S3, the ester exchange treatment specifically includes: placing the upper layer material containing EPA ethyl ester in an enzyme reactor, adding glycerol and lipase, stirring and reacting at 30-140°C and 100-800r / min under vacuum conditions for 2-14h, filtering to remove the lipase in the system, and taking the supernatant to obtain tri-EPA triglyceride.

[0028] Further optionally, in one embodiment of the present invention, in step S3, the molar ratio of the upper layer material containing EPA ethyl ester (also referred to as substrate oil in the present invention) to glycerol is 3: 1-15: 1. Further optionally, in one embodiment of the present invention, in step S3, the molar ratio of the upper layer material containing EPA ethyl ester (substrate oil) to glycerol is 9: 1-12: 1.

[0029] Further optionally, in one embodiment of the present invention, in step S3, the lipase is selected from at least one of LipozymeTL IM, Lipozyme 435, CL "Amano" IM, DF "Amano" IM, CALA and CALB. Further optionally, in one embodiment of the present invention, in step S3, the lipase is selected from CL "Amano" IM.

[0030] Further optionally, in one embodiment of the present invention, in step S3, the mass proportion of the lipase in the upper layer material (i.e., substrate oil) is 4-20% (w / w). Further optionally, in one embodiment of the present invention, in step S3, the mass proportion of the lipase in the upper layer material (i.e., substrate oil) is 16% (w / w). Further optionally, in one embodiment of the present invention, in step S3, the reaction is stirred at 100-800r / min at 30-140°C under vacuum conditions for 2-14h. Further optionally, in one embodiment of the present invention, in step S3, the reaction is stirred at 100-800r / min at 90°C-110°C under vacuum conditions for 5-9h. Optionally, in one embodiment of the present invention, in step S3, the number of cycles is not less than 3 times.

[0031] Further optionally, in one embodiment of the present invention, the number of cycles is 3-10 times.

[0032] The second object of the present invention is to provide a high-purity sn-2EPA MAG oil, wherein the high-purity 2-EPA MAG refers to an oil in which EPA is located at the sn-2 position of glycerol, the purity is above 70%, and the sn-2 MAG content is above 70%. The efficient preparation of sn-2EPA MAG refers to a preparation method in which the yield of 2-EPA MAG is as high as above 70% and the purity of EPA is above 70%.

[0033] The key to the present invention is to further transesterify the EPA ethyl ester product after the alcoholysis of the first step tri-EPA triglyceride to obtain high-yield and high-purity tri-EPA triglyceride, and then conduct a new round of alcoholysis and transesterification. The conventional transesterification method for preparing 2-MAG generally optimizes the reaction parameters to obtain the highest 2-MAG content. The present invention obtains the highest triglyceride content by optimizing the reaction parameters of the transesterification reaction, and then synthesizes 2-EPA MAG through triglycerides. The difference is that we have adopted a different reaction route. Compared with the direct synthesis of 2-MAG through transesterification, the present invention is more efficient. The content of 2-MAG in the traditional method is extremely low (about 5%), and the recovery rate of EPA in 2-MAG is extremely low (about 5%). The present invention significantly improves the content of 2-MAG (92.19%) and the recovery rate of EPA in 2-MAG (72.08%), and achieves high recovery rate, high purity and low cost for the preparation of sn-2EPA MAG.

[0034] Beneficial effects: The present invention has the following advantages: Compared with existing oils and fats, the sn-2EPA MAG provided by the present invention has a higher EPA purity, can improve the absorption effect of EPA, and enhance the lipid-lowering effect of EPA, and improve bioavailability. The sn-2EPA MAG prepared by the sn-2EPA MAG preparation technology provided by the present invention has a high yield, high EPA purity, low cost, and simple operation, which can avoid nausea, vomiting and other side effects in patients with hypertriglyceridemia due to taking high-dose EPA ethyl ester drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Process flow chart of the preparation method of the present invention.

[0036] Figure 2 The cyclic alcoholysis-ester exchange reaction equation of the present invention.

[0037] Figure 3 Experimental technical roadmap of the embodiments of the present invention.

[0038] Figure 4 Effect of different alcoholysis reaction conditions on the lipid composition of the products.

[0039] Figure 5 Effect of different alcoholysis reaction conditions on the fatty acid composition of the products.

[0040] Figure 6 Effects of different lipase types on triglyceride synthesis via transesterification.

[0041] Figure 7 Effects of different reaction times on triglyceride synthesis by lipase transesterification.

[0042] Figure 8 Effects of lipase addition, temperature and glycerol substrate molar ratio on triglyceride synthesis in transesterification reaction.

[0043] Fig. 9 Fatty acid levels in plasma of rats in different treatment groups.

[0044] Fig.10 EPA pharmacokinetic curves in rats of different treatment groups. DETAILED DESCRIPTION

[0045] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0046] For the purpose of this specification and the appended claims, unless otherwise stated, all numbers expressing amounts, percentages or ratios and other numerical values ​​used in this specification and the appended claims are understood to be modified in all cases by the term "about". In addition, all ranges disclosed herein are inclusive and independently combinable.

[0047] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0048] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0049] The yield of sn-2-EPA-MAG prepared by the prior art is low, and the EPA purity of sn-2-EPA-MAG is low. In order to solve the problem of low EPA purity, the present invention, on the one hand, examines the raw materials on the market and selects tri-EPA triglyceride with higher purity and lower cost, and on the other hand, optimizes parameters such as enzyme ratio, ethanol molar amount, reaction time, reaction temperature, etc., to increase the EPA purity to 94%. In order to solve the problem of low sn-2-EPA-MAG yield, the present invention, on the one hand, adopts the combined enzymatic hydrolysis technology of triglyceride alcoholysis and ethyl glyceride ester exchange, and on the other hand, the triglyceride obtained by ester exchange is re-subjected to alcoholysis and ester exchange for cyclic enzymatic hydrolysis, and the cyclic enzymatic hydrolysis three times increases the EPA recovery rate in sn-2MAG to 72.08%. The research technical route of the present invention is as follows: Figure 3 As shown, the specific steps include:

[0050] In the first step, the tri-EPA triglyceride raw material is subjected to alcoholysis

[0051] The preparation technology of high-purity tri-EPA triglyceride is mature and low-cost, so this study selected tri-EPA triglyceride raw materials with EPA purity of 80-100% for different alcoholysis reactions.

[0052] In some examples, the specific method for alcoholysis of tri-EPA triglyceride raw material includes: weighing a certain mass of high-purity tri-EPA-triglyceride into an enzyme reactor, adding ethanol and lipase, wherein the molar ratio of ethanol / oil (ethanol / tri-EPA-triglyceride) is 1:15-1:120, and the mass ratio of enzyme to substrate oil is 3-20% (w / w) of lipase, reacting at 35°C and stirring at 100-800r / min for 2-12h, removing the lipase in the system by suction, removing the solvent in the reaction system by rotary evaporation at 40-65°C, determining the content of sn-2MAG in the initial product by a high performance liquid chromatography differential refractometer, and determining the EPA content in the product by gas chromatography.

[0053] The second step is liquid-liquid extraction by solvent extraction

[0054] A large amount of EPA ethyl ester products are generated in the initial alcoholysis product, which reduces the yield of 2-MAG. Therefore, the present invention reuses the EPA ethyl ester product separated after liquid-liquid extraction to exchange with glycerol ester to generate tri-EPA triglyceride, which will recycle the EPA raw material.

[0055] In some examples, the specific method of performing liquid-liquid extraction by solvent extraction includes: extracting and separating using n-hexane and 85% ethanol-water solution.

[0056] In the third step, EPA ethyl ester undergoes transesterification to generate tri-EPA triglyceride

[0057] The present invention reuses the EPA ethyl ester product separated after liquid-liquid extraction and exchanges it with glycerol to generate tri-EPA triglyceride, so that the EPA raw material can be recycled to increase the yield of 2-MAG.

[0058] In some examples, the specific method of ester exchange of EPA ethyl ester to produce tri-EPA triglyceride includes: placing a certain mass of EPA ethyl ester in an enzyme reactor, adding glycerol with a molar ratio of EPA ethyl ester / glycerol of 1:1-1:30, and lipase with an enzyme ratio of 4-20%, reacting at 30-140°C and stirring at 100-800r / min for 2-14h under vacuum conditions, filtering out the lipase in the system, taking the supernatant to obtain a high-yield tri-EPA triglyceride, and reusing the tri-EPA triglyceride to synthesize sn-2EPA MAG for cyclic enzymatic hydrolysis.

[0059] The present invention is described in more detail below using specific examples, and the present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0060] Reaction material information: High-purity tri-EPA triglyceride and commercially available fish oil were purchased from the OmegaVia flagship store.

[0061] Example 1 Preparation of 2-EPA MAG by Enzymatic Alcoholysis

[0062] The preparation technology of high-purity tri-EPA triglyceride is mature and low in cost. In this study, tri-EPA triglyceride raw material with EPA purity of 94.73% was selected for different alcoholysis reactions, and the specific component test results are shown in Table 1 below.

[0063] Table 1 Fatty acid and lipid composition of tri-EPA triglycerides (%) w / w

[0064]

[0065] It can be seen from Table 1 that TAG accounts for 95.23% w / w, EPA accounts for 94.73% w / w, and tri-EPA triglyceride accounts for 79.7% w / w in the tri-EPA triglyceride raw material.

[0066] In the embodiment of the present invention, the reaction equation for synthesizing 2-MAG by alcoholysis reaction is as follows:

[0067]

[0068] Specific method: weigh a certain mass of high-purity tri-EPA-triglyceride into an enzyme reactor, add ethanol with an ethanol / oil molar ratio of 1:15-1:120 and lipase with an enzyme ratio of 3-20%, react at 35°C and 300-600r / min with stirring for 2-10h, remove the lipase in the system by suction filtration, remove the solvent in the reaction system by rotary evaporation at 45°C, and determine the content of 2-MAG in the initial product by HPLC-RID.

[0069] Using the above method, the reaction parameters such as lipase type, reaction substrate mole, reaction time, reaction temperature and enzyme ratio were optimized through parameter setting.

[0070] (1) Effect of lipase type on enzymatic alcoholysis preparation of 2-MAG Figure 4 A)

[0071] Seven common immobilized lipases, NS40086, Lipozyme 435, Lipozyme RM, LipozymeTL IM, CL "Amano" IM and DF "Amano" IM, were selected to catalyze alcoholysis to prepare 2-MAG. The reaction conditions were: ethanol and oil (here refers to tri-EPA-triglyceride, the same below) molar ratio of 60:1, reaction time 5h, enzyme addition amount 12% (total substrate mass, w / w), reaction temperature 35°C, stirring at 600r / min for 5h.

[0072] from Figure 4 As can be seen in A, the content of 2-MAG in the product of Lipozyme 435 lipase is the highest, so Lipozyme 435 lipase was selected for further optimization. The next ones are CL "Amano" IM, TL "Amano" IM and NS40086. This shows that the type of lipase has a significant effect on the preparation of 2-MAG by enzymatic alcoholysis.

[0073] (2) Effect of substrate molar ratio on the preparation of 2-MAG by enzymatic alcoholysis ( Figure 4 E)

[0074] The molar ratio of ethanol to oil was selected as 7.5:1, 15:1, 30:1, 60:1 and 120:1. The reaction conditions were: 12% addition of lipase Lipozyme 435, 35°C reaction temperature, stirring at 600 r / min, and reaction time of 5 h.

[0075] from Figure 4It can be seen from E that when the molar ratio of ethanol to oil is 60:1, the content of 2-MAG in the alcoholysis product is the highest, so the molar ratio of ethanol to oil is 60:1 as the optimal molar ratio of ethanol to oil. The next ones are 120:1 and 30:1. This shows that the molar ratio of the reaction substrate has a significant effect on the preparation of 2-MAG by enzymatic alcoholysis.

[0076] (3) Effect of reaction time on the preparation of 2-MAG by enzymatic alcoholysis ( Figure 4 D)

[0077] The product within 0 to 12 hours of the reaction process was taken to determine the 2-MAG content. Other reaction conditions were: ethanol to oil molar ratio 60:1, reaction temperature 35°C, lipase Lipozyme 435 addition amount 12% (total substrate mass), stirring at 600 r / min.

[0078] from Figure 4 As can be seen from D, 2-MAG reaches the highest content at 4h and 5h, and the content of 2-MAG gradually decreases after 6h. In order to save time, 4h is selected as the optimal reaction time. This shows that the reaction time has a significant effect on the preparation of 2-MAG by enzymatic alcoholysis.

[0079] (4) Effect of reaction temperature on the preparation of 2-MAG by enzymatic alcoholysis ( Figure 4 B)

[0080] The reaction temperatures were selected as 25°C, 30°C, 35°C, 40°C, and 45°C. Other reaction conditions were: stirring at 600 r / min, reaction time 4 h, oil to ethanol molar ratio 60:1, and lipase Lipozyme 435 addition amount 12% (accounting for the total mass of the substrate).

[0081] from Figure 4 It can be seen from B that when the reaction temperature rises to 35°C, the content of 2-MAG is the highest. When the temperature continues to rise to 40°C, there is no significant difference in the content of 2-MAG. Therefore, 35°C is selected as the optimal reaction condition. The next best reaction conditions are 40°C and 30°C. This indicates that the reaction temperature has a significant effect on the preparation of 2-MAG by enzymatic alcoholysis.

[0082] (5) Effect of enzyme dosage on the preparation of 2-MAG by enzymatic alcoholysis ( Figure 4 C)

[0083] The enzyme dosage of Lipozyme 435 was selected as 6%, 8%, 10%, 12%, and 14% (w / w). Other reaction conditions were: reaction time 4h, ethanol to oil molar ratio 60:1, reaction temperature 35°C, stirring at 600r / min.

[0084] from Figure 4As can be seen from C, with the increase of enzyme addition, there is no significant difference in 2-MAG in the 6%-10% group product, and the 12% (w / w) enzyme addition significantly increases the 2-MAG content compared with other groups. Therefore, the 12% (w / w) enzyme addition is selected as the optimal enzyme amount. The second is 14% (w / w). This shows that the amount of enzyme added has a significant effect on the preparation of 2-MAG by enzymatic alcoholysis.

[0085] Example 2: Separation and purification of 2-MAG by solvent extraction

[0086] After the alcoholysis reaction of Example 1 is completed, the reaction system contains 2-MAG, 1-MAG, 1,2-DAG, 1,3-DAG, EE and a small amount of unreacted TAG. 2-MAG is separated and purified by solvent extraction. According to the difference in polarity between the target product 2-MAG and impurities, 2-MAG is extracted and separated using n-hexane and 85% ethanol-water solution.

[0087] Specific method: First, add 10 mL of 85% ethanol-water solution and 10 mL of n-hexane to 1 g of the product after the solvent is removed, and then transfer to a separatory funnel for shaking and separation.

[0088] The mixed solution was divided into two layers: the upper layer was the n-hexane phase, containing TAG, EE and DAG with weaker polarity; the lower layer was the 85% ethanol-water phase, containing MAG with stronger polarity. The lower layer containing MAG was collected and washed twice with n-hexane. The solvent was removed by rotary evaporation at 40°C and stored at -20°C for later use.

[0089] Based on the optimization results of Example 1 and Example 2, the optimal alcoholysis reaction conditions of the present invention are selected as follows: 435 enzyme, 12% (w / w) enzyme, 60:1 ethanol / oil ratio, 35°C temperature, and 4h reaction time. Figure 4 ), the TAG conversion rate reached 96.35%, the product 2-MAG content reached 26.60%, and the EPA content in the 2-MAG of the reaction product was higher than 90% ( Figure 5 ).

[0090] Example 3 Enzymatic esterification of glycerol with EPA-EE

[0091] A large amount of EPA ethyl ester products are generated in the initial alcoholysis product, which reduces the yield of 2-MAG. The EPA ethyl ester product separated after liquid-liquid extraction is reused to exchange with glycerol to generate tri-EPA triglyceride, which will recycle the EPA raw material. The reaction equation is shown below:

[0092]

[0093] In an embodiment of the present invention, a specific method for exchanging an EPA ethyl ester product (i.e., an upper layer containing EPA ethyl ester, also referred to as substrate oil) with glycerol to generate tri-EPA triglyceride includes: placing a certain mass of EPA ethyl ester product in an enzyme reactor, adding glycerol with a glycerol / EPA ethyl ester product molar ratio of 1:1-1:30, and lipase with an enzyme ratio of 4-20% (w / w), reacting at 30-140°C and stirring at 100-800 r / min for 2-14 hours, removing the lipase in the system by suction filtration, taking the supernatant to obtain a high-yield tri-EPA triglyceride, and reusing the tri-EPA triglyceride to synthesize sn-2EPA MAG for cyclic enzymatic hydrolysis.

[0094] The above method was used to further synthesize triglycerides by esterification reaction based on the extraction and removal of monoglycerides from the alcoholysis reaction products, and 2-EPA MAG was prepared by cyclic enzymatic hydrolysis. The reaction parameters such as lipase type, reaction time, enzyme ratio, reaction temperature and glycerol substrate oil molar ratio were optimized by setting the following parameters.

[0095] (1) Effect of lipase type on the preparation of tri-EPA triglyceride Figure 6 )

[0096] Fourteen common immobilized lipases: DSM A1, CL "Amano", longkete, TL100L, Lipozyme CALA, KDN, Lipozyme CALB, Palatase 20000L, Lipozyme TL IM, Lipozyme RM, DF "Amano" IM, Lipozyme 435, CL "Amano" IM, Lipura flex and glycerol with an ethyl ester / glycerol molar ratio of 12:1 were selected in an enzyme reactor and reacted at 30-110°C under vacuum conditions for 2-13 hours.

[0097] Taking into account the cost and TAG yield, we initially screened out Lipozyme TL IM, Lipozyme 435, CL "Amano" IM, DF "Amano" IM with a TAG ratio greater than 10%, and CALA (2 yuan / g) and CALB (5 yuan / g) with lower costs to further optimize the reaction conditions ( Figure 6 ). This indicates that the type of lipase has a significant effect on the preparation of tri-EPA triglyceride.

[0098] (2) Effect of reaction time of different lipases on the preparation of tri-EPA triglyceride ( Figure 7 )

[0099] The product of the reaction within 0-13h was taken to determine the TAG content therein. The other reaction conditions were: ethyl ester / glycerol molar ratio of 1:9, and the lipases were the six types screened above: CL "Amano" IM, DF "Amano" IM, Lipozyme435, Lipozyme TL IM, Lipozyme CALB and Lipozyme CALA.

[0100] CL "Amano" IM has the highest TAG conversion rate among all enzymes (greater than 90%) ( Figure 7 ), and 95.4% EE conversion rate was achieved in 5h, so CL "Amano" IM enzyme reaction was selected for 5h to optimize the conditions of temperature, substrate oil / glycerol molar ratio, and enzyme addition amount. This shows that the reaction time of different lipases has a significant effect on the preparation of tri-EPA triglyceride.

[0101] (3) Effect of enzyme dosage on the preparation of tri-EPA triglyceride ( Figure 8 A)

[0102] The addition amount of lipase CL "Amano" IM was selected to be 4-20% (w / w), and the other reaction conditions were: reaction time 5h, molar ratio of substrate oil to glycerol 1:9, and reaction temperature 70°C.

[0103] from Figure 8 As can be seen from A, the triglyceride content is the highest when the enzyme addition amount is 16% (w / w), and 16% (w / w) is selected as the optimal enzyme addition amount, indicating that the enzyme addition amount has a significant effect on the preparation of tri-EPA triglyceride.

[0104] (4) Effect of reaction temperature on the preparation of tri-EPA triglyceride Figure 8 C)

[0105] The reaction temperature was selected to be 30-110° C. Other reaction conditions were: reaction time 5 h, substrate oil glycerol molar ratio 1:9, lipase CL “Amano” IM addition amount 16% (w / w).

[0106] from Figure 8 As can be seen from Figure C, the content of triglycerides in the product is the highest at 90°C, so 90°C is selected as the optimal reaction temperature. This shows that the amount of enzyme added has a significant effect on the preparation of tri-EPA triglycerides.

[0107] (5) Effect of glycerol substrate molar ratio on the preparation of tri-EPA triglyceride ( Figure 8 B)

[0108] The ethyl ester / glycerol substrate molar ratio was selected to be 3:1-15:1, and other reaction conditions were as follows: reaction time 5 h, and lipase CL "Amano" IM addition amount 16%.

[0109] from Figure 8 It can be seen from B that when the substrate oil / glycerol molar ratio is 12:1, the triglyceride content is the highest. As the substrate oil / glycerol molar ratio continues to increase, the triglyceride content gradually decreases. Therefore, 12:1 is selected as the optimal substrate oil / glycerol molar ratio.

[0110] 5. Calculation of conversion rate of EPA ethyl ester in transesterification reaction

[0111]

[0112] Based on the optimization results of Example 1, Example 2 and Example 3, the present invention selects CL "Amano" IM enzyme, reaction time 5h, enzyme ratio 16%, 90°C, substrate oil / glycerol molar ratio 12:1 as the optimal transesterification reaction conditions ( Figure 7 , Figure 8 ), the reaction product TAG reached 88.74%, EE conversion rate was 97.5%, and EPA content was 94.42% (Table 2).

[0113] Table 2 Lipid and fatty acid composition of triglyceride products before and after synthesis (% w / w)

[0114]

[0115]

[0116] Example 4 Cyclic alcoholysis and transesterification reaction (Table 3)

[0117] The tri-EPA triglyceride obtained by ester exchange is subjected to the above alcoholysis reaction again. After the alcoholysis product is subjected to rotary evaporation to remove the solvent and liquid-liquid extraction, the yield of 2-EPA MAG in the entire reaction is calculated. The obtained EPA ethyl ester is subjected to a secondary ester exchange reaction to generate tri-EPA triglyceride. The reaction equation is as follows: cyclic alcoholysis-ester exchange reaction:

[0118]

[0119] Test Example 1 Recovery rate of 2-MAG after alcoholysis reaction

[0120] The recovery rate of 2-MAG after each alcoholysis reaction was calculated. If the yield was lower than , the transesterification reaction of triglycerides and glycerol was continued until the yield of 2-EPA MAG reached more than 70%.

[0121] The calculation of EPA recovery rate in 2-MAG during alcoholysis reaction was carried out by the following method:

[0122]

[0123] The calculation method for the recovery rate of 2-EPA MAG by cyclic alcoholysis transesterification is:

[0124] Calculation of the recovery rate of EPA in 2-MAG after n alcoholysis:

[0125]

[0126] The TAG obtained from the ester exchange was recycled for alcoholysis to prepare 2-EPA-MAG. After three cycles, the EPA recovery rate in 2-MAG reached 70.64% (Table 3).

[0127] Table 3 Recovery rate of 2-MAG at different cycle times

[0128]

[0129]

[0130] Test Example 2 Recycling of lipase

[0131] Under the optimal conditions of alcoholysis, the recyclability of Lipozyme 435 and Lipozyme TL IM was investigated using the content of 2-MAG in the product as an indicator. After the reaction, the immobilized lipase was collected by vacuum filtration and washed twice with anhydrous ethanol and n-hexane. After removing the solvent, the lipase was placed in a 40°C oven for 10 hours and then taken out for the next round of reaction to evaluate the stability of lipase recycling.

[0132] Lipozyme 435 and CL "Amano" IM lipase can be recycled at least 10 times ( Figure 8 D. Figure 8 E).

[0133] Test Example 3: Oil and fat composition detection and analysis

[0134] (1) Gas chromatography analysis of fatty acid composition ( Figure 5 )

[0135] Fatty acid methyl esterification: refer to AOCS standard method 996.01, and convert fatty acids into corresponding methyl esters for gas phase analysis. Take about 50 mg of free fatty acids and mix them with 2 mL of 14% boron trifluoride-methanol solution, and heat them in a constant temperature water bath at 70°C for 3 minutes until they are completely derivatized. After cooling, extract the fatty acid methyl esters with 2 mL of chromatographically pure n-hexane, and add 2 mL of saturated sodium chloride solution to accelerate stratification. Collect the n-hexane phase, add anhydrous sodium sulfate, centrifuge at 10,000 r / min for 5 minutes, and pass through an organic membrane for analysis.

[0136] Gas chromatography conditions: The detection of fatty acid methyl esters uses a hydrogen flame ionization detector (FID), and the chromatographic column is an Agilent DB-Fast FAME capillary column (60m×0.25mm×2.5μm). The gas chromatography column temperature program is 80℃ for 0.5min, then heated to 165℃ at a rate of 40℃ / min and maintained for 1min, and then heated to 230℃ at 2℃ / min and maintained for 2min. The carrier gas is nitrogen with a flow rate of 1mL / min. The split ratio is 20:1. The column temperature and the injection port temperature are both 250℃. Each fatty acid is qualitatively identified by comparing the retention time of the standard, and the relative composition is calculated by the area normalization method.

[0137] (2) High performance liquid chromatography analysis of lipid composition

[0138] HPLC-RID was used to quantitatively analyze the lipid components in the reaction system. The chromatographic conditions were as follows: chromatographic column Sepax HP-Silica (4.6mm×250mm×5μm), column temperature 30°C; sample concentration 10mg / mL, injection volume 20μL; mobile phase ratio of n-hexane: isopropanol: formic acid 15:1:0.03 (v / v / v), flow rate 1mL / min. Each lipid component was qualitatively analyzed by standard, sample concentration and peak area were linearly related, and the relative composition of each substance was expressed by area normalization method (%).

[0139] (3) Analysis of triglyceride composition by liquid chromatography-tandem mass spectrometry

[0140] (3.1) Mass spectrometry conditions

[0141] The data acquisition mode was an electrospray ion source, with positive and negative ion modes respectively collected; the Auto-MSe mode collected primary and secondary mass spectrometry dissociation fragments; the electrospray voltage was 2500V, and the ESI ion source temperature was 100°C; the cone gas flow rate was 50L / H, and the desolvation gas flow rate was 500L / H; the primary collision energy was 6.0eV, and the secondary collision dissociation energy was 30eV-40eV; the data acquisition mass range was 100-650Da.

[0142] (3.2) Chromatographic conditions

[0143] Chromatographic conditions: Waters C 18 (2.1×100mm, 1.7μm) ultra-high performance liquid chromatography column; mobile phase A is 0.05% acetic acid aqueous solution; mobile phase B is methanol and acetonitrile, injection volume is 2.00μL, flow rate is 0.3mL / min, column oven temperature: 40℃; automatic sampler temperature: 4℃. When mobile phase B is methanol, the chromatographic gradient is: 0-3.0min, 45%-60% organic phase; 3.0-14.0min, 60%-65% organic phase; 14.0-16.0min, 65%-85% organic phase; 16.0-19.0min, 85%-95% organic phase; 19.0-23.0min, 95% organic phase; 23.0-23.1min, 95%-45% organic phase; 23.1-26min, 45% organic phase. Mobile phase B is acetonitrile chromatographic gradient: 0-6.0min, 25%-40% organic phase; 6.0-16.0min, 40%-60% organic phase; 16.0-20.0min, 60%-95% organic phase; 20.0-23.0min, 95% organic phase; 23.0-23.1min, 95%-25% organic phase; 23.1-26min, 25% organic phase.

[0144] Test Example 4 Determination of Physical and Chemical Indexes of Oils and Fats

[0145] (1) Peroxide value

[0146] Refer to GB5009.227-2016 National Food Safety Standard Determination of Peroxide Value in Food.

[0147] (2) Determination of anisidine value

[0148] The determination of anisidine value in oils and fats shall be in accordance with GB / T 24304-2009.

[0149] The results of test examples 3 and 4 are shown in Table 4 below.

[0150] Table 4 Lipid and fatty acid composition of 2-MAG before and after purification after 3 cycles (%) w / w

[0151]

[0152]

[0153] After extraction, the 2-MAG content was 91.50% (Table 4), the EPA content was 92.19%, the peroxide value was 5.3316 mmol / kg, and the anisidine value was 11.8596.

[0154] Test Example 5 Pharmacokinetics of 2-EPA MAG Absorption in Rats

[0155] (1) Raising of SD rats

[0156] Thirty-six SD rats with high triglyceride model were selected and divided into 6 groups: EPA ethyl ester (dose 360 ​​mg / kg), EPA monoglyceride (410 mg / kg), tri-EPA triglyceride (343 mg / kg), EPA ethyl ester and statin (180 mg / kg+1.8 mg / kg), EPA monoglyceride and statin (205+1.8 mg / kg), and normal saline group (300 mg / kg), with 6 rats in each group. The EPA dose for rats was calculated according to the human dose of 4 g / d. The 40 model rats were gavaged once after fasting for 12 hours, and fasted for 24 hours after gavage. Blood was collected every 4 hours, once before gavage, at 0h, 4h, 8h, 12h, 16h, 20h, and 24h, for a total of 7 blood draws, 200 μL of blood each time, and the plasma was stored at -80℃.

[0157] (2) Gas chromatography determination of plasma fatty acids

[0158] Plasma fatty acids were determined by the chloroform-methanol method. About 50 mg of plasma tissue sample was weighed into a glass tube with a screw cap, 30 μL of tridecanonic acid (C13:0) was added as an internal standard, and then 500 μL of chloroform-methanol solution and 600 μL of chromatographically pure n-hexane were added. The mixture was methylated in a metal bath at 100°C for 10 min, then cooled to room temperature, 3 mL of saturated saline was added, and the fatty acid methyl esters were extracted by shaking for 10 s. After adding 1 mL of saturated sodium chloride solution, anhydrous sodium sulfate was added for dehydration, and the supernatant was collected by centrifugation at 4000 rpm / min at 20°C for 5 min, and the supernatant was filtered and analyzed by gas phase detection.

[0159] The gas chromatography conditions were based on the previous methods of this laboratory.

[0160] Data statistical analysis: GraphPad Prism 9.0 was used to plot the experimental results and SPSS17.0 was used for data analysis. Each group of experiments was repeated three times. One-way ANOVA and Duncan test were used to analyze the significant differences between the data. Each group of experiments was repeated three times, and the results were expressed as mean ± standard deviation. Different letters indicate significant differences at the p < 0.05 level.

[0161] The results are as follows Figure 8 As shown in the figure, it can be seen that compared with EPA-EE, 2-EPA-MAG can reduce the concentration of saturated fatty acids in plasma ( Figure 8 ).

[0162] Comparison of Sn-2 MAG and EE forms of EPA revealed that ( Fig.10 ), sn-2MAG can increase the concentration of EPA in plasma, and the plasma concentration of EPA in the form of EE is still higher when the oral dose of sn-2MAG is reduced to 1 / 2.

[0163] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing high-purity sn-2 eicosapentaenoic acid monoglyceride oil, characterized in that: The following steps are involved: S1, alcoholysis reaction of tri-EPA triglyceride; S2. The product after alcoholysis is subjected to liquid-liquid extraction by solvent extraction, and the lower layer is separated to obtain sn-2-EPA-MAG, and the upper layer obtained by separation contains EPA ethyl ester; S3. The upper layer after liquid-liquid extraction is subjected to transesterification with glyceride to generate tri-EPA triglyceride, and steps S1-S3 are repeated in a recycling process. During each cycle, the sn-2-EPA-MAG obtained after liquid-liquid extraction in step S2 is collected.

2. The method according to claim 1, characterized in that In step S1, the purity of the tri-EPA triglyceride is not less than 75%.

3. The method according to claim 1, characterized in that In step S1, the alcoholysis reaction method includes: adding ethanol and lipase to tri-EPA-triglyceride in an enzyme reactor, stirring at 30°C-40°C and 100-800r / min for 2-12h, removing the lipase in the system by suction filtration, and removing the solvent in the reaction system by rotary evaporation at 40-65°C.

4. The method according to claim 3, characterized in that In step S1, the molar ratio of ethanol to tri-EPA-triglyceride is 1:30-1:120, and / or Preferably, in step S1, the mass percentage of lipase to substrate tri-EPA-triglyceride is 6-14% w / w, and / or Preferably, in step S1, the lipase is selected from at least one of Lipozyme 435 lipase, CL "Amano" IM, Lipozyme TL IM and NS40086, and / or Preferably, in step S1, the reaction is carried out at 35°C-40°C with stirring at 300-600 r / min for 3-8 hours, and / or Preferably, in step S1, the solvent in the reaction system is removed by rotary evaporation at 45-60°C.

5. The method according to claim 1, characterized in that In step S2, the liquid-liquid extraction method includes: using n-hexane and 85% ethanol-water solution for extraction and separation.

6. The method according to claim 5, characterized in that The ratio of n-hexane, 85% ethanol-water solution and alcoholysis product is 1:10 w / w respectively, and the alcoholysis product refers to the product obtained by removing the solvent from the alcoholysis product.

7. The method according to claim 1, characterized in that In step S3, the transesterification treatment specifically includes: adding glycerol and lipase to the upper layer containing EPA ethyl ester in an enzyme reactor, stirring and reacting at 30-140°C and 100-800r / min for 2-14h under vacuum conditions, removing the lipase in the system by suction filtration, and taking the supernatant to obtain tri-EPA triglyceride.

8. The method according to claim 7, characterized in that The molar ratio of the upper layer material to glycerol is 3:1-15:1, and / or Preferably, the mass percentage of lipase in the upper layer is 4-20% (w / w), and / or Preferably, the reaction is carried out under vacuum conditions at 90-110° C. and 100-800 r / min with stirring for 5-9 hours.

9. The method according to claim 1, characterized in that: In step S3, the number of cycles is not less than 3 times, and / or Preferably, the number of cycles is 3-10 times.

10. The sn-2 eicosapentaenoic acid monoglyceride prepared according to the method of claims 1-9, characterized in that: The sn-2 eicosapentaenoic acid monoglyceride is recorded as 2-EPA MAG, which refers to oil with EPA at the sn-2 position of glycerol, a purity of more than 70%, and a sn-2 MAG content of more than 70%.

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