CN117604047A

CN117604047A - Preparation method of high-purity DHA ethyl ester

Info

Publication number: CN117604047A
Application number: CN202311571923.8A
Authority: CN
Inventors: 李家琪; 王永华; 罗日明; 林森; 王卫飞; 杨博
Original assignee: Guangdong Yueshan Special Medical Nutrition Technology Co ltd; South China University of Technology SCUT
Current assignee: Guangdong Yueshan Special Medical Nutrition Technology Co ltd; South China University of Technology SCUT
Priority date: 2023-11-22
Filing date: 2023-11-22
Publication date: 2024-02-27

Abstract

The invention discloses a preparation method of high-purity DHA ethyl ester, which comprises the steps of adding lipase and pH buffer solution into raw oil containing DHA, stirring for a period of time at 30-60 ℃, adding short-chain alcohol for esterification reaction, separating and purifying to obtain a DHA ethyl ester crude product, and then obtaining the high-purity DHA ethyl ester through tertiary molecular distillation; the lipase is preferably immobilized lipase MAS1. Performing transesterification by using an enzymatic method instead of a chemical method, adding a phosphate buffer in advance, stirring, regulating and controlling the reaction temperature, the mass ratio of lipase to raw oil and the mole ratio of short-chain alcohol to raw oil in the reaction process, and improving the conversion rate of DHA, thereby improving the content of DHA ethyl ester products; the method is simple to operate and reduces the use of toxic organic solvents. Meanwhile, the high-purity DHA ethyl ester product is obtained by three-stage molecular distillation, the high-purity DHA ethyl ester product is prepared, and the DHA ethyl ester content in the final product reaches 80%.

Description

Preparation method of high-purity DHA ethyl ester

Technical Field

The invention belongs to a method for preparing DHA ethyl ester by taking DHA grease as a raw material, and particularly relates to a method for preparing high-purity DHA ethyl ester by taking DHA algae oil as a raw material.

Background

Docosahexaenoic acid (DHA) is an important active ingredient in algae oil, belongs to omega-3 polyunsaturated fatty acid, and has the functions of improving brain function, promoting brain cell growth and development, and benefiting vision. At present, DHA is widely added to infant milk powder for use, and high-purity DHA is becoming a potential candidate drug for treating various diseases. Unlike fish oil, the DHA content in algae oil is more, and can reach 60% at maximum. In addition, the fatty acid composition of the algae oil is relatively simple, which is greatly beneficial to separating DHA with high purity. Most unrefined oils extracted from microalgae contain DHA in combination with glycerol. In general, methods for increasing the DHA content of triglyceride algae oils are very limited, requiring conversion of the triglyceride oil to the Fatty Acid Ethyl Ester (FAEE) form. At present, more literature still selects a chemical method to carry out ethyl esterification on algae oil, and few literature reports that immobilized lipase catalyzes the transesterification of algae oil and ethanol to prepare fatty acid ethyl ester in a solvent-free system in one step, in particular to algae oil containing a large amount of long-chain polyunsaturated fatty acid (> 50%). Therefore, the efficient conversion of algae oils containing large amounts of long chain polyunsaturated fatty acids into fatty acid ethyl esters and the realization of the preparation of high purity DHA ethyl esters remains a problem to be solved.

Chinese patent CN103864614B discloses a method for separating and purifying DHA from DHA grease produced by microbial fermentation, using cryptococcus equi-DHA grease as raw material, using KOH as catalyst to make ethylation, then using urea embedding-gradient freeze crystallization method and secondary molecular distillation method to make purification so as to obtain high-purity ethyl DHA. Chinese patent CN103880672B discloses a preparation method of high-purity DHA algae oil ethyl ester and glyceride converted from the DHA algae oil ethyl ester, wherein the Crypthecodinium algae DHA oil is taken as a raw material, and the high-purity DHA ethyl ester is obtained by alkali process ethyl esterification and then purification by a secondary molecular distillation method. When the method is used for preparing the high-purity DHA ethyl ester, the chemical method is usually adopted for preparing the fatty acid ethyl ester and then separating the fatty acid ethyl ester. However, this process requires the use of a variety of toxic and harmful organic solvents and is complicated in steps.

Chinese patent CN111943837B discloses a process for preparing high-purity EPA or DHA ethyl ester by combining molecular distillation and dynamic axial chromatography, wherein the crude EPA or DHA is treated with enzyme-base combined ultrasonic transesterification, concentration to remove ethanol, then two-step molecular distillation is carried out, and finally two-dimensional DAC refining and third-step molecular distillation or rectification are carried out to prepare high-purity ethyl ester type EPA or DHA. The enzymatic method has mild reaction conditions for catalyzing the ethylation, and the obtained product has good quality and does not need post-treatment, thus being a more suitable process method. However, the esterification rate of the single enzyme method is lower than that of the chemical method, the reaction time is long, and the esterification rate is improved by combining the chemical method or ultrasonic technology. Therefore, a lipase with strong exchange capacity for polyunsaturated fatty acid esters is developed, which can reduce the use of toxic solvents and realize the high ethyl ester conversion of DHA.

Chinese patent CN109880857a discloses a method for obtaining free fatty acid by enzymatic hydrolysis of crude oil obtained by microbial fermentation, and obtaining high-purity DHA by enzymatic enrichment, molecular distillation and column chromatography coupling enrichment. CN107216252B discloses a preparation method of high content Omega-3 fatty acid ethyl ester, which uses fish oil ethyl ester as raw material, and receives EPA ethyl ester and DHA ethyl ester components at the 2 nd and 3 rd molecular distillation light phase outlets respectively through a 3 rd molecular distillation combined process, so as to obtain high content EPA ethyl ester and DHA ethyl ester products. Molecular distillation is a common purification technique from the conventional purification process, but it is difficult to obtain high purity DHA by secondary molecular distillation, and combined purification is usually required in combination with other methods. Therefore, finding a method which has simple process and low operation cost and can realize industrialized production of high-purity DHA ethyl ester is a difficult problem to be solved.

In view of the prior art, further research is still needed to improve the high esterification conversion rate of DHA and prepare high-purity DHA.

Disclosure of Invention

In view of the above, the invention aims to provide a preparation method of high-purity DHA ethyl ester, which aims at overcoming the defects existing in the prior art, realizes high esterification conversion rate of DHA, remarkably improves the purity of DHA ethyl ester products, and improves the DHA content from 60.18% to more than 80.01%.

In a first aspect of the invention, a method for preparing DHA ethyl ester is provided.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a method for preparing DHA ethyl ester, comprising the following steps:

s1) ethylation of DHA-containing raw oil: adding lipase MAS1 into raw oil containing DHA, wherein the addition amount of the lipase is 200-600U/g of oil calculated according to the enzyme activity, adding a phosphate buffer solution with the concentration of 20mmol/L and the pH of 5.0-8.0, accounting for 0.25-8 wt% of the MASs of substrate oil, stirring for 15min-1h, adding short-chain alcohol according to the mole ratio of the short-chain alcohol to the raw oil of (1-10): 1, carrying out esterification reaction at the temperature of 30-60 ℃, and separating and purifying to obtain a DHA ethyl ester crude product;

s2) performing tertiary molecular distillation on the obtained DHA ethyl ester crude product to obtain high-purity DHA ethyl ester.

In some embodiments, the feedstock oil comprises at least one of algae oil or fish oil; preferably algae oil.

In some of these embodiments, the stirring time is 30min-1h; preferably 30-45min.

In some of these embodiments, the esterification reaction time is 1 to 12 hours; preferably 5-7h.

In some of these embodiments, the lipase MAS1 is at least one of a liquid lipase MAS1 or an immobilized lipase MAS1, preferably an immobilized lipase MAS1.

In some preferred embodiments, the immobilization of the immobilized lipase MAS1 comprises at least one of physical adsorption, covalent bonding, cross-linking, entrapment, copolymerization, and immobilization of empty vector; the immobilization method is preferably a gel embedding method.

In some embodiments, the phosphate buffer is added in an amount of 0.5wt% to 6wt% based on the mass of the substrate oil; preferably 0.5wt% to 4wt%.

In some of these embodiments, the short-chain alcohol is added in an amount such that the molar ratio of the short-chain alcohol to the feedstock is (1-6): 1; the preferred molar ratio of the alcohol oil is (1-3): 1; more preferably, the molar ratio of the alcohol oil is (1.5-2): 1.

In some of these embodiments, the esterification reaction system temperature is from 30 to 50 ℃, preferably from 30 to 45 ℃.

In some of these embodiments, the immobilized lipase is added in an amount of 300 to 450U/g of oil, preferably 300 to 400U/g of oil, calculated as enzyme activity.

In some of these embodiments, the short chain alcohol is selected from at least one of the alcohols of C1-C6 (methanol, ethanol, propanol, butanol, propanol, hexanol); preferably one selected from C1-C3 alcohols (methanol, ethanol, propanol); more preferably ethanol.

In some of these embodiments, step S2) comprises: the DHA ethyl ester crude product enters a molecular distillation device at a feeding speed of 1.0-20 g/min for primary molecular distillation, the primary molecular distillation is carried out under the conditions of 60-80 ℃ and a system pressure of 0.2-10 Pa and a film distillation scraper rotating speed of 250-350 rpm, and the heavy components are collected; performing secondary molecular distillation on the heavy component obtained by the primary molecular distillation at a feeding speed of 1.0-20 g/min and a system pressure of 0.2-10 Pa at a temperature of 90-100 ℃, and collecting the heavy component under the condition that the rotating speed of a thin film distillation scraper is 250-350 rpm; and (3) carrying out tertiary molecular distillation on the heavy component obtained by the secondary molecular distillation, removing triglyceride in the mixture obtained in the previous stage, thereby obtaining high-purity DHA ethyl ester with high ethyl ester content, wherein the condition is that the feeding speed is 1.0-20 g/min, the tertiary molecular distillation is carried out under the conditions that the temperature is 160-170 ℃ and the system pressure is 0.2-10 Pa, and the rotating speed of a thin film distillation scraper is 250-350 rpm, and taking out the light component to obtain the DHA ethyl ester product.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention uses an enzymatic method to replace a chemical method for transesterification, and the inventor finds that lipase is adopted as immobilized lipase MAS1, and under the condition of adding phosphate buffer in advance and stirring, the reaction temperature, the MASs ratio of the lipase to the raw oil and the mole ratio of short-chain alcohol to the raw oil in the reaction process are correspondingly regulated and controlled, so that the conversion rate of DHA is greatly improved, and the DHA ethyl ester product content is improved; the method is simple to operate and reduces the use of toxic organic solvents.

(2) According to the invention, three-stage molecular distillation is adopted to obtain the high-purity DHA ethyl ester product, and the DHA ethyl ester content in the final product reaches 80 percent.

(3) The preparation method is suitable for preparing high-purity DHA ethyl ester from DHA-containing raw oil, particularly DHA algae oil with high unsaturated fatty acid content, and the difference of DHA content in the raw oil can not obviously influence the ethyl ester conversion rate and purity of the product.

Drawings

FIG. 1 is a comparative graph of the catalytic ethylation of algae oil with lipase MAS1 of example 1 of the present invention under different pretreatment conditions.

FIG. 2 is a graph showing the comparison of the ethylation of algal oils catalyzed by different lipases in example 2 of the present invention.

FIG. 3 is a graph showing the comparison of the ability of liquid enzymes MAS1 and solid enzymes MAS1 to catalyze the ethyl esterification of algae oil in example 3 of the present invention.

FIGS. 4 and 5 are graphs showing the comparison of the effect of the immobilized enzymes MAS1 and Novozym435 on catalyzing different feedstock oils in example 4 of the present invention.

FIG. 6 is a graph showing the analysis of the components before and after the preparation of the materials in example 5 of the present invention.

FIG. 7 is a graph comparing the effect of substrate molar ratio on ethyl ester conversion of catalytic algae oil in example 6 of the present invention.

FIG. 8 is a graph showing the effect of the addition amount of phosphate buffer solution on ethyl ester conversion of catalytic algae oil in example 7 of the present invention.

FIG. 9 is a graph comparing the effect of different temperatures on ethyl ester conversion of catalytic algae oil in example 8 of the present invention.

FIG. 10 is a graph showing the comparison of the effect of different enzyme addition amounts on ethyl ester conversion of catalytic algae oil in example 9 of the present invention.

FIG. 11 is a graph showing the comparison of the effect of alcohols of different chain lengths on ethyl ester conversion of catalytic algae oil in example 10 of the present invention.

FIG. 12 is a graph showing the comparison of the effect of alcohols of different chain lengths on ethyl ester conversion of catalytic algae oil in example 10 of the present invention.

Detailed Description

The technical scheme of the invention is further described by the following specific examples. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The immobilized lipase MAS1 used in the following examples was immobilized by gel entrapment.

"x U/g oil" means that one gram of oil in the substrate corresponds to the addition of an enzyme having an enzyme activity of x U.

Note that: in a specific example, the method for measuring the enzyme activity is as follows:

4.0114g of lauric acid, 1.202g of n-propanol and 160. Mu.L of distilled water were added to a 25mL Erlenmeyer flask and placed in a gas bath thermostatic shaker at a speed of 150rpm and a temperature of 60℃for 10min for preheating; respectively adding 15mg of immobilized enzyme, and reacting for 10min; immediately, 30. Mu.L of the reaction product was dissolved in 970. Mu.L of n-heptane, and the contents of lauric acid and laurate were measured by Gas Chromatography (GC).

One enzyme activity unit (U) is defined as: under certain reaction conditions, the immobilized enzyme catalyzes the esterification of the substrate to produce 1 mu mol of propyl laurate per minute.

The calculation formula of the esterification activity:

m: initial amounts of 1-propanol and lauric acid, mol

C: lauric acid esterification rate

W: mass of immobilized enzyme, g

t: reaction time, min.

The present invention will be described in further detail with reference to specific examples.

Example 1

5 250mL round bottom flasks were prepared, 100g of algae oil was added to each flask, and 300U/g of oil immobilized lipase MAS1 was then added to each reaction system separately. Under the protection of nitrogen, the round bottom flask is placed in a heat-collecting type constant temperature magnetic stirrer, the rotating speed is adjusted to be 200rpm, and the esterification reaction temperature is adjusted to be 40 ℃. Experimental group: 0.5g of phosphate buffer (0.5 wt% of the mass of the substrate oil) of pH7.0 at a concentration of 20mmol/L was added, and the mixture was stirred for 15min, 30min and 1h, respectively, and then ethanol was added to carry out esterification reaction at 40 ℃. Control group: 0.5g of phosphate buffer (0.5 wt% based on the mass of the substrate oil) having a concentration of 20mmol/L and pH7.0 was added thereto, and the esterification reaction was started by directly adding ethanol at a temperature of 40℃without stirring in advance. 31.6g of ethanol (ethanol/algae oil molar ratio 2:1) was added to the esterification reaction system in four portions. The reaction was stopped after 12 hours. Subsequently, the lipase was removed by centrifugation, and ethanol was recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was determined and the results are shown in FIG. 1.

As can be seen from FIG. 1, the immobilized lipase MAS1 did not undergo transesterification in the control group, and at this time, the ethyl ester conversion was lower than 2%. The immobilized lipase MAS1 has super-strong transesterification capability in an experimental group, the ethyl ester content reaches 96% at 6h, and the transesterification reaction rates of stirring for 30min and 1h are not obviously different. Therefore, the lipase MAS1 is stirred for a period of time only by adding a certain amount of buffer before adding ethanol, and the stirring effect is best for 30min. Therefore, in the following examples, the experiment was performed by adding the buffer and stirring for 30min before adding ethanol.

Example 2

Immobilized lipases MAS1, novozyme 435, lipozyme TL IM and Lipozyme RM IM produced by Guangdong Uyose Co., ltd were weighed out according to the same enzyme activities (300U/g oil), respectively. 5 250mL round bottom flasks were prepared, 100g of algae oil was added to each flask, and then a plurality of weighed immobilized lipases were added to each reaction system. Under the protection of nitrogen, the round bottom flask is placed in a heat-collecting constant temperature magnetic stirrer, and the rotating speed is adjusted to be 200rpm, and the temperature is adjusted to be 40 ℃. 0.5g of phosphate buffer (0.5 wt% based on the mass of the substrate oil) with a concentration of 20mmol/L and pH7.0 was added and stirred for 30min, and 31.6g of ethanol (ethanol/algae oil molar ratio 2:1) was added four times to the esterification reaction system at 40 ℃. The reaction was stopped after 12 hours. Subsequently, the lipase was removed by centrifugation, and ethanol was recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was determined and the results are shown in FIG. 2.

As can be seen from FIG. 2, immobilized lipase MAS1 had a higher ethyl ester conversion, wherein the ethyl ester conversion of immobilized lipase MAS1 was about 96%, and the ethyl ester conversion of Novozym435, lipozyme TL IM, and Lipozyme RM IM were all lower than 65%. Meanwhile, immobilized lipase MAS1 had a faster ethyl ester conversion rate, wherein immobilized lipase MAS1 reached 96% at 6h, and at this time ethyl ester conversion rates of Novozym435, lipozyme TL IM, and Lipozyme RM IM were 22%, 43%, and 31%, respectively. The lipase MAS1 can reach the highest ethyl ester conversion rate in the shortest time and has better transesterification capability than commercial enzymes. Therefore, in the following examples, experiments were performed using the lipase MAS1.

Example 3

The liquid enzyme MAS1 and the immobilized lipase MAS1 are weighed according to the same enzyme activity (300U/g oil), wherein the immobilization mode of the immobilized enzyme MAS1 comprises any one or a combination of several of physical adsorption, covalent bond combination, crosslinking, embedding, copolymerization and empty carrier immobilization. The immobilization of immobilized lipase MAS1 in this example was performed by gel embedding, preparing 2 250mL round-bottomed flasks, adding 100g of algae oil to each flask, and then adding the weighed liquid enzyme MAS1 and immobilized lipase MAS1 to each flask. Under the protection of nitrogen, the round bottom flask is placed in a heat-collecting constant temperature magnetic stirrer, and the rotating speed is adjusted to be 200rpm, and the temperature is adjusted to be 40 ℃. 0.5g of phosphate buffer (0.5 wt% based on the mass of the substrate oil) with a concentration of 20mmol/L Ph7.0 was added, stirred for 30min, and 31.6g of ethanol (ethanol/algae oil molar ratio 2:1) was added four times to the esterification reaction system at 40 ℃. The reaction was stopped after 12 hours. Subsequently, the lipase was removed by centrifugation, and ethanol was recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was determined and the results are shown in FIG. 3.

The results show that the ethyl ester conversion of immobilized lipase MAS1 is better than that of liquid enzyme MAS1; the experiments were performed using immobilized lipase MAS1 in the following examples.

Example 4

100g of algae oil (DHA content: 60.13%) and 100g of soybean oil (DHA content: 0%) were weighed out, and added to a 250mL reaction flask, and 300U/g of immobilized lipase MAS1 and Novozym435 were added to the reaction flask, respectively. Under the protection of nitrogen, the round bottom flask is placed in a heat-collecting constant temperature magnetic stirrer, the rotating speed is adjusted to be 200rpm, and the temperature is 40 ℃. 0.5g of 20mmol/L phosphate buffer (0.5 wt% based on the mass of the substrate oil) with pH7.0 is added, stirred for 30min, and 31.6g of ethanol (ethanol/algae oil molar ratio 2:1) is added into the esterification reaction system at 40℃four times. After 6h of reaction, the lipase was removed by centrifugation and ethanol was recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was determined, and the results are shown in fig. 4 and 5.

The result shows that for immobilized enzyme MAS1, after the treatment of raw oil with obvious DHA content difference, the ethyl ester conversion rate of the finally obtained mixture has small variation range, and the reaction balance time is only different; therefore, the difference of DHA content in the raw oil has no obvious influence on the final ethyl ester conversion rate of the grease ethyl esterification catalyzed by the immobilized enzyme MAS1.

Example 5

Purification of the product by molecular distillation

500g of algae oil (DHA content 60.10%) was first weighed into a 1000mL round bottom flask, followed by 300U/g of oil immobilized lipase MAS1. Under the protection of nitrogen, the rotating speed is regulated to 200rpm, the temperature is 40 ℃, 5.0g of phosphate buffer solution (accounting for 0.5 weight percent of the mass of substrate oil) with the concentration of 20mmol/L and pH of 7.0 is added, stirring is carried out for 30min, and 158g of ethanol (the mol ratio of ethanol to raw oil is 2:1) is added into an esterification reaction system with the temperature of 40 ℃ for four times. After 12h of reaction, the lipase was removed by centrifugation, and ethanol was recovered by distillation under reduced pressure.

The resulting mixture was subjected to tertiary molecular distillation under the following conditions: performing primary molecular distillation at 80 ℃ under the system pressure of 0.2-0.3 Pa and the film distillation scraper rotating speed of 300rpm, and collecting heavy components; performing secondary molecular distillation on the heavy component obtained by the primary molecular distillation at a feeding speed of 1.0g/min and a system pressure of 0.2-0.3 Pa at a temperature of 100 ℃ under the condition that the rotating speed of a film distillation scraper is 300rpm, and collecting the heavy component; performing three-stage molecular distillation on the heavy component obtained by the two-stage molecular distillation at the feeding speed of 1.0g/min and the system pressure of 0.2-0.3 Pa at 160-170 ℃ under the condition that the rotating speed of a film distillation scraper is 300rpm, and taking out the light component to obtain the high-purity DHA ethyl ester. The results before and after the preparation of the materials are shown in fig. 6 and 7.

The results show that the content of ethyl DHA in the collected heavy phase can reach 75.94 percent by the first-stage molecular distillation, and the heavy phase yield is 71.24. And then carrying out secondary molecular distillation at the feed rate of 1.0g/min and the system pressure of 0.2-0.3 Pa and the film distillation scraper rotation speed of 300rpm to obtain the product with DHA content of 80.04% and the heavy phase yield of 79.87%. At this time, only 88.45% ethyl ester was present in the heavy phase, and the content of triglycerides, free fatty acids and other glycerides was 5.59%, 1.94% and 4.02%, respectively. Therefore, a further molecular distillation is required to obtain ethyl ester of high purity. The third stage molecular distillation temperature was selected to be 170℃and the light phase was collected. And after the third-stage molecular distillation treatment, collecting a light phase. The ethyl ester content in the light phase reached 96.52%, no triglycerides, and the free fatty acids and other glycerides were 2.36% and 1.12%, respectively. At this time, the ethyl ester content of DHA in the light phase was 80.01%, and enrichment of DHA in ethyl ester form was achieved.

Example 6

To 5 250mL round bottom flasks, 100g of algae oil was added, and 300U/g of oil immobilized lipase MAS1 was added. Under the protection of nitrogen, the round bottom flask is placed in a heat-collecting constant temperature magnetic stirrer, and the rotating speed is adjusted to be 200rpm, and the temperature is adjusted to be 40 ℃. 0.5g of phosphate buffer (0.5 wt% based on the mass of the substrate oil) having a concentration of 20mmol/L pH7.0 was added thereto and stirred for 30 minutes. And then according to ethanol: the algae oil is prepared by respectively weighing ethanol with corresponding mass according to the mole ratio of 1:1, 1.5:1, 3:1, 6:1 and 9:1, adding the ethanol into an esterification reaction system with the temperature of 40 ℃ for four times, and starting the reaction. After 12h of reaction, the lipase was removed by centrifugation, and ethanol was recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was measured, and the result is shown in FIG. 8.

The result shows that the ethanol with proper dosage is favorable for improving the conversion rate of the ethyl ester, and the mol ratio of the ethanol to the algae oil (1-10) is more than 80 percent when the mol ratio of the ethanol to the algae oil is 1; when the mole ratio of the ethanol to the algae oil is 1-3, the ethyl ester conversion rate can reach more than 90 percent, and when the mole ratio of the ethanol to the algae oil is 1.5:1, the ethyl ester conversion rate of the obtained mixture is measured to be the best effect.

Example 7

To 6 250mL round bottom flasks, 100g of algae oil was added, and 300U/g of oil immobilized lipase MAS1 was added. Under the protection of nitrogen, adding 0.25wt%,0.5wt%,2wt%,4wt%,6wt% and 8wt% of a phosphate buffer solution with the concentration of 20mmol/L and the pH of 7.0 into an esterification reaction system with the temperature of 40 ℃ according to the mass ratio of the ethanol to the raw oil of 1.5:1 after stirring for 30min. The reaction flask was placed in a heat-collecting type constant temperature magnetic stirrer with a rotation speed of 200rpm at 40℃and the reaction was started. After 12h of reaction, the lipase was removed by centrifugation, and ethanol was recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was measured, and the result is shown in FIG. 9.

The result shows that when the addition amount of the phosphate buffer solution is less than 0.5wt%, the increase of the addition amount of the phosphate buffer solution is beneficial to the improvement of the ethyl ester conversion rate, but when the addition amount of the phosphate buffer solution is more than 0.5wt%, the ethyl ester conversion rate is not obviously changed; the ethyl ester conversion was slightly decreased when the amount of phosphate buffer solution added was increased to 6wt%, but when it was increased to 8wt%, the ethyl ester conversion was still 95%. Therefore, when the adding amount of the phosphate buffer solution is 0.25wt percent to 8wt percent, the ethyl ester conversion rate can reach more than 95 percent, wherein the effect is optimal from 0.5wt percent to 4wt percent.

Example 8

To 5 250mL round bottom flasks, 100g of algae oil was added, and 300U/g of oil immobilized lipase MAS1 was added. Under the protection of nitrogen, adding a phosphate buffer solution with the concentration of 20mmol/L and the pH of 7.0 according to the weight of 0.5 percent of the substrate oil, stirring for 30min, and adding 23.7g of ethanol (the mol ratio of the ethanol to the raw oil is 1.5:1) into an esterification reaction system with the temperature of 40 ℃ for four times. The reaction flask was placed in a heat-collecting type constant temperature magnetic stirrer with a rotation speed of 200rpm and different temperatures, the temperatures were set at 30, 40, 50, 60, 70 ℃, and the reaction was started. After 12h of reaction, the lipase was removed by centrifugation, and ethanol was recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was measured, and the result is shown in FIG. 10.

The results show that there is a significant increase in ethyl ester conversion after heating from 30 to 40 ℃, and there is no significant change in ethyl ester conversion after heating from 40 to 60 ℃, but the ethyl ester conversion gradually becomes lower after heating above 60 ℃. Meanwhile, when the reaction system is heated to 30-60 ℃, the ethyl ester conversion rate can reach more than 85%, the ethyl ester conversion rate is obviously reduced after the reaction system is heated to more than 70 ℃, and the ethyl ester conversion rate can still reach more than 80%. The highest conversion rate of ethyl ester at 40 ℃ is 96.5%, and the comparison shows that the esterification reaction temperature is set to be 40 ℃, 50 ℃ and 60 ℃ respectively, and the experimental results show that the conversion rate of ethyl ester is not obvious, but the energy consumption at 40 ℃ is less, and the optimal reaction temperature at 40 ℃ is selected from the viewpoint of saving energy consumption.

Example 9

To 7 250mL round bottom flasks, 100g of algae oil was added, 100U/g of oil, 200U/g of oil, 300U/g of oil, 400U/g of oil, 450U/g of oil, 500U/g of oil, 600U/g of oil immobilized lipase MAS1, respectively. Under the protection of nitrogen, adding a phosphate buffer solution with the concentration of 20mmol/L and pH7.0 accounting for 0.5wt% of the substrate oil into the reaction system, stirring for 30min, and adding 23.7g of ethanol (the mol ratio of the ethanol to the raw oil is 1.5:1) into the esterification reaction system at the temperature of 40 ℃ for four times. The round bottom flask was placed in a heat-collecting thermostatic magnetic stirrer with a rotation speed of 200rpm and a temperature of 40℃and the reaction was started. After 12h of reaction, the lipase was removed by centrifugation, and ethanol was recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was measured, and the result is shown in FIG. 11.

The results showed that the ethylation efficiency was not good enough at enzyme addition of 100U/g oil and 200U/g oil (where the data for 100U/g oil was not shown because of the insufficient effect). It was found that the ethyl esterification rate was greatly improved when increased to 300U/g oil and was optimal from the overall ethyl ester conversion. The ethyl ester conversion did not change significantly between 300U/g oil and 450U/g oil, at which point the ethyl ester conversion was about 95%. And when the enzyme addition amount is increased to 500U/g of oil, the ethyl ester conversion rate is reduced to 91%, and when the enzyme addition amount of the reaction system reaches 600U/g of oil, the ethyl ester conversion rate is reduced to 85%. It can be seen that when the lipase MAS1 is selected, the selection of its enzyme amount is important.

Example 10

To 6 250mL round bottom flasks was added 100g of algae oil, 300U/g of immobilized lipase MAS1, respectively. Under the protection of nitrogen, adding a phosphate buffer solution with the concentration of 20mmol/LpH 7.0.0 accounting for 0.5wt% of the substrate oil quality, stirring for 30min, and adding C1-C6 alcohol (methanol, ethanol, propanol, butanol, propanol and hexanol) with the molar ratio of alcohol to oil of 1.5:1 into an esterification reaction system with the temperature of 40 ℃ for four times. The round bottom flask was placed in a heat-collecting thermostatic magnetic stirrer with a rotation speed of 200rpm and a temperature of 40℃and the reaction was started. After 12 hours of reaction, the reaction mixture reaches equilibrium, the lipase is removed by centrifugation, and ethanol is recovered by distillation under reduced pressure. Finally, the ethyl ester conversion of the resulting mixture was measured, and the result is shown in FIG. 12.

The results show that when methanol, ethanol and propanol are added, the ethyl esterification rate is over 90 percent, wherein the ethyl esterification rate of the ethanol is the highest. While as the chain length of the alcohol increases, the ethylation starts to be less than 90%, but the ethylation rate is still higher than 80%.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The preparation method of the high-purity DHA ethyl ester is characterized by comprising the following steps of:

s1) ethylation of DHA-containing raw oil: adding lipase MAS1 into raw oil containing DHA, wherein the addition amount of the lipase is 200-600U/g of oil calculated according to the enzyme activity, adding a phosphate buffer solution with the concentration of 20mmol/L and the pH of 5.0-8.0 accounting for 0.25-8 wt% of the MASs of substrate oil, stirring for 15min-1h, adding short-chain alcohol according to the mole ratio of the short-chain alcohol to the raw oil of (1-10): 1, carrying out esterification reaction at the temperature of 30-60 ℃, and separating and purifying to obtain a DHA ethyl ester crude product;

2. The method for preparing high purity ethyl DHA according to claim 1, wherein S2) the tertiary molecular distillation comprises the steps of: the DHA ethyl ester crude product enters a molecular distillation device at a feeding speed of 1.0-20 g/min for primary molecular distillation, the primary molecular distillation is carried out under the conditions of 60-80 ℃ and a system pressure of 0.2-10 Pa and a film distillation scraper rotating speed of 250-350 rpm, and the heavy components are collected; performing secondary molecular distillation on the heavy component obtained by the primary molecular distillation at a feeding speed of 1.0-20 g/min and a system pressure of 0.2-10 Pa at a temperature of 90-100 ℃, and collecting the heavy component under the condition that the rotating speed of a thin film distillation scraper is 250-350 rpm; and (3) carrying out tertiary molecular distillation on the heavy component obtained by the secondary molecular distillation, removing triglyceride in the mixture obtained in the previous stage, thereby obtaining high-purity DHA ethyl ester with high ethyl ester content, wherein the condition is that the feeding speed is 1.0-20 g/min, the tertiary molecular distillation is carried out under the conditions that the temperature is 160-170 ℃ and the system pressure is 0.2-10 Pa, and the rotating speed of a thin film distillation scraper is 250-350 rpm, and taking out the light component to obtain the DHA ethyl ester product.

3. The method for preparing high-purity DHA ethyl ester according to claim 1, wherein the addition amount of the immobilized lipase is 300-450U/g oil; preferably 300 to 400U/g oil.

4. A process for the preparation of high purity ethyl DHA according to any of claims 1-3, wherein the lipase MAS1 is immobilized lipase MAS1; the MAS1 immobilization mode of the immobilized lipase comprises at least one of physical adsorption, covalent bond combination, crosslinking, embedding, copolymerization and empty carrier immobilization; preferably, the immobilization mode is a gel embedding method.

5. A process for the preparation of high purity ethyl DHA according to any of claims 1-3, wherein the short chain alcohol is selected from at least one of methanol, ethanol, propanol, butanol, propanol, hexanol; preferably at least one selected from methanol, ethanol and propanol; more preferably ethanol.

6. The method for producing high-purity ethyl DHA ester according to claim 5, wherein the short-chain alcohol is added in an amount such that the molar ratio of the short-chain alcohol to the raw oil is (1-6) 1; the preferred molar ratio is (1-3): 1; more preferably, the molar ratio is (1.5-2): 1.

7. A method for preparing high purity ethyl DHA according to any of claims 1-3, wherein the phosphate buffer is added in an amount of 0.5wt% to 6wt% based on the mass of the substrate oil; preferably 0.5wt% to 4wt%.

8. A method for preparing high purity ethyl DHA according to any one of claims 1-3, wherein the stirring time is 30min-1h, the esterification reaction system temperature is 30-50 ℃, and the esterification reaction time is 1-12h; preferably, the stirring time is 30-45min, the temperature of the esterification reaction system is 30-45 ℃, and the reaction time is 5-7h.

9. A method for preparing high purity ethyl ester of DHA according to any one of claims 1-3, wherein the raw oil comprising DHA comprises at least one of algae oil, fish oil; preferably algae oil.

10. The high purity ethyl DHA produced by the production process according to any one of claims 1-9.