CN115478084A - Preparation method of polyunsaturated fatty acid ester - Google Patents

Preparation method of polyunsaturated fatty acid ester Download PDF

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CN115478084A
CN115478084A CN202110662785.9A CN202110662785A CN115478084A CN 115478084 A CN115478084 A CN 115478084A CN 202110662785 A CN202110662785 A CN 202110662785A CN 115478084 A CN115478084 A CN 115478084A
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fatty acid
polyunsaturated fatty
phospholipid
lipase
ester
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李翔宇
汪志明
王维曼
马凡提
陆姝欢
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Cabio Biotech Wuhan Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6481—Phosphoglycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6454—Glycerides by esterification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

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Abstract

The invention relates to the technical field of synthesis of polyunsaturated fatty acid esters, and particularly relates to a preparation method of polyunsaturated fatty acid esters. In the preparation method provided by the invention, the phospholipid type polyunsaturated fatty acid is prepared by enzymatic reaction of the following raw materials: the polyunsaturated fatty acid ester is characterized by comprising polyunsaturated fatty acid ester, polyunsaturated fatty acid salt, phospholipid and lipase, wherein the polyunsaturated fatty acid corresponding to the polyunsaturated fatty acid ester is the same as the polyunsaturated fatty acid corresponding to the polyunsaturated fatty acid salt in type. The method has wide raw material selection range, does not depend on organic solvent, is suitable for various reaction environments, can realize higher fatty acid access rate in a system without organic solvent, obviously improves the content of polyunsaturated fatty acid in the prepared phospholipid polyunsaturated fatty acid, and is suitable for large-scale popularization and application.

Description

Preparation method of polyunsaturated fatty acid ester
Technical Field
The invention relates to the technical field of polyunsaturated fatty acid ester synthesis, in particular to a preparation method of phospholipid type polyunsaturated fatty acid.
Background
Polyunsaturated fatty acid (PUFA) ester products are generally classified into ethyl ester type, triglyceride type, and phospholipid type. The physiological functions of PUFAs vary depending on their molecular form in fat. The PUFA existing in the form of phospholipid has physiological functions of phospholipid and PUFA, has more stable structure, is not easy to oxidize and is more beneficial to absorption of human bodies. Research shows that the absorption rate of ethyl ester type DHA in human body is only about 20%, the human body absorption rate of glycerol type DHA can be improved to 70%, and the absorption rate of phospholipid type DHA in human body is the highest and exceeds 99%.
The natural phospholipid rich in EPA/DHA exists mainly in marine animals, the yield of the phospholipid is far from meeting the requirements of people, and the artificial synthesis of phospholipid type PUFA becomes another acquisition way. The fatty acid composition of the phospholipid is changed, and generally, transesterification or acidolysis can be selected. The ester exchange reaction is to select the ester compound of the target fatty acid and the fatty acid on the phospholipid to exchange under the enzymatic action; the acidolysis reaction is carried out by exchanging free fatty acids with fatty acids on phospholipids. At present, a satisfactory fatty acid access rate can be obtained under the acidolysis reaction, and the enzymatic transesterification reaction is more green and environment-friendly, but the access effect is difficult to achieve the same effect as the acidolysis reaction.
The Chinese invention patent CN 104962590A discloses microbial phospholipid type polyunsaturated fatty acid oil and a preparation method thereof, and the method utilizes the lipid containing docosahexaenoic acid and powdered phospholipid to carry out lipase-catalyzed transesterification to prepare the microbial phospholipid type polyunsaturated fatty acid oil and fat. However, the method involves a two-step enzyme method, and has the disadvantages of complex reaction and high production cost.
The Chinese invention patent CN106893747A discloses a method for preparing n-3 polyunsaturated fatty acid phospholipid by catalyzing n-3 polyunsaturated fatty acid or ester derivatives thereof and soybean phospholipid ester exchange through an enzyme method, but the enzymatic reaction can only play a good effect in the presence of an organic solvent, and the access rate in the absence of the solvent is less than 10%.
Disclosure of Invention
The invention aims to provide a preparation method of polyunsaturated fatty acid ester, in particular to a preparation method of phospholipid type polyunsaturated fatty acid. The method does not depend on the addition of an organic solvent in the process of preparing the phospholipid polyunsaturated fatty acid, and has a good fatty acid access effect.
Aiming at the problems that the preparation of the phospholipid polyunsaturated fatty acid by the ester exchange reaction in the prior art mostly depends on a solvent system formed by an organic solvent, and the better fatty acid access rate cannot be ensured after the organic solvent is removed, the invention develops a high-efficiency method for preparing the phospholipid polyunsaturated fatty acid without depending on the organic solvent system. In the research and development process, the organic solvent system is very critical to ensure the high access rate of the fatty acid in the preparation process of the phospholipid polyunsaturated fatty acid, and after the organic solvent is removed, the fatty acid access rate under the condition of the organic solvent is difficult to achieve even if the reaction system is optimized in multiple aspects and substances which are disclosed in the prior art and are possibly beneficial to promoting the ester exchange reaction are added. The invention unexpectedly discovers that the introduction of the polyunsaturated fatty acid salt corresponding to the polyunsaturated fatty acid ester into the reaction system can obviously improve the access rate of the fatty acid in a solvent-free system, and further improve the content of the fatty acid in the prepared phospholipid type polyunsaturated fatty acid. The invention also finds that the introduction of the polyunsaturated fatty acid salt can realize better fatty acid access rate under the conditions of a reaction system without a solvent or with a solvent, and does not need to depend on the addition or selection of the solvent in the system.
Specifically, the invention provides the following technical scheme:
the invention provides a preparation method of phospholipid type polyunsaturated fatty acid, which is prepared by the enzymatic reaction of the following raw materials: the polyunsaturated fatty acid ester is characterized by comprising polyunsaturated fatty acid ester, polyunsaturated fatty acid salt, phospholipid and lipase, wherein the polyunsaturated fatty acid corresponding to the polyunsaturated fatty acid ester is the same as the polyunsaturated fatty acid corresponding to the polyunsaturated fatty acid salt in type.
The invention discovers that in order to ensure the fatty acid access rate of a solvent-free system, the polyunsaturated fatty acid ester, the polyunsaturated fatty acid salt and the phospholipid in the reaction raw materials are indispensable substrates, and the removal of one of the substrates or the replacement of the substrate with other similar substrates can obviously reduce the fatty acid access rate.
In the above-mentioned raw material system, the type of the polyunsaturated fatty acid in the polyunsaturated fatty acid salt is the same as the type of the polyunsaturated fatty acid in the polyunsaturated fatty acid ester. The polyunsaturated fatty acid salt can be a single polyunsaturated fatty acid salt or a mixture of polyunsaturated fatty acid salts, and likewise the polyunsaturated fatty acid ester can be a single polyunsaturated fatty acid ester or a mixture of polyunsaturated fatty acid esters. If one of the polyunsaturated fatty acid ester and the polyunsaturated fatty acid salt is a mixture, the polyunsaturated fatty acid with the highest content in the polyunsaturated fatty acid salt is of the same type as the polyunsaturated fatty acid with the highest content in the polyunsaturated fatty acid ester. For example: the polyunsaturated fatty acid ester raw material contains 70% of EPA ethyl ester, and the polyunsaturated fatty acid salt is EPA metal salt.
In the present invention, the polyunsaturated fatty acid esters include, but are not limited to, polyunsaturated fatty acid alkyl esters, polyunsaturated fatty acid glycerides.
In one embodiment, the phospholipid-type polyunsaturated fatty acid is prepared from a raw material that does not contain an organic solvent.
As another embodiment, the raw material further comprises an organic solvent, and the mass-to-volume ratio of the polyunsaturated fatty acid ester to the organic solvent in the raw material, g: mL is 1: (2-5).
For the above two embodiments, preferably, the mass ratio of the polyunsaturated fatty acid ester to the polyunsaturated fatty acid salt in the feedstock is (15-1500): 1.
further preferably, the polyunsaturated fatty acid ester is a polyunsaturated fatty acid ethyl ester or a polyunsaturated fatty acid glyceride. In this case, the mass ratio of the polyunsaturated fatty acid ethyl esters or polyunsaturated fatty acid glycerides to the polyunsaturated fatty acid salts in the feedstock is preferably (20-200): 1.
The ratio of the polyunsaturated fatty acid ester to the polyunsaturated fatty acid salt is controlled within the above range, which is more favorable for the improvement of the fatty acid incorporation rate.
In the present invention, the polyunsaturated fatty acid salt may be added to the reaction system alone, or the polyunsaturated fatty acid ester and the polyunsaturated fatty acid salt may be simultaneously provided by adding a raw material for fat or oil containing the polyunsaturated fatty acid ester and the polyunsaturated fatty acid salt.
Preferably, the lipase is 1,3-specific lipase.
The invention discovers that the 1,3-specific lipase used in the reaction system can obviously improve the fatty acid access rate compared with non-directional lipase.
Further preferably, the mass ratio of the lipase to the raw materials other than the lipase in the raw materials is 10 to 30%.
Preferably, the mass ratio of the phospholipid to the polyunsaturated fatty acid ester in the raw materials is 1 (3-10).
In the preparation method, the raw material also comprises water, and the mass ratio of the water to the lipase in the raw material is 10-20%.
The polyunsaturated fatty acid of the present invention may be one or more selected from the group consisting of omega-3 fatty acids, omega-6 fatty acids, docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, octadecatrienoic acid, octadecadienoic acid, and octadecatetraenoic acid.
The polyunsaturated fatty acid content of the polyunsaturated fatty acid ester used in the present invention is preferably controlled to not less than 65%.
The polyunsaturated fatty acid salt used in the present invention is preferably a metal salt corresponding to the polyunsaturated fatty acid, including but not limited to sodium salt, potassium salt, and the like.
The phospholipid used in the present invention is preferably a glycerophospholipid. The state of the phospholipid is preferably in a powdery state.
In the preparation method, the reaction temperature of the enzymatic reaction is 45-65 ℃. The preferred reaction time is 4-24h.
Specifically, the invention provides a method for preparing phospholipid type polyunsaturated fatty acid by a solvent-free system, which comprises the following steps:
(1) Mixing polyunsaturated fatty acid ester and polyunsaturated fatty acid salt with phospholipid and water, wherein the mass ratio of the polyunsaturated fatty acid ester to the polyunsaturated fatty acid salt is (15-1500): 1, the mass ratio of the phospholipid to the polyunsaturated fatty acid ester is 1 (3-10);
(2) Mixing the mixed material obtained in the step (1) with 1,3-specific lipase, wherein the mass ratio of the lipase to reaction raw materials except the lipase is 10-30%, the mass ratio of water to the lipase is 10-20%, and performing enzymatic transesterification reaction for 4-24h at 45-65 ℃;
(3) Separating the oil mixture to obtain phospholipid type polyunsaturated fatty acid.
Preferably, in step (1), the mixing is at room temperature. More preferably by sonication or stirring at room temperature.
And (3) after the reaction is finished, centrifuging or filtering to remove the lipase, and collecting the grease mixture.
The invention has the beneficial effects that: the preparation method of the phospholipid type polyunsaturated fatty acid provided by the invention has wide selection of raw materials, does not depend on an organic solvent system, can realize better fatty acid access rate no matter under the conditions of a solvent-free reaction system or a solvent-containing reaction system, and particularly can realize great improvement of the fatty acid access rate in the solvent-free reaction system, and the content of the polyunsaturated fatty acid in the prepared phospholipid type polyunsaturated fatty acid is remarkably improved. The method can be suitable for various reaction environments, and the preparation method of the solvent-free system also has the advantages of environment-friendly production conditions and high safety, and is suitable for large-scale popularization and application.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The following examples use the following feed parameters: randomized lipase 435 is Novozym435; the lipase TL is Novovern lipozyme TLIM; the immobilized lipase RMIM is Novovirin lipozyme RM IM; the powder phospholipid is neovadose (acetone insoluble 95%); the EPA ethyl ester oil is obtained by extracting and transesterifying nannochloropsis; the DHA ethyl ester oil is obtained by extracting and transesterification of schizochytrium limacinum.
Example 1
This example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g of EPA ethyl ester oil (area content 70.1%), 0.36g of sodium EPA, 6g of powdered phospholipid and 0.72g of water, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the oil mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the area content of EPA (eicosapentaenoic acid) of 28.0%.
Example 2
This example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g of EPA ethyl ester oil (area content 70.1%), 0.36g of EPA sodium, 6g of powdered phospholipid and 0.72g of water, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of lipase TL into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the oil mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the area content of EPA (eicosapentaenoic acid) of 18.5%.
Example 3
This example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g of EPA ethyl ester oil (EPA content: 80%), 0.60g of sodium EPA, 4.5g of powdered phospholipid and 0.72g of water, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) 3.6g of immobilized lipase RM IM were added to the substrate of step (1), placed in a water bath at 65 ℃ and stirred at 300rpm for 18h.
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the oil mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the area content of EPA (eicosapentaenoic acid) of 28.2%.
Example 4
This example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g EPA ethyl ester oil (area content 70.1%), 0.18g EPA sodium, 3.6g powdered phospholipid and 0.36g water, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the grease mixture, and performing methyl esterification and gas chromatography detection to obtain the phospholipid with the EPA content of 27.4%.
Example 5
This example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g of DHA ethyl ester oil (area content 71%), 0.58g of DHA potassium, 6g of powdered phospholipid and 0.72g of water, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the grease mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the DHA area content of 25.6%.
Example 6
This example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing the mixture oil (containing 18g EPA ethyl ester oil (area content 70.1%) and 0.09g EPA sodium and 0.72g water) with 6g powdered phospholipid, and performing ultrasonic dispersion for 5min to obtain substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the grease mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the EPA content of 23.5%.
Example 7
This example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 25.2g EPA glyceride (area content 70.1%), 0.36g EPA sodium, 0.72g water and 6g powder phospholipid, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the grease mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the EPA content of 7.2%.
Example 8
This example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g of EPA ethyl ester oil (area content 70.1%), 0.36g of EPA sodium, 6g of powdered phospholipid and 0.72g of water, adding 50ml of n-hexane, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the oil mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the area content of EPA (eicosapentaenoic acid) of 27.3%.
Comparative example 1
This comparative example provides a process for producing a phospholipid type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g of EPA ethyl ester oil (area content 70.1%), 0.72g of water and 6g of powdered phospholipid, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the oil mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the area content of EPA (eicosapentaenoic acid) of 10.3%.
Comparative example 2
This comparative example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g EPA fatty acid, 0.36g EPA sodium, 6g powder phospholipid and 0.72g water, and dispersing with ultrasound for 5min to obtain substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the oil mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the area content of EPA (eicosapentaenoic acid) of 5.0%.
Comparative example 3
This comparative example provides a process for producing a phospholipid type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g of EPA ethyl ester oil (area content 70.1%), 0.36g of EPA sodium, 0.72g of water and 6g of powdered phospholipid, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of randomized lipase 435 to the substrate of step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the oil mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the EPA area content of 6.8%.
Comparative example 4
This comparative example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 17.5g of DHA ethyl ester (DHA content is 71%) with 5g of soybean phospholipid raw material, and adding 40ml of n-hexane to obtain a grease mixture;
(2) Preheating the grease mixture obtained in the step (1) to 52 ℃;
(3) Adding 0.604g of DHA potassium salt, 0.06g of water and 3g of immobilized lipase RM IM into the reaction mixture obtained in the step (2), and performing ultrasonic dispersion; reacting at 55 deg.C for 8 hr to obtain oil composition;
and (3) separating phospholipid in the grease mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the DHA area content of 7.4%.
Comparative example 5
This comparative example provides a method for producing a phospholipid-type polyunsaturated fatty acid, comprising the steps of:
(1) Mixing 18g of EPA sodium, 6g of powdered phospholipid, 50mL of n-hexane and 0.72g of water, and performing ultrasonic dispersion for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM into the substrate in the step (1), placing the substrate in a water bath at 65 ℃, and stirring the substrate at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5min, the lower immobilized enzyme was removed, and the oil-and-fat mixture was collected.
And (3) separating phospholipid in the grease mixture, and performing methyl esterification and then performing gas chromatography detection to obtain the phospholipid with the EPA content of 3.0%.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims (10)

1. The preparation method of the phospholipid type polyunsaturated fatty acid is characterized in that the phospholipid type polyunsaturated fatty acid is prepared by the enzymatic reaction of the following raw materials: polyunsaturated fatty acid esters, polyunsaturated fatty acid salts, phospholipids and lipases,
wherein the polyunsaturated fatty acid corresponding to the polyunsaturated fatty acid ester is of the same type as the polyunsaturated fatty acid corresponding to the polyunsaturated fatty acid salt.
2. The method according to claim 1, wherein the raw material does not contain an organic solvent.
3. The method according to claim 1, wherein the raw material further comprises an organic solvent, and the mass-to-volume ratio of the polyunsaturated fatty acid ester to the organic solvent in the raw material is g: mL is 1: (2-5).
4. The method according to any one of claims 1 to 3, wherein the mass ratio of the polyunsaturated fatty acid ester to the polyunsaturated fatty acid salt in the starting material is (15-1500): 1.
5. The method of producing the phospholipid-type polyunsaturated fatty acid according to claim 4, wherein the polyunsaturated fatty acid ester is a polyunsaturated fatty acid ethyl ester or a polyunsaturated fatty acid glyceride, and the mass ratio of the polyunsaturated fatty acid ethyl ester or the polyunsaturated fatty acid glyceride to the polyunsaturated fatty acid salt in the raw material is (20-200): 1.
6. The method for producing the phospholipid-based polyunsaturated fatty acid according to any one of claims 1 to 5, wherein the lipase is a 1,3-specific lipase;
preferably, the mass ratio of the lipase to the raw materials except the lipase in the raw materials is 10-30%.
7. The method for producing a phospholipid-type polyunsaturated fatty acid according to any one of claims 1 to 6, wherein the mass ratio of the phospholipid to the polyunsaturated fatty acid ester is 1 (3-10).
8. The method for producing the phospholipid-based polyunsaturated fatty acid according to any one of claims 1 to 7, wherein the raw material further comprises water, and the mass ratio of water to the lipase is 10 to 20%.
9. The method for producing the phospholipid-based polyunsaturated fatty acid according to any one of claims 1 to 8, wherein the polyunsaturated fatty acid is one or more selected from the group consisting of an omega-3 fatty acid, an omega-6 fatty acid, docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, octadecatrienoic acid, octadecadienoic acid and octadecatetraenoic acid.
10. The method for producing the phospholipid-based polyunsaturated fatty acid according to any one of claims 1 to 9, wherein the reaction temperature is 45 to 65 ℃.
CN202110662785.9A 2021-06-15 2021-06-15 Preparation method of polyunsaturated fatty acid ester Pending CN115478084A (en)

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Citations (5)

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