CN115478084B - A kind of preparation method of polyunsaturated fatty acid ester - Google Patents
A kind of preparation method of polyunsaturated fatty acid ester Download PDFInfo
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Abstract
本发明涉及多不饱和脂肪酸酯合成技术领域，具体涉及一种多不饱和脂肪酸酯的制备方法。本发明提供的制备方法中，磷脂型多不饱和脂肪酸由包括如下组分的原料经酶促反应制备得到：多不饱和脂肪酸酯、多不饱和脂肪酸盐、磷脂和脂肪酶，其中，所述多不饱和脂肪酸酯对应的多不饱和脂肪酸与所述多不饱和脂肪酸盐对应的多不饱和脂肪酸的类型相同。该方法对于原料的选择较为宽泛，不依赖于有机溶剂，适用于多种反应环境，能够在不含有机溶剂的体系中实现较高的脂肪酸接入率，制备得到的磷脂型多不饱和脂肪酸中多不饱和脂肪酸的含量显著提高，适于大规模推广应用。The present invention relates to the technical field of polyunsaturated fatty acid ester synthesis, and in particular to a method for preparing polyunsaturated fatty acid ester. In the preparation method provided by the present invention, phospholipid-type polyunsaturated fatty acids are prepared by enzymatic reaction of raw materials including the following components: polyunsaturated fatty acid esters, polyunsaturated fatty acid salts, phospholipids and lipase, wherein the polyunsaturated fatty acids corresponding to the polyunsaturated fatty acid esters are of the same type as the polyunsaturated fatty acids corresponding to the polyunsaturated fatty acid salts. The method has a wide selection of raw materials, is independent of organic solvents, is applicable to a variety of reaction environments, can achieve a high fatty acid access rate in a system without organic solvents, and the content of polyunsaturated fatty acids in the prepared phospholipid-type polyunsaturated fatty acids is significantly improved, which is suitable for large-scale promotion and application.
Description
Technical Field
The invention relates to the technical field of polyunsaturated fatty acid ester synthesis, in particular to a preparation method of phospholipid polyunsaturated fatty acid.
Background
Ester products of polyunsaturated fatty acids (PUFAs) are generally classified as ethyl ester, triglyceride and phospholipid types. The physiological function of PUFAs varies due to their molecular form in fat. The PUFA in the form of phospholipid has the physiological functions of phospholipid and PUFA, and has more stable structure, is not easy to oxidize and is more beneficial to the absorption of human bodies. The research shows that the absorption rate of the ethyl DHA in the human body is only about 20%, the human body absorption rate of the glycerol DHA can be improved to 70%, and the absorption rate of the phospholipid DHA in the human body is the highest and exceeds 99%.
Natural phospholipids rich in EPA/DHA are mainly found in marine animals, and their yields are far from satisfactory for human demand, and the artificial synthesis of phospholipid-type PUFAs is another route of acquisition. The fatty acid composition of the phospholipid is changed, and transesterification or acidolysis can be generally selected. The ester exchange reaction is to exchange the ester compound of the target fatty acid with the fatty acid on the phospholipid under the enzymatic action, and the acidolysis reaction is to exchange the free fatty acid with the fatty acid on the phospholipid. At present, under acidolysis reaction, a satisfactory fatty acid access rate can be obtained, and the enzymatic transesterification reaction is more green and environment-friendly, but the access effect is difficult to achieve the same effect as that of acidolysis reaction.
The Chinese patent CN 104962590A discloses a phospholipid type polyunsaturated fatty acid grease from microorganism and a preparation method, and the method utilizes the grease containing docosahexaenoic acid to carry out lipase enzymatic transesterification with powdery phospholipid to prepare the phospholipid type polyunsaturated fatty acid grease from microorganism. However, the method involves a two-step enzyme method, has complex reaction and high production cost.
Chinese patent No. 106893747A discloses a method for preparing n-3 polyunsaturated fatty acid phosphatide by enzymatic transesterification of n-3 polyunsaturated fatty acid or its ester derivatives and soybean phosphatide, but the enzymatic reaction only plays a good role in the participation of organic solvent, and the access rate in the absence of 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 polyunsaturated fatty acid. The method is independent of the addition of organic solvents in the process of preparing phospholipid polyunsaturated fatty acids, and has a good fatty acid access effect.
Aiming at the problem that the prior art needs to rely on a solvent system formed by an organic solvent for preparing phospholipid polyunsaturated fatty acid by transesterification, and the better fatty acid access rate cannot be ensured after the organic solvent is removed, the invention develops a method for preparing phospholipid polyunsaturated fatty acid with high efficiency and without relying on the organic solvent system. In the research and development process, the organic solvent system is very critical for ensuring the high access rate of fatty acid in the preparation process of phospholipid polyunsaturated fatty acid, and after the organic solvent is removed, even if the reaction system is optimized in many aspects and substances which are disclosed in the prior art and are possibly beneficial to promoting the transesterification reaction are added, the access rate of fatty acid under the condition of existence of the organic solvent is difficult to achieve. According to the invention, the polyunsaturated fatty acid salt corresponding to the polyunsaturated fatty acid ester is introduced into the reaction system, so that the access rate of the fatty acid in a solvent-free system can be remarkably improved, and the content of the fatty acid in the prepared phospholipid polyunsaturated fatty acid can be further improved. The invention also discovers that the polyunsaturated fatty acid salt can achieve better fatty acid access rate under the condition of a solvent-free reaction system or a solvent-containing reaction system, 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 from the following raw materials by an enzymatic reaction, wherein the polyunsaturated fatty acid comprises polyunsaturated fatty acid esters, polyunsaturated fatty acid salts, phospholipids and lipase, and the polyunsaturated fatty acid corresponding to the polyunsaturated fatty acid esters has the same type as the polyunsaturated fatty acid corresponding to the polyunsaturated fatty acid salts.
The present invention has found that polyunsaturated fatty acid esters, polyunsaturated fatty acid salts and phospholipids are essential substrates in the above reaction materials to ensure fatty acid access in solvent-free systems, and removal of one or replacement of the other substrate with a similar substrate significantly reduces fatty acid access.
In the above raw material system, the type of polyunsaturated fatty acid in the polyunsaturated fatty acid salt is the same as the type of polyunsaturated fatty acid in the polyunsaturated fatty acid ester. The polyunsaturated fatty acid salt may be a single polyunsaturated fatty acid salt or a mixture of polyunsaturated fatty acid salts, and likewise the polyunsaturated fatty acid ester may be a single polyunsaturated fatty acid ester or a mixture of polyunsaturated fatty acid esters. If one of the polyunsaturated fatty acid esters and the polyunsaturated fatty acid salts is a mixture, the polyunsaturated fatty acid having the highest content in the polyunsaturated fatty acid salts is of the same type as the polyunsaturated fatty acid having the highest content in the polyunsaturated fatty acid esters. For example, if the polyunsaturated fatty acid ester feedstock contains 70% EPA ethyl ester, the polyunsaturated fatty acid salt is EPA metal salt.
In the present invention, polyunsaturated fatty acid esters include, but are not limited to, polyunsaturated fatty acid alkyl esters, polyunsaturated fatty acid glycerides.
As an embodiment, the raw material for preparing the phospholipid polyunsaturated fatty acid does not contain an organic solvent.
As another embodiment, the raw material further comprises an organic solvent, wherein the mass volume ratio g of the polyunsaturated fatty acid ester to the organic solvent in the raw material is 1 (2-5).
For both embodiments, the mass ratio of the polyunsaturated fatty acid ester to the polyunsaturated fatty acid salt in the starting material is preferably (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 ester or polyunsaturated fatty acid glyceride to the polyunsaturated fatty acid salt in the raw material is preferably (20-200): 1.
The ratio of polyunsaturated fatty acid ester to polyunsaturated fatty acid salt is controlled within the above range, which is more favorable for improving the fatty acid access 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 supplied by adding a fat or oil raw material containing the polyunsaturated fatty acid ester and the polyunsaturated fatty acid salt.
Preferably, the lipase is a1, 3-specific lipase.
The present inventors have found that the use of a1, 3-specific lipase in the reaction system of the present invention can significantly increase the fatty acid access rate compared to a non-directional lipase.
Further preferably, the mass ratio of the lipase to the raw materials other than lipase in the raw materials is 10 to 30%.
Preferably, the mass ratio of phospholipid to polyunsaturated fatty acid ester in the raw material is 1 (3-10).
In the preparation method, the raw materials also comprise water, and the mass ratio of the water to the lipase in the raw materials is 10-20%.
The polyunsaturated fatty acid according to the present invention may be one or more selected from omega-3 fatty acids, omega-6 fatty acids, docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, stearidonic acid or stearidonic acid.
The polyunsaturated fatty acid content in the polyunsaturated fatty acid ester used in the present invention is preferably controlled to not less than 65%.
The polyunsaturated fatty acid salts used in the present invention are preferably metal salts corresponding to the polyunsaturated fatty acids, including but not limited to sodium salts, potassium salts, and the like.
The phospholipid used in the present invention is preferably glycerophospholipid. The phospholipid is preferably in the form of a powder.
In the preparation method of the invention, the reaction temperature of the enzymatic reaction is 45-65 ℃. The preferred reaction time is from 4 to 24 hours.
Specifically, the invention provides a method for preparing phospholipid polyunsaturated fatty acid by using a solvent-free system, which comprises the following steps:
(1) Mixing polyunsaturated fatty acid ester, polyunsaturated fatty acid salt, phospholipid and water, wherein the mass ratio of polyunsaturated fatty acid ester to polyunsaturated fatty acid salt is (15-1500): 1, and the mass ratio of phospholipid to polyunsaturated fatty acid ester is (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 the reaction raw materials except the lipase is 10-30%, the mass ratio of water to the lipase is 10-20%, and performing enzymatic transesterification for 4-24 hours at 45-65 ℃;
(3) Separating the oil mixture to obtain phospholipid polyunsaturated fatty acid.
Preferably, in step (1), the mixing is at room temperature. More preferably at room temperature by ultrasonic or stirring.
In the step (3), after the reaction is finished, the lipase is removed by centrifugation or suction filtration, and the oil mixture is collected.
The preparation method of the phospholipid polyunsaturated fatty acid has the beneficial effects that the preparation method of the phospholipid polyunsaturated fatty acid is wide in raw material selection, does not depend on an organic solvent system, can realize good fatty acid access rate under the condition of a solvent-free reaction system or a solvent-containing reaction system, and can particularly realize the great improvement of the fatty acid access rate in the solvent-free system, and the polyunsaturated fatty acid content in the prepared phospholipid polyunsaturated fatty acid is obviously improved. The method can be suitable for various reaction environments, wherein the preparation method of the solvent-free system also has the advantages of friendly production conditions, high safety and suitability for large-scale popularization and application.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The raw material parameters used in the following examples were as follows, randomized lipase 435 was Novozym435, lipase TL was NovozymeTLIM, immobilized lipase RMIM was Novozyme LipozymeRM IM, powdered phospholipids were new hundred vitamins (acetone insoluble 95%), EPA ethyl ester oil was obtained by extraction and transesterification of Nanochloropsis, and DHA ethyl ester oil was obtained by extraction and transesterification of Schizochytrium.
Example 1
The embodiment provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gEPA ethyl ester oil (area content 70.1%), 0.36gEPA sodium, 6g of powdery phospholipid and 0.72g of water, and dispersing by ultrasonic treatment for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA area content in the phospholipid of 28.0%.
Example 2
The embodiment provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gEPA ethyl ester oil (area content 70.1%), 0.36gEPA sodium, 6g of powdery phospholipid and 0.72g of water, and dispersing by ultrasonic treatment for 5min to obtain a substrate;
(2) Adding 3.6g lipase TL to the substrate of step (1), placing in a 65 ℃ water bath and stirring at 300rpm for 18h;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA with an area content of 18.5% in the phospholipid.
Example 3
The embodiment provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gEPA ethyl ester oil (EPA content is 80%), 0.60gEPA sodium, 4.5g powder phospholipid and 0.72g water, and dispersing by ultrasonic treatment for 5min to obtain substrate;
(2) 3.6g of immobilized lipase RM IM was added to the substrate of step (1), placed in a 65℃water bath, and stirred at 300rpm for 18 hours.
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA area content in the phospholipid of 28.2%.
Example 4
The embodiment provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gEPA ethyl ester oil (area content 70.1%), 0.18gEPA sodium, 3.6g powder phospholipid and 0.36g water, and dispersing by ultrasonic treatment for 5min to obtain substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA area content in the phospholipid of 27.4%.
Example 5
The embodiment provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gDHA ethyl ester oil (area content 71%), 0.58gDHA potassium, 6g of powdery phospholipid and 0.72g of water, and dispersing by ultrasonic treatment for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
Separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the DHA area content in the phospholipid of 25.6%.
Example 6
The embodiment provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing the mixture oil (containing 18gEPA ethyl ester oil (70.1% of area content) and 0.09gEPA sodium and 0.72g of water) with 6g of powder phospholipid, and dispersing by ultrasonic treatment for 5min to obtain substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA with an area content of 23.5% in the phospholipid.
Example 7
The embodiment provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 25.2gEPA glyceride (area content 70.1%), 0.36gEPA sodium, 0.72g water and 6g powder phospholipid, and dispersing by ultrasonic for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA area content in the phospholipid of 7.2%.
Example 8
The embodiment provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gEPA ethyl ester oil (area content 70.1%), 0.36gEPA sodium, 6g of powdery phospholipid and 0.72g of water, adding 50ml of n-hexane, and dispersing by ultrasonic treatment for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA area content in the phospholipid of 27.3%.
Comparative example 1
The comparative example provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gEPA ethyl ester oil (area content 70.1%), 0.72g water and 6g powder phospholipid, and dispersing by ultrasonic for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA with an area content of 10.3% in the phospholipid.
Comparative example 2
The comparative example provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18g of EPA fatty acid, 0.36gEPA g of sodium, 6g of powder phospholipid and 0.72g of water, and dispersing by ultrasonic treatment for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA with an area content of 5.0% in the phospholipid.
Comparative example 3
The comparative example provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gEPA ethyl ester oil (area content 70.1%), 0.36gEPA sodium, 0.72g water and 6g powder phospholipid, and dispersing by ultrasonic treatment for 5min to obtain a substrate;
(2) Adding 3.6g of randomized lipase 435 to the substrate of step (1), placing in a 65 ℃ water bath, and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain EPA area content in the phospholipid of 6.8%.
Comparative example 4
The comparative example provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) 17.5g of DHA ethyl ester (DHA content is 71%) and 5g of soybean phospholipid raw material are mixed, and 40ml of normal hexane is added to obtain an oil mixture;
(2) Preheating the fat mixture obtained in the step (1) to 52 ℃;
(3) Adding 0.604gDHA potassium salt and 0.06g of water, adding 3g of immobilized lipase RM IM into the reaction mixture obtained in the step (2), and performing ultrasonic dispersion, and reacting at 55 ℃ for 8 hours to obtain an oil composition;
separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the DHA area content in the phospholipid of 7.4%.
Comparative example 5
The comparative example provides a preparation method of phospholipid polyunsaturated fatty acid, which comprises the following steps:
(1) Mixing 18gEPA sodium, 6g of powdery phospholipid, 50mL of normal hexane and 0.72g of water, and dispersing by ultrasonic treatment for 5min to obtain a substrate;
(2) Adding 3.6g of immobilized lipase RM IM to the substrate in the step (1), placing in a water bath at 65 ℃ and stirring at 300rpm for 18 hours;
(3) After the reaction was completed, the mixture obtained by the reaction was centrifuged at 6000rpm for 5 minutes, and the lower immobilized enzyme was removed to collect the oil-and-fat mixture.
And (3) separating phospholipid in the oil mixture, and performing gas chromatography detection after methyl esterification to obtain the EPA with an area content of 3.0% in the phospholipid.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
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| Title |
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| Incorporation of omega-3 polyunsaturated fatty acids into soybean lecithin: effect of amines and divalent cations on transesterification by lipases;Nabil Marsaoui 等;World J Microbiol Biotechnol;20130609;第2233–2238页 * |
| Synthesis of DHA/EPA-rich phosphatidylcholine by immobilized phospholipase A1: effect of water addition and vacuum condition;Daoming Li 等;Bioprocess Biosyst Eng;20160423;第1305-1314页 * |
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| CN115478084A (en) | 2022-12-16 |
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