Enzymatic enrichment of high-purity nannochloropsis polar lipids and preparation method thereof

Abstract

The present application relates to the technical field of functional lipid preparation, and particularly relates to a method for enriching high-purity Nannochloropsis polar lipid by enzyme method and a preparation method thereof. The immobilized Lip2 lipase is used to specifically and selectively hydrolyze triglyceride to generate glycerol and free fatty acid, and the polar lipid component is reserved and enriched, and then high-purity Nannochloropsis polar lipid is obtained through centrifugal filtration and molecular distillation. The present application provides a method for preparing high-purity polar lipid by enzymolysis of Nannochloropsis oil using the lipase Lip2 produced by Yarrowia lipolytica. The specific catalytic effect of the Lip2 lipase maximally reserves the polar lipid, and high-purity polar lipid product is obtained. The present application provides a method for high-efficiency enrichment of Nannochloropsis polar lipid, which is mild in reaction condition, friendly to environment, simple in steps and high in purity. The prepared polar lipid product is extremely high in purity, and fills the blank of high-efficiency preparation of Nannochloropsis oil polar lipid in the prior art.

Description

Technical Field

[0001] This invention relates to the field of functional lipid preparation technology, specifically to an enzymatic enrichment method for high-purity polar lipids from *Microcystis aeruginosa* and its preparation method. Background Technology

[0002] Polar lipids are a class of lipids whose molecular structure contains both polar groups (hydrophilic ends) and nonpolar groups (hydrophobic ends). This "amphiphilic" structure is the core characteristic that distinguishes them from neutral lipids (such as triglycerides) and determines their crucial role in life activities such as biomembrane construction, substance transport, and signal transduction. Therefore, polar lipids are a class of lipid molecules with important physiological functions, mainly including phospholipids, glycolipids, sterol lipids, and betaine esters. Their molecular structure contains polar groups and nonpolar fatty acid chains, exhibiting good emulsifying, antioxidant, and biological activity. In the food industry, they can be used as natural emulsifiers; in the health supplement field, they can regulate blood lipids and enhance immunity; in cosmetics, they can moisturize and repair the skin barrier; and in the pharmaceutical field, they can serve as drug carriers, showing a wide range of applications. Moreover, compared with the neutral form, n-3 polyunsaturated fatty acids (PUFAs) combined with polar lipids (phospholipids and glycolipids) have higher bioavailability and nutritional activity. Currently, research and product development on polar lipids mainly focus on phospholipids of different sources and structures, while other types of polar lipids lack in-depth development.

[0003] Nannochloropsis, a marine microalga, has a high lipid content of 20%-50%, and is rich in n-3 polyunsaturated fatty acids (>50%). Because Nannochloropsis oil contains not only triglycerides but also phospholipids, glyceroglycolipids, and betaine esters, among other polar lipid components, with phospholipids and glyceroglycolipids each exceeding 20%, it is a high-quality raw material for preparing high-purity polar lipids. However, triglycerides typically account for 30%-50% of the total lipids in Nannochloropsis oil. Therefore, effectively removing triglycerides from Nannochloropsis oil to achieve the enrichment and purification of polar lipids has become a key research focus in this field.

[0004] Current research on the enrichment of polar lipids mainly focuses on physical methods (such as low-temperature crystallization), solvent extraction, and column chromatography. For example, in the industrial production of egg yolk lecithin, triple countercurrent ethanol extraction can achieve a phospholipid purity of over 95%; while column chromatography using silica gel columns can recover 86.6% of the polar lipids in algal oil. For instance, Chinese patent CN108486177A, "A Method for Preparing Phospholipids Rich in ω-3 Fatty Acids from Algal Oil," utilizes a silica gel column with ethanol elution to separate and remove phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid; then, ethanol elution is used to separate and obtain phosphatidylcholine, which is then synthesized. However, physical methods suffer from low separation efficiency, high energy consumption, and limited product purity. Solvent methods are simple to operate and easy to industrialize. For example, Chinese patent CN104962590A, "A Phospholipid-type Polyunsaturated Fatty Acid Oil from Microorganisms and its Preparation Method," involves breaking down the cell walls of microorganisms and then extracting them with organic solvents to obtain an organic phase containing polyunsaturated fatty acid oils. However, it has the disadvantages of high solvent consumption and high production costs. Column chromatography offers high purity but has low loading capacity, high cost, and also uses organic solvents, which does not meet green production standards. Enzymatic hydrolysis, due to its advantages of mild reaction conditions, high specificity, minimal product damage, and environmental friendliness, is gradually becoming the preferred technology in the field of oil refining. However, current enzymatic hydrolysis technology only focuses on destroying the structure of algal cells and releasing their contents. For example, Chinese patent CN117044940A, "Krill Oil from Haematococcus pluvialis and its Preparation Method," involves further decomposing and destroying the structure of algal cells after the initial enzymatic hydrolysis, thereby releasing more astaxanthin. Cholesterol esterase is used in the secondary enzymatic hydrolysis process, which further breaks down the remaining lipids and disrupts the structure of algal cells. Further innovation and optimization are needed to develop novel, high-purity, green enrichment technologies for polar lipids using enzyme engineering. An environmentally friendly, high-purity *Microcystis aeruginosa* polar lipid preparation technology using substrate-specific lipases for efficient enrichment has not yet been reported. Summary of the Invention

[0005] The technical problem this invention aims to solve is how to develop a novel green enrichment technology for high-purity polar lipids using enzyme engineering technology, which requires further innovation and optimization; and there are no reports yet on an environmentally friendly high-purity microalgae polar lipid preparation technology using substrate-specific lipases for efficient enrichment.

[0006] To address the problems of existing technologies, this invention provides a method for preparing high-purity polar lipids from *Nyctaginus chrysogenum* oil by enzymatic hydrolysis using the lipase Lip2 produced by *Yarrowia lipolytica*. This method, through the specific catalytic action of Lip2 lipase, maximizes the retention of polar lipids, yielding a high-purity polar lipid product. It provides a method for the efficient enrichment of polar lipids from *Nyctaginus chrysogenum* with mild reaction conditions, environmental friendliness, simple steps, and high purity. The prepared polar lipid product has extremely high purity, filling the gap in the efficient preparation of polar lipids from *Nyctaginus chrysogenum* oil in existing technologies, and has broad application prospects in the food, health product, cosmetic, and pharmaceutical fields.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: a method for preparing high-purity microalgae polar lipids by enzymatic enrichment, which utilizes immobilized Lip2 lipase to specifically and selectively hydrolyze triglycerides to generate glycerol and free fatty acids, retains and enriches polar lipid components, and then obtains high-purity microalgae polar lipids by centrifugation, filtration and molecular distillation.

[0008] Furthermore, this includes the following steps:

[0009] (1) Dissolve *Chlorella vulgaris* oil in ethanol, add 0.1-1% (by weight of *Chlorella vulgaris* oil) of immobilized Lip2 lipase, and carry out a specific hydrolysis reaction at 35-55℃ for 3-12 hours; this allows the triglycerides in the *Chlorella vulgaris* oil to be specifically hydrolyzed; for example... Figure 5 and Figure 6 As shown, the amount of enzyme added is a crucial factor affecting enzyme reactions; too little enzyme leads to low reaction efficiency, while too much wastes enzyme resources. Figure 3 and Figure 4 As shown, temperature is an important factor affecting enzyme reactions; both excessively low and excessively high temperatures can lead to decreased enzyme activity and reduced reaction efficiency.

[0010] (2) After centrifugation and filtration, the upper ethanol phase is collected; centrifugation can remove immobilized enzymes and impurities, and the upper ethanol phase enriched with polar lipids is collected.

[0011] (3) Molecular distillation is performed, and the resulting heavy phase is high-purity microalgae polar lipids. Molecular distillation can effectively separate neutral lipids from polar lipids. The light phase consists of glycerol, free fatty acids, and specific pro-inflammatory mediators (SPMs), while the resulting heavy phase is high-purity polar lipids.

[0012] Furthermore, the microalgae oil in step (1) is crude oil obtained by extracting microalgae powder or refined oil after decolorization.

[0013] Furthermore, in step (1), the ratio of algal oil to ethanol is 1:2 to 1:10. The appropriate addition of ethanol solvent helps the effective contact and reaction between microalgal lipids and lipases. If the ethanol ratio exceeds 10 times, the contact area and probability with lipases will decrease, resulting in a decrease in the catalytic effect of the reaction; while if it is less than 2 times, the algal oil has low fluidity, poor stirring and catalytic reaction effect, resulting in a decrease in yield.

[0014] Furthermore, the immobilized Lip2 lipase described in step (1) is *Yersinia lipolytica* lipase Lip2. It was purchased from Beijing Kaitai New Century Biotechnology Co., Ltd., with an enzyme activity ≥1000 U / g. *Yersinia lipolytica* lipase Lip2 exhibits extremely strong specific catalytic activity against ester bonds in triglycerides, while showing almost no catalytic activity against lipid molecules containing polar groups such as phosphate groups and glycosyl groups (e.g., phospholipids, glyceroglycolipids, betaine esters). This invention utilizes the specificity of Lip2 lipase to selectively hydrolyze triglycerides in *Microcystis aeruginosa* oil, thereby achieving the enrichment and purification of polar lipids.

[0015] Furthermore, in step (1), the temperature of the enzymatic hydrolysis reaction involving the immobilized Lip2 lipase is 35-55℃, the reaction pH is 5.6-9.5, and magnetic or mechanical stirring with a stirring rate of 150-500r / min is used during the reaction. Stirring allows the enzyme and substrate to come into full contact and accelerates the occurrence of the enzyme reaction. Too low a rate will cause the enzyme and substrate to not come into full contact and react, while too high a rate will easily lead to the breakage of the immobilized enzyme and damage to the enzyme activity.

[0016] Furthermore, in step (2), centrifugation is carried out at 3000-8000 rpm for 10-30 minutes, and filtration is performed using a 0.45 μm microporous membrane. The above parameters can effectively remove solid impurities in the reaction, such as solid enzymes and fragments. Too low a centrifugation speed and too large a filter membrane pore size will not effectively remove most of the solid impurities, while too high a speed will lead to a waste of resources.

[0017] Furthermore, the conditions for the molecular distillation reaction in step (3) are a vacuum degree of less than 10 Pa and a temperature of 120-150 °C. The vacuum degree and temperature of the molecular distillation reaction can effectively separate neutral lipids and polar lipids. Too high a temperature and vacuum degree will cause polar lipids to enter the light phase, resulting in a decrease in yield, while too low a temperature and vacuum degree will prevent neutral lipids from fully entering the light phase, resulting in a decrease in purity.

[0018] A high-purity microalgae polar lipid prepared by the above method has a total polar lipid purity of ≥85% and its hygiene indicators meet food-grade or cosmetic-grade standards; wherein the phospholipid content is ≥35%, the glycerol glycolipid content is ≥40%, and the betaine ester content is ≥3%.

[0019] The beneficial effects of this invention are as follows:

[0020] (1) This invention promotes the efficient degradation of *Microcystis globulus* triglycerides through a Lip2 lipase-mediated enzymatic hydrolysis reaction, but does not mediate the hydrolysis of *Microcystis globulus* polar lipids, thus preserving them. The mechanism lies in the fact that Lip2 lipase has a very strong specific catalytic effect on the ester bonds in triglycerides, while having almost no catalytic activity on lipid molecules containing polar groups such as phosphate groups and glycosyl groups (e.g., phospholipids, glyceroglycolipids, betaine esters). Figure 1 and Figure 2 As shown, compared with other lipases, Lip2 lipase can effectively improve the extraction rate and purity of polar lipids.

[0021] (2) This invention optimizes the enrichment process (e.g. Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown in the figure, a preferred temperature of 35-55℃ and an enzyme dosage of 0.1% or higher result in high extraction rates and purity of polar lipids. Furthermore, molecular distillation of the enzymatic hydrolysis products of *Microcystis aeruginosa* oil yields polar lipids containing *Microcystis aeruginosa*, with a polar lipid extraction rate exceeding 80%. The total polar lipid content in the product can reach up to 92.35%, including phospholipids (41.3 g / 100g lipid extract), glyceroglycolipids (47.1 g / 100g lipid extract), and betaine esters (3.95 g / 100g lipid extract), significantly improving purity compared to existing technologies. Attached Figure Description

[0022] Figure 1 The effect of various lipases on the extraction rate of polar lipids is shown in the figure. Among them, TLIM is an immobilized lipase derived from *Thermophila spp.*, RMIM is an immobilized lipase derived from *Rhizopus oryzae*, Lip2 is a lipase produced by *Yarrowia lipolytica*, PLA1 is phospholipase A1, and Novozymes 435 is Novozymes lipase 435.

[0023] Figure 2 The effect of various lipases on the proportion of polar lipids in total lipids is shown in the figure. Among them, TLIM is an immobilized lipase from *Thermophila spp.*, RMIM is an immobilized lipase from *Rhizopus micranthum*, Lip2 is a lipase produced by *Yarrowia lipolytica*, PLA1 is phospholipase A1, and Novozymes 435 is Novozymes lipase 435.

[0024] Figure 3 The effect of Lip2 enzymatic hydrolysis temperature on the extraction rate of polar lipids is shown in the figure.

[0025] Figure 4 The effect of Lip2 enzymatic hydrolysis temperature on the proportion of polar lipids in total lipids is shown in the figure.

[0026] Figure 5 The effect of enzyme dosage on the extraction rate of polar lipids in the Lip2 enzymatic hydrolysis reaction is shown in the figure.

[0027] Figure 6 The effect of the amount of enzyme added in the Lip2 enzymatic hydrolysis reaction on the proportion of polar lipids in the total lipids is shown in the figure. Detailed Implementation

[0028] The following description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0029] The following embodiments can be understood as illustrating a part of the structure or method of the present invention individually, or as combining embodiments to explain the broader structure or method of the present invention. Unless otherwise specified, the instruments, reagents, materials, etc., involved in the following embodiments are all conventional instruments, reagents, materials, etc., already existing in the prior art and obtainable through legitimate commercial channels. Unless otherwise specified, the experimental methods, detection methods, etc., involved in the following embodiments are all conventional experimental methods, detection methods, etc., already existing in the prior art.

[0030] In the following examples, the raw material used was microalgae oil (APE-BO, purchased from Xiaozao Agricultural Technology Co., Ltd.).

[0031] The ethanol and 0.45μm microporous filter membrane used were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0032] The immobilized Lip2 lipase used was purchased from Beijing Kaitai New Century Biotechnology Co., Ltd. This enzyme has a very strong specific catalytic effect on the ester bonds in triglycerides and diglycerides, but it has no specific hydrolytic ability on phospholipids, glycolipids, and betaine esters.

[0033] Example 1: Accurately weigh 50g of *Chlorella vulgaris* oil and place it in a 1L reactor. Add 250mL of ethanol and heat to 40°C in a constant-temperature magnetic stirrer. Add 0.1% immobilized Lip2 lipase and stir at 500rpm for 5 hours at 40°C. After the reaction, quickly centrifuge the mixture at 8000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation apparatus with the evaporation chamber temperature set to 150°C and the condensation chamber temperature set to 35°C, and simultaneously activate the vacuum system. The system maintains a stable vacuum of <10 Pa and uses a peristaltic pump to feed the pretreated raw materials into the evaporation chamber, collecting the light phase and heavy phase products. The lipid composition of these products is determined. The heavy phase product contains 4.7 g / 100g of free fatty acids, 6.4 g / 100g of glycerides, 38.4 g / 100g of phospholipids, 46.6 g / 100g of glycolipids, and 3.9 g / 100g of betaine esters. The total polar lipid purity is as high as 88.9%, and the total polar lipid extraction rate is 85.6%.

[0034] Example 2: Accurately weigh 50g of *Chlorella vulgaris* oil and place it in a 1L reactor. Add 250mL of ethanol and heat to 40°C in a constant-temperature magnetic stirrer. Add 0.5% immobilized Lip2 lipase and stir at 500rpm for 5 hours at 40°C. After the reaction, quickly centrifuge the mixture at 8000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation apparatus with the evaporation chamber temperature set to 150°C and the condensation chamber temperature set to 35°C, and simultaneously activate the vacuum system. The system maintains a stable vacuum of <10 Pa and uses a peristaltic pump to feed the pretreated raw materials into the evaporation chamber, collecting the light phase and heavy phase products. The lipid composition of these products is determined. The heavy phase product contains 3.2 g / 100g of free fatty acids, 5.9 g / 100g of glycerides, 39.6 g / 100g of phospholipids, 47.2 g / 100g of glycolipids, and 3.8 g / 100g of betaine esters. The total polar lipid purity is as high as 90.6%, and the total polar lipid extraction rate is 88.1%.

[0035] Example 3: Accurately weigh 50g of *Microcystis aeruginosa* oil and place it in a 1L reactor. Add 250mL of ethanol and heat to 45°C in a constant-temperature magnetic stirrer. Add 0.3% immobilized Lip2 lipase and stir at 500rpm for 5 hours at 45°C. After the reaction, quickly centrifuge the mixture at 8000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation apparatus with the evaporation chamber temperature set to 140°C and the condensation chamber temperature set to 30°C. A vacuum system was used to stably control the vacuum level at <10 Pa. The pretreated raw materials were fed into the evaporation chamber by a peristaltic pump, and the light phase product and heavy phase product were collected. The lipid composition of the above products was determined. Among them, the heavy phase product contained 5.1 g of free fatty acids per 100 g of lipid extract, 9.8 g of glycerides per 100 g of lipid extract, 36.2 g of phospholipids per 100 g of lipid extract, 45.4 g of glyceroglycolipids per 100 g of lipid extract, and 3.5 g of betaine per 100 g of lipid extract. The purity of total polar lipids was as high as 85.1%, and the extraction rate of total polar lipids was 83.2%.

[0036] Example 4: Accurately weigh 50g of *Chlorella vulgaris* oil and place it in a 1L reactor. Add 250mL of ethanol and heat to 45°C in a constant-temperature magnetic stirrer. Add 0.1% immobilized Lip2 lipase and stir at 500rpm for 5 hours at 45°C. After the reaction, quickly centrifuge the mixture at 8000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation apparatus with the evaporation chamber temperature set to 140°C and the condensation chamber temperature set to 30°C. Simultaneously start the vacuum distillation process. An empty system was used to stably control the vacuum level at <10 Pa. The pretreated raw materials were fed into the evaporation chamber by a peristaltic pump, and the light phase product and heavy phase product were collected. The lipid composition of the above products was determined. Among them, the heavy phase product contained 3.9 g / 100g of free fatty acids, 10.9 g / 100g of glycerides, 36.5 g / 100g of phospholipids, 44.8 g / 100g of glycolipids, and 3.9 g / 100g of betaine esters. The purity of total polar lipids was as high as 85.2%, and the extraction rate of total polar lipids was 86.0%.

[0037] Example 5: Accurately weigh 50g of *Chlorella vulgaris* oil and place it in a 1L reactor. Add 250mL of ethanol and heat to 40°C in a constant-temperature magnetic stirrer. Add 0.05%, 0.1%, 0.3%, 0.5%, and 1% immobilized Lip2 lipase, respectively. Stir and react for 5 hours at 500rpm and 40°C. After the reaction, quickly centrifuge the mixture at 8000rpm for 10 minutes. Then, further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation apparatus, setting the evaporation chamber temperature to 130°C and the condensation chamber temperature to 35°C. Simultaneously, start the vacuum system and stabilize the vacuum at <10Pa. Then, use peristalsis... The pump delivers the pretreated raw materials into the evaporation chamber, collecting the light phase and heavy phase products. The lipid composition of these products is determined. Under 0.05% enzyme addition, the heavy phase product contains 17.5 g / 100g free fatty acids, 27.7 g / 100g glycerides, 16.4 g / 100g phospholipids, 35.6 g / 100g glycolipids, and 2.8 g / 100g betaine esters, with a total polar lipid purity of 54.8% and a total polar lipid extraction rate of 61.5%. Under 0.1% enzyme addition, the heavy phase product contains 4.65 g / 100g free fatty acids and 10.1 g / 100g glycerides. The lipid extract contained 36.7 g / 100g phospholipids, 44.8 g / 100g glycerol glycolipids, and 3.5 g / 100g betaine esters, with a total polar lipid purity of 85.1% and an extraction rate of 88.2%. Under 0.3% enzyme addition, the heavy phase product contained 5.4 g / 100g free fatty acids, 9.2 g / 100g glycerol esters, 36.6 g / 100g phospholipids, 45.1 g / 100g glycerol glycolipids, and 3.6 g / 100g betaine esters, with a total polar lipid purity of 85.3% and an extraction rate of 86.0%. (0.5%) Under the enzyme-added conditions, the heavy phase product contained 5.3 g / 100g of free fatty acids, 9.3 g / 100g of glycerides, 37.1 g / 100g of phospholipids, 45.6 g / 100g of glyceroglycolipids, and 3.7 g / 100g of betaine, with a total polar lipid purity of 86.4% and a total polar lipid extraction rate of 87.9%. Under the 1% enzyme-added conditions, the heavy phase product contained 6.6 g / 100g of free fatty acids, 8.0 g / 100g of glycerides, 36.9 g / 100g of phospholipids, 44.9 g / 100g of glyceroglycolipids, and 3 g / 100g of betaine.A 5g / 100g lipid extract showed a total polar lipid purity of up to 85.3% and a total polar lipid extraction rate of 86.9%. Single-factor experiments revealed that under enzyme addition conditions of 0.1%-1%, the total polar lipid purity could reach over 85%, ensuring the completeness of the reaction and the high purity of the product.

[0038] Comparative Example 1: Accurately weigh 50g of *Microcystis aeruginosa* oil and place it in a 1L reactor. Add 250mL of ethanol and heat to 40℃ in a constant-temperature magnetic stirrer. Add 0.1% of TL IM, RM IM, PLA1, and Novozymes 435 lipase (purchased from Novozymes (China) Biotechnology Co., Ltd.). Stir and react for 5 hours at 500rpm and 40℃. After the reaction, the mixture is rapidly centrifuged at 8000rpm for 10 minutes and further filtered under pressure using a 0.45μm microporous membrane (pressure 0.2MPa). Start the molecular distillation apparatus with the evaporation chamber temperature set to 150℃ and the condensation chamber temperature set to 30℃. Start the vacuum system with a vacuum degree <10Pa. Use a peristaltic pump to send the pretreated raw material into the evaporation chamber and collect the light phase product and the heavy phase product. Determine the lipid composition of the above product, including TL... Under the catalytic action of IM, free fatty acids accounted for 36.2 g / 100g of lipid extract, glycerides for 16.7 g / 100g of lipid extract, phospholipids for 19.7 g / 100g of lipid extract, glyceroglycolipids for 20.6 g / 100g of lipid extract, and betaine esters for 6.8 g / 100g of lipid extract. The purity of total polar lipids was only 47.1%, with a small increase in purity, and the extraction rate of total polar lipids was 15.6%. RM Under the catalysis of IM, free fatty acids accounted for 34.3 g / 100g of lipid extract, glycerides for 20.1 g / 100g of lipid extract, phospholipids for 15.9 g / 100g of lipid extract, glyceroglycolipids for 22.9 g / 100g of lipid extract, and betaine esters for 6.8 g / 100g of lipid extract, with a total polar lipid purity of 45.6% and a total polar lipid extraction rate of 20.7%. Under the catalysis of PLA1, free fatty acids accounted for 27.8 g / 100g of lipid extract, glycerides for 25.9 g / 100g of lipid extract, and phospholipids for 14.7 g / 100g of lipid extract. The total lipid extract contained 25.4 g of glyceroglycolipids and 6.2 g of betaine esters, with a total polar lipid purity of 46.3% and a total polar lipid extraction rate of 6.6%. Catalyzed by Novozymes 435 lipase, the total lipid extract contained 27.8 g of free fatty acids, 25.9 g of glycerides, 14.7 g of phospholipids, 25.4 g of glyceroglycolipids, and 6.2 g of betaine esters, with a total polar lipid purity of 51.2% and a total polar lipid extraction rate of 17.4%. Compared to Examples 1-5, the enzymatic enrichment effects of TL IM, RM IM, PLA1, and Novozymes 435 lipase were all lower than those of Lip2 lipase (e.g., ...). Figure 1 and Figure 2 As shown in the figure, this demonstrates the superior effect of the Lip2 lipase method in enriching polar lipids.

[0039] Comparative Example 2: Accurately weigh 50g of *Chlorella vulgaris* oil and place it in a 1L reactor. Add 250mL of ethanol and heat in a constant-temperature magnetic stirrer. Add 0.5% immobilized Lip2 lipase and maintain a stirring speed of 500rpm. Stir the mixture at 25, 30, 35, 40, 45, 50, 55, and 60℃ for 5 hours respectively. After the reaction, quickly centrifuge the mixture at 8000rpm for 10 minutes and further filter it under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation apparatus, setting the evaporation chamber temperature to 140℃ and the condensation chamber temperature to 30℃. Simultaneously, start the vacuum system and stabilize the vacuum degree at <10Pa. Use a peristaltic pump to transfer the pre-evaporated oil to the condenser. The processed raw materials were sent to an evaporation chamber, and the light phase and heavy phase products were collected. The lipid composition of these products was determined. At 25°C, free fatty acids accounted for 10.5 g / 100g of the lipid extract, glycerides for 48.5 g / 100g, phospholipids for 23.5 g / 100g, glycolipids for 10.6 g / 100g, and betaine esters for 6.9 g / 100g. The total polar lipid purity was only 41.0%, and the total polar lipid extraction rate was 37.5%. At 30°C, free fatty acids accounted for 11.8 g / 100g of the lipid extract, glycerides for 41.1 g / 100g, and phospholipids for 20.3 g / 100g. The total lipid extract contained 21.7 g / 100g of glycerol glycolipids and 5.1 g / 100g of betaine esters, with a total polar lipid purity of only 47.1% and a total polar lipid extraction rate of 54.5%. At 40℃, the extract contained 3.2 g / 100g of free fatty acids, 5.9 g / 100g of glycerides, 39.6 g / 100g of phospholipids, 47.2 g / 100g of glycerol glycolipids, and 3.8 g / 100g of betaine esters, with a total polar lipid purity as high as 90.6% and a total polar lipid extraction rate of 88.1%. At 45℃, the extract contained 3.9 g / 100g of free fatty acids and 6.4 g / 100g of glycerides. The lipid extract contained 39.7g / 100g of phospholipids, 45.9g / 100g of glyceroglycolipids, and 3.5g / 100g of betaine, with a total polar lipid purity of 89.1% and a total polar lipid extraction rate of 89.7%. At 50℃, the lipid extract contained 7.8g / 100g of free fatty acids, 3.8g / 100g of glycerides, 38.3g / 100g of phospholipids, 42.9g / 100g of glyceroglycolipids, and 3.8g / 100g of betaine, with a total polar lipid purity of 88.4% and a total polar lipid extraction rate of 90.5%. At 55℃, the lipid extract contained 8g / 100g of free fatty acids.The total lipid extract was 0 g / 100g, containing 6.4 g / 100g of glycerides, 36.1 g / 100g of phospholipids, 45.1 g / 100g of glyceroglycolipids, and 4.4 g / 100g of betaine. The total polar lipid purity was 85.6%, and the total polar lipid extraction rate was 75.1%. However, at 60℃, the total lipid extract contained 5.9 g / 100g of free fatty acids, 29.1 g / 100g of glycerides, 30.5 g / 100g of phospholipids, 32.9 g / 100g of glyceroglycolipids, and 7.5 g / 100g of betaine. The total polar lipid purity was only 65.0%, and the total polar lipid extraction rate was 30.5%. The comparative examples above show that enzymatic hydrolysis at temperatures between 35-55℃ results in higher purity and extraction rate of total polar lipids. Temperatures of 25, 30, or 60℃ all negatively impact the enzyme reaction, leading to a decrease in purity and extraction rate.

[0040] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0041] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0042] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A method for preparing high-purity polar lipids from *Microcystis aeruginosa* using enzymatic enrichment, characterized in that: Immobilized Lip2 lipase was used to specifically and selectively hydrolyze triglycerides to generate glycerol and free fatty acids, retaining and enriching polar lipid components. After centrifugation, filtration and molecular distillation, high-purity microalgae polar lipids were obtained. The immobilized Lip2 lipase was produced by Yersinia lipase Lip2.

2. The preparation method according to claim 1, characterized in that... Includes the following steps: (1) Dissolve the microspiral oil in ethanol, add 0.1-1% of the mass of the microspiral oil immobilized Lip2 lipase and carry out a specific hydrolysis reaction at 35-55℃ for 3-12 hours; the immobilized Lip2 lipase is Lip2 produced by Yeastia lipolytica. (2) After centrifugation and filtration, the upper ethanol phase is collected; (3) Molecular distillation is performed, and the resulting heavy phase is high-purity microalgae polar lipid.

3. The preparation method according to claim 2, characterized in that: The microalgae oil in step (1) is crude oil obtained by extracting microalgae powder or refined oil after decolorization.

4. The preparation method according to claim 2, characterized in that: In step (1), the ratio of algal oil to ethanol is 1:2 to 1:

10.

5. The preparation method according to claim 2, characterized in that: In step (1), the pH of the enzymatic hydrolysis reaction involving immobilized Lip2 lipase is 5.6-9.5, and magnetic or mechanical stirring with a stirring rate of 150-500 r / min is used during the reaction.

6. The preparation method according to claim 2, characterized in that: Step (2) Centrifugation is carried out at 3000-8000 rpm for 10-30 minutes, and the filtration is carried out using a 0.45μm microporous membrane.

7. The preparation method according to claim 2, characterized in that: The conditions for the molecular distillation reaction in step (3) are a vacuum of less than 10 Pa and a temperature of 120-150 °C.

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