Enzymatic enrichment of high-purity glycolipids from microalgae and preparation method thereof

Abstract

This invention relates to the field of functional lipid preparation technology, specifically to an enzymatic enrichment method for high-purity microalgal glycerol glycolipids and its preparation. This invention provides an enzymatic enrichment method for high-purity microalgal glycerol glycolipids and its preparation, utilizing the substrate specificity of Candida antarctica lipase B, which selectively hydrolyzes triglycerides and phospholipids while retaining and enriching the indigestible glycerol glycolipid components. This maximizes the retention of glycerol glycolipids, resulting in high-purity microalgal glycerol glycolipid products. This provides a method for the efficient enrichment of microalgal glycerol glycolipids that is mild, environmentally friendly, simple, and yields high purity.

Description

Technical Field

[0001] This invention relates to the field of functional lipid preparation technology, specifically to an enzymatic enrichment method for high-purity microalgal glycerol glycolipids and its preparation method. Background Technology

[0002] Glycerol glycolipids, as a class of lipids containing glycosidic groups, may not receive as much attention as proteins and carbohydrates, but they possess unique and important nutritional functions, and their research has profound significance for the health field. Due to their unique chemical structure and biological activity, in addition to being a core component of plant and microbial cell membranes, glycerol glycolipids exhibit activity and application potential in anti-aging (related to neurodegenerative diseases), anti-inflammation, and immune regulation, making them of significant research value in the field of health foods. After ingestion, they can participate in the construction of the body's own cell membranes, enhancing membrane stability and fluidity, ensuring cell signal transduction and substance transport, and especially maintaining the integrity of the intestinal mucosal barrier, reducing the penetration of harmful substances. Simultaneously, some glycerol glycolipids alleviate chronic inflammation and inhibit cancer cell proliferation by regulating the release of inflammatory factors, supporting immune function and making them one of the important functional lipids. Precisely because of their unique biological activities, such as antioxidant, antibacterial, antitumor, antiviral, and immune-enhancing activities, they have broad application prospects in scientific research, pharmaceutical preparation, food and cosmetic development, and other fields.

[0003] Microalgae are important food and novel food raw material resources. Their nutritional value stems from their rich bioactive components, including functional lipids, proteins, and various functional compounds, which synergistically endow microalgae foods with unique nutritional and health benefits. Glycerol glycolipids are important functional lipids from microalgae, mainly including monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and thioisorhamnosyldiacylglycerol (SQDG). Compared with higher plants, using microalgae to produce glycerol glycolipids has more advantages and broad application prospects in functional lipid production. Microalgae such as *Micrococcus pluvialis* and *Haematococcus pluvialis* are rich in glycerol glycolipids and, as important commercially available microalgae for food raw materials, have the potential to become the substrate organisms for functional lipid production. However, the lack of extraction and purification methods for microalgal glycerol glycolipids restricts their application in the food industry.

[0004] Currently, there are few studies and patents on the extraction of glycolipids from natural sources. Previous studies have employed combined column chromatography to prepare high-purity monogalactosylglycerol from microalgae; or used a combination of Q Sepharose and hydrophobic columns to extract thioisorhamnosylglycerol. For example, Chinese patent CN117642219A, "Method for Separation and / or Purification of Glycolipids," discloses a method of contacting an adsorbent with a pre-selected recovery solution to recover the predetermined desired type of glycolipid from the loaded material on the adsorbent; and obtaining the recovered predetermined desired type of glycolipid from the processing unit. However, while these techniques produce high-purity products, they still suffer from complex purification processes (requiring the sequential use of two chromatographic columns), high costs, and safety and environmental concerns due to the reliance on organic solvents such as chloroform during extraction, leading to challenges in large-scale production.

[0005] Enzymatic hydrolysis, with its advantages of mild reaction conditions, high specificity, minimal product damage, and environmental friendliness, has gradually become the preferred technology in the field of oil refining. Therefore, further innovation and optimization are needed to develop novel, green enrichment technologies for high-purity glycerol glycolipids using enzyme engineering, while simultaneously exploring alternatives to low-toxicity solvents such as ethanol to reduce production costs and environmental risks. Summary of the Invention

[0006] The technical problem this invention aims to solve is how to develop a novel, high-purity, green enrichment technology for glycerol glycolipids using enzyme engineering technology. Further innovation and optimization are needed, while alternative solutions such as ethanol with low toxicity need to be explored to reduce production costs and environmental risks.

[0007] To address the problems of existing technologies, this invention provides an enzymatic enrichment method for high-purity microalgal glycerol glycolipids and its preparation method. It utilizes the substrate specificity of Candida antarctica lipase B, which selectively hydrolyzes triglycerides and phospholipids while retaining and enriching the indigestible glycerol glycolipid components. This maximizes the retention of glycerol glycolipids, resulting in high-purity microalgal glycerol glycolipid products. This provides a method for the efficient enrichment of microalgal glycerol glycolipids that is mild, environmentally friendly, simple, and yields high purity.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: a method for enzymatically enriching high-purity microalgal glycerol glycolipids, which utilizes Candida antarcticis lipase B to specifically and selectively hydrolyze triglycerides and phospholipids, retains and enriches glycerol glycolipids, and then obtains high-purity microalgal glycerol glycolipids after centrifugation filtration, molecular distillation, and water washing.

[0009] Furthermore, this includes the following steps:

[0010] (1) Dissolve the crude microalgae oil raw material in ethanol, add 5%~40% of Candida antarcticis lipase B (in liquid and immobilized forms), and carry out a specific hydrolysis reaction at 35~65℃ for 5-16 hours. The mechanism is that Candida antarcticis lipase B has substrate specificity, which can rapidly hydrolyze triglycerides and phospholipids in microalgae, while having almost no catalytic activity on glycolipids. The amount of enzyme added and the temperature are important factors affecting the enzyme reaction. Too little enzyme will lead to low enzyme reaction efficiency, while too much will waste enzyme resources. Too low or too high temperature will lead to a decrease in enzyme activity and low reaction efficiency, while 35~60℃ can ensure the hydrolytic activity of Candida antarcticis lipase B.

[0011] (2) After the reaction, centrifuge and filter to remove lipase and collect the ethanol phase; remove lipase and impurities and collect the upper ethanol phase enriched with polar lipids.

[0012] (3) Molecular distillation is carried out under conditions of vacuum degree less than 10 Pa and temperature of 110-150℃, and the resulting heavy phase is collected. Molecular distillation can effectively separate neutral lipids and polar lipids. Excessive temperature and vacuum degree will cause the free fatty acids produced by hydrolysis to also enter the heavy phase, resulting in a decrease in yield. Excessive temperature and vacuum degree will prevent glycerol glycolipids from fully entering the heavy phase, resulting in a decrease in purity.

[0013] (4) After the obtained heavy phase is evaporated to remove ethanol, water is added and stirred. Then, the phase is allowed to stand and separate into layers. Water-soluble impurities are removed, and the oil phase is collected to obtain high-purity microalgae glycerol glycolipid.

[0014] Furthermore, the crude oil raw material of microalgae in step (1) includes crude oil or refined oil obtained by ethanol extraction from one or more of the following: *Microcystis aeruginosa*, *Haematococcus pluvialis*, *Spirulina*, *Chlorella vulgaris*, *Euglena*, *Dunaliella salina*, *Chlamydomonas reinhardtii*, and *Scenedesmus*.

[0015] Furthermore, in step (1), the ratio of algal oil to ethanol is 1:3 to 1:20. The appropriate addition of ethanol solvent helps the effective contact and reaction between microalgal lipids and lipases. If the ethanol ratio exceeds 20 times, the contact area and probability with Candida antarcticis lipase B decrease, resulting in a decrease in the catalytic effect of the hydrolysis reaction; while if it is less than 3 times, the algal oil has low fluidity, poor stirring and hydrolysis catalytic reaction effect, resulting in a decrease in yield.

[0016] Furthermore, in step (1), a magnetic or mechanical stirring speed of 100-600 r / min is used during the reaction. Stirring allows the enzyme and substrate to come into full contact, accelerating the enzyme reaction. Too low a speed will prevent the enzyme and substrate from coming into full contact and reacting, while too high a speed will easily lead to the breakage of the immobilized enzyme and damage to enzyme activity.

[0017] Furthermore, in step (2), centrifugation is carried out at 1500-6000 rpm for 5-20 minutes; filtration is performed using a 0.45 μm microporous membrane. Filtration and centrifugation 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 cannot effectively remove most of the solid impurities, while too high a speed will lead to waste of resources.

[0018] Furthermore, the washing conditions in step (4) are a temperature of 40-60℃, a water volume of 20-40% of the oil volume, and a stirring time of 10-30 minutes. After stirring, the mixture is allowed to stand and separate into layers to remove water-soluble impurities. The oil phase is then collected to obtain a high-purity microalgae glycerol glycolipid product. The washing temperature of 40-60℃ ensures that water-soluble impurities are effectively dissolved. If the water volume is less than 20%, the impurity removal effect will be poor. Stirring for 10-30 minutes can fully dissolve water-soluble impurities and achieve effective impurity removal through layering.

[0019] A microalgal glycerol glycolipid prepared by the above method has a total glycolipid content of ≥75% and its hygiene indicators meet food-grade or cosmetic-grade standards. The microalgal glycerol glycolipid comprises monogalactose diacylglycerol (MGDG), digalactose diacylglycerol (DGDG), and thioisorhamnosyldiacylglycerol (SQDG).

[0020] The beneficial effects of this invention are as follows:

[0021] (1) This invention utilizes an enzymatic hydrolysis reaction mediated by Candida antarctica lipase B to rapidly hydrolyze fatty acids of triglycerides and phospholipids in microalgae, but it cannot specifically hydrolyze microalgal glycolipids (MGDG and DGDG, such as...). Figure 1 and Figure 2 As shown in the figure, microalgal glycerol glycolipids are retained, achieving further enrichment. The mechanism lies in the substrate specificity of *Candida antarcticus* lipase B, while exhibiting almost no catalytic activity towards glycerol glycolipids. In the preferred embodiments, *Candida antarcticus* lipase B (CALB) and its immobilized form (Novozymes 435) show the best enzymatic enrichment effects (e.g., ...). Figure 3 and Figure 4 As shown in the figure, it has the highest yield and purity.

[0022] (2) This invention further purifies the product through molecular distillation and water washing to obtain high-purity glycerol glycolipids. The highest extraction rate of glycerol glycolipids is 91.3%, and the highest content of glycerol glycolipids in the product can reach 87.6%, which is a significant improvement in purity compared with the prior art. For example, the preferred temperature is 35-60℃, under which the purity of glycerol glycolipids is relatively high, reaching more than 82% (e.g. Figure 5 As shown), the preferred enzyme dosage is 5%-30%, under which conditions the purity of glycerol glycolipids is high, reaching over 79% (e.g. Figure 6 (As shown). Attached Figure Description

[0023] Figure 1 The results of the specificity of various lipases for the hydrolysis of glycerol glycolipid MGDG are shown in the figure. Among them, TL IM is the immobilized lipase TLIM from *Thermophilus spp.*, RM IM is the immobilized lipase RMIM from *Rhizopus oryzae*, LowP is the phospholipase Quara® Low P, PLA1 is the phospholipase A1, TL100L is the *Aspergillus oryzae* lipase TL100L, Palatase 20000L is the Novozymes Palatase 20000L cheese flavor lipase, CALB is the *Candida antarcticis* lipase B, and Novozymes 435 is the Novozymes lipase 435.

[0024] Figure 2 The results of the specificity of various lipases for the hydrolysis of glycerol glycolipid DGDG are shown in the figure. Among them, TL IM is the immobilized lipase TLIM from *Thermophilus spp.*, RM IM is the immobilized lipase RMIM from *Rhizopus oryzae*, LowP is the phospholipase Quara® Low P, PLA1 is the phospholipase A1, TL100L is the *Aspergillus oryzae* lipase TL100L, Palatase 20000L is the Novozymes Palatase 20000L cheese flavor lipase, CALB is the *Candida antarcticis* lipase B, and Novozymes 435 is the Novozymes lipase 435.

[0025] Figure 3 The effect of various lipases on the extraction rate of glycerol glycolipids from Haematococcus pluvialis is shown in the figure. Among them, TL IM is an immobilized lipase derived from *Thermophilus spp.*, RM IM is an immobilized lipase derived from *Rhizopus oryzae*, LowP is phospholipase Quara® Low P, PLA1 is phospholipase A1, TL100L is *Aspergillus oryzae* lipase TL100L, Palatase 20000L is Novozymes Palatase 20000L cheese flavor lipase, CALB is *Candida antarcticis* lipase B, and Novozymes 435 is Novozymes lipase 435.

[0026] Figure 4The effect of various lipases on the purity of enriched Haematococcus pluvialis glycerol glycolipids is shown in the figure. Among them, TL IM is the immobilized lipase TLIM from *Thermophilus spp.*, RM IM is the immobilized lipase RMIM from *Rhizopus oryzae*, LowP is phospholipase Quara® Low P, PLA1 is phospholipase A1, TL100L is *Aspergillus oryzae* lipase TL100L, Palatase 20000L is Novozymes Palatase 20000L cheese flavor lipase, CALB is *Candida antarcticis* lipase B, and Novozymes 435 is Novozymes lipase 435.

[0027] Figure 5 Figure showing the effect of CALB enzymatic hydrolysis temperature on the purity of enriched Haematococcus pluvialis glycerol glycolipids.

[0028] Figure 6 The effect of enzyme dosage on the purity of Haematococcus pluvialis glycerol glycolipids enriched in the CALB enzymatic hydrolysis reaction is shown in the figure. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] The raw materials used in this invention include crude oil of Haematococcus pluvialis (Yunnan Aierfa Biotechnology Co., Ltd., XQSRM0711) and crude oil of Micrococcus pluvialis (Guangxi Xiaozao Agricultural Technology Co., Ltd., APEEEBW).

[0032] The ethanol and 0.45μm microporous filter membrane used in this invention were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0033] The Antarctic Candida lipase B used in this invention was purchased from Novozymes (China) Biotechnology Co., Ltd., S35565. This enzyme has a very strong specific catalytic effect on ester bonds in triglycerides, diglycerides, and phospholipids (including PC / PE / PS / PI / PA), but it does not have a specific hydrolytic ability on ester bonds in glycolipids.

[0034] Example 1: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 40°C in a constant-temperature magnetic stirrer. Add 5% of Candida antarctica lipase B and stir at 300rpm for 5 hours at 40°C. After the reaction, quickly centrifuge the mixture at 5000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation equipment with the evaporation chamber temperature set to 150°C and the condensation chamber temperature set to 35°C. Simultaneously, start the vacuum system and stabilize the vacuum at <10Pa. Use a peristaltic pump to send the pretreated raw material into the evaporation chamber and collect the light and heavy phase products. Evaporate the heavy phase product to remove ethanol, heat to 60°C, add distilled water (20% of the weight of algal oil), stir and hydrate for 30 minutes, then allow it to stand and separate into layers to remove water-soluble impurities. Collect the oil phase to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed that the product contained 82.2g of glycerol glycolipids per 100g of lipid extract, with an extraction rate of 77.1%.

[0035] Example 2: Accurately weigh 50g of *Candida albicans* oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 40°C in a constant-temperature magnetic stirrer. Add 5% of *Candida antarcticis* lipase B and stir at 300rpm for 5 hours at 40°C. After the reaction, quickly centrifuge the mixture at 5000rpm for 10 minutes and further filter under pressure using a 0.45μm microporous membrane (pressure 0.2MPa). Start the molecular distillation equipment with the evaporation chamber temperature set to 150°C and the condensation chamber temperature set to 35°C. Simultaneously, start the vacuum system and stabilize the vacuum at <10Pa. Use a peristaltic pump to send the pretreated raw material into the evaporation chamber and collect the light and heavy phase products. Evaporate the heavy phase product to remove ethanol, heat to 60°C, add distilled water (20% of the weight of algal oil), stir and hydrate for 30 minutes, allow it to stand and separate into layers to remove water-soluble impurities, and collect the oil phase to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed that the product contained 89.6g of glycerol glycolipids per 100g of lipid extract, with an extraction rate of 76.8%.

[0036] Example 3: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 40°C in a constant-temperature magnetic stirrer. Add 30% of Candida antarctica lipase B and stir at 300rpm and 40°C for 5 hours. After the reaction, quickly centrifuge the mixture at 5000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation equipment with the evaporation chamber temperature set to 150°C and the condensation chamber temperature set to 35°C. Simultaneously, start the vacuum system and stabilize the vacuum at <10Pa. Use a peristaltic pump to send the pretreated raw material into the evaporation chamber and collect the light and heavy phase products. Evaporate the heavy phase product to remove ethanol, heat to 60°C, add distilled water (20% of the weight of algal oil), stir and hydrate for 30 minutes, then allow it to stand and separate into layers to remove water-soluble impurities. Collect the oil phase to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed that the product contained 89.8g of glycerol glycolipids per 100g of lipid extract, with an extraction rate of 85.2%.

[0037] Example 4: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 60°C in a constant-temperature magnetic stirrer. Add 20% of Candida antarctica lipase B and stir at 300rpm and 60°C for 5 hours. After the reaction, quickly centrifuge the mixture at 5000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation equipment with the evaporation chamber temperature set to 150°C and the condensation chamber temperature set to 35°C. Simultaneously, start the vacuum system and stabilize the vacuum at <10Pa. Use a peristaltic pump to send the pretreated raw material into the evaporation chamber and collect the light and heavy phase products. Evaporate the heavy phase product to remove ethanol, heat to 60°C, add distilled water (20% of the weight of algal oil), stir and hydrate for 30 minutes, then allow it to stand and separate into layers to remove water-soluble impurities. Collect the oil phase to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed that the product contained 83.8g of glycerol glycolipids per 100g of lipid extract, with an extraction rate of 81.7%.

[0038] Example 5: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 40°C in a constant-temperature magnetic stirrer. Add 30% of Candida antarctica lipase B and stir at 300rpm and 40°C for 12 hours. After the reaction, the mixture is quickly centrifuged at 5000rpm for 10 minutes and further filtered under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation equipment with the evaporation chamber temperature set to 130°C and the condensation chamber temperature set to 35°C. Simultaneously, start the vacuum system and stabilize the vacuum at <10Pa. Use a peristaltic pump to send the pretreated raw material into the evaporation chamber and collect the light and heavy phase products. Evaporate the heavy phase product to remove ethanol, heat to 60°C, add distilled water (20% of the weight of algal oil), stir and hydrate for 30 minutes, and allow it to stand and separate into layers to remove water-soluble impurities. Collect the oil phase to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed that the product contained 82.9g of glycerol glycolipids per 100g of lipid extract, with an extraction rate of 84.8%.

[0039] Example 6: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 50°C in a constant-temperature magnetic stirrer. Add 20% of Candida antarctica lipase B and stir at 300rpm and 50°C for 16h. After the reaction, the mixture is quickly centrifuged at 5000rpm for 10min and further filtered under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the molecular distillation equipment with the evaporation chamber temperature set to 150°C and the condensation chamber temperature set to 35°C. Simultaneously, start the vacuum system and stabilize the vacuum at <10Pa. Use a peristaltic pump to send the pretreated raw material into the evaporation chamber and collect the light and heavy phase products. Evaporate the heavy phase product to remove ethanol, heat to 60°C, add distilled water (20% of the weight of algal oil), stir and hydrate for 30 minutes, and allow it to stand and separate into layers to remove water-soluble impurities. Collect the oil phase to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed that the product contained 91.3g of glycerol glycolipids per 100g of lipid extract, with an extraction rate of 87.6%.

[0040] Comparative Example 1: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 40℃ in a constant temperature magnetic stirrer. Add 30% lipase (including lipase TLIM, lipase RMIM, phospholipase Low P, phospholipase A1, lipase TL100L, and cheese flavor lipase Palatase). 20,000 L of lipase CALB and lipase 435 (both CALB and 435 belong to the Candida antarcticis lipase B) were stirred and reacted at 300 rpm and 40°C for 5 hours. After the reaction, the mixture was quickly centrifuged at 5,000 rpm for 10 minutes and then further filtered under pressure (0.2 MPa) using a 0.45 μm microporous membrane. The molecular distillation equipment was started, with the evaporation chamber temperature set to 150°C and the condensation chamber temperature set to 35°C. The vacuum system was started and the vacuum degree was stably controlled at <10 Pa. The pretreated raw material was sent into the evaporation chamber by a peristaltic pump, and the light phase product and the heavy phase product were collected. The heavy phase product was evaporated to remove ethanol, heated to 50°C, and distilled water (20% of the weight of algal oil) was added. After stirring and hydrating for 30 minutes, the mixture was allowed to stand and separate into layers to remove water-soluble impurities. The oil phase was collected to obtain a high-purity microalgal glycerol glycolipid product. After testing, the following results were obtained: Under the action of lipase TLIM, the product contained 65.1g / 100g of glycerol glycolipids in the lipid extract, with an extraction rate of 12.8%; under the action of lipase RMIM, the product contained 46.6g / 100g of glycerol glycolipids in the lipid extract, with an extraction rate of 14.7%; under the action of phospholipase LowP, the product contained 36.0g / 100g of glycerol glycolipids in the lipid extract, with an extraction rate of 44.7%; under the action of phospholipase A1, the product contained 38.4g / 100g of glycerol glycolipids in the lipid extract, with an extraction rate of 26.6%; and under the action of lipase TL100L, the product contained 35.7g / 100g of glycerol glycolipids in the lipid extract. The extraction rate of glycerol glycolipids was 8.7%; with the action of Palatase 20000L lipase, the glycerol glycolipid content in the product was 47.4g / 100g of lipid extract, and the extraction rate was 34.0%; conversely, with the action of free Candida antarcticis lipase B (i.e., CALB lipase), the glycerol glycolipid content in the product was 84.5g / 100g of lipid extract, and the extraction rate was 82.6%; with the action of immobilized Candida antarcticis lipase B (i.e., lipase 435 lipase), the glycerol glycolipid content in the product was 86.2g / 100g of lipid extract, and the extraction rate was 82.3%. The above data prove that Candida antarcticis lipase B has a significant improvement in the extraction rate and purity of glycerol glycolipids.

[0041] Comparative Example 2: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 50℃ in a constant-temperature magnetic stirrer. Add 20% of Candida antarctica lipase B and stir at 300rpm for 16h at 50℃. After the reaction, quickly centrifuge the mixture at 5000rpm for 10min and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. The distillation equipment was set with the evaporation chamber at 100℃ and the condensation chamber at 35℃. A vacuum system was simultaneously activated to maintain a stable vacuum of <10Pa. Pretreated raw materials were fed into the evaporation chamber via a peristaltic pump, and the light and heavy phase products were collected. The heavy phase product was evaporated to remove ethanol, heated to 40-60℃, and distilled water (20% of the algal oil weight) was added. After stirring and hydrating for 10-30 minutes, the mixture was allowed to stand and separate into layers to remove water-soluble impurities. The oil phase was then collected to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed that the product contained 61.2g / 100g of glycerol glycolipids and 29.4g / 100g of free fatty acids, with a glycerol glycolipid extraction rate of 71.6%. However, the free fatty acids were not effectively removed.

[0042] Comparative Example 3: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat in a constant-temperature magnetic stirrer. Add 20% of Candida antarctica lipase B and stir for 16 hours at 25-65℃ and 300rpm. After the reaction, quickly centrifuge the mixture at 5000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the reaction. The distillation equipment is set with the evaporation chamber temperature at 150℃ and the condensation chamber temperature at 35℃. The vacuum system is activated simultaneously to stabilize the vacuum at <10Pa. The pretreated raw material is fed into the evaporation chamber via a peristaltic pump, and the light and heavy phase products are collected. The heavy phase product is evaporated to remove ethanol, heated to 40-60℃, and distilled water (20% of the weight of algal oil) is added. After stirring and hydrating for 10-30 minutes, the mixture is allowed to stand and separate into layers to remove water-soluble impurities. The oil phase is then collected to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed the following results: At 25℃, the product contained 24.8 g / 100g of glycerol glycolipids in the lipid extract, with an extraction rate of 25.7%; at 30℃, the extraction rate was 45.4 g / 100g, with an extraction rate of 46.1%; at 35℃, the extraction rate was 81.0 g / 100g, with an extraction rate of 81.5%; at 40℃, the extraction rate was 83.1 g / 100g, with an extraction rate of 79.8%; and at 45℃, the extraction rate was... The extraction rates were as follows: 89.3 g / 100g lipid extract, with an extraction rate of 84.6%; at 50℃, 86.7 g / 100g lipid extract, with an extraction rate of 82.1%; at 55℃, 86.9 g / 100g lipid extract, with an extraction rate of 86.0%; at 60℃, 86.4 g / 100g lipid extract, with an extraction rate of 88.1%; and at 65℃, 60.9 g / 100g lipid extract, with an extraction rate of 54.6%. These data demonstrate that *Candida antarcticis* lipase B exhibits high efficiency in its specific hydrolysis reaction at temperatures between 35-65℃, and the purity of glycerol glycolipids in the product is greater than 80%.

[0043] Comparative Example 4: Accurately weigh 50g of Haematococcus pluvialis oil and place it in a 1L reactor. Add 500mL of ethanol and heat to 40℃ in a constant-temperature magnetic stirrer. Add 1-40% of Candida antarctica lipase B and stir at 300rpm for 5 hours at 40℃. After the reaction, quickly centrifuge the mixture at 5000rpm for 10 minutes and then further filter under pressure (0.2MPa) using a 0.45μm microporous membrane. Start the reaction. The distillation equipment is set with the evaporation chamber temperature at 150℃ and the condensation chamber temperature at 35℃. The vacuum system is activated simultaneously to stabilize the vacuum at <10Pa. The pretreated raw material is fed into the evaporation chamber via a peristaltic pump, and the light and heavy phase products are collected. The heavy phase product is evaporated to remove ethanol, heated to 40-60℃, and distilled water (20% of the weight of algal oil) is added. After stirring and hydrating for 10-30 minutes, the mixture is allowed to stand and separate into layers to remove water-soluble impurities. The oil phase is then collected to obtain a high-purity microalgal glycerol glycolipid product. Testing revealed the following concentrations of glycerol glycolipids in the product: 1% *Candida antarcticis* lipase B; 2%; 5%; 10%; 2%; 2%; 2%; 2%; 30%; and 40%. The above data proves that when the amount of Candida antarcticis lipase B added is between 5% and 40%, the purity of glycerol glycolipids in the product is greater than 80%, and the enrichment effect is optimal.

[0044] 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.

[0045] 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.

[0046] 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 microalgal glycerol glycolipids by enzymatic enrichment, characterized in that: High-purity microalgal glycerol glycolipids were obtained by using Candida antarctica lipase B to specifically and selectively hydrolyze triglycerides and phospholipids, retaining and enriching glycerol glycolipids, and then obtaining them through centrifugation, filtration, molecular distillation, and washing.

2. The preparation method according to claim 1, characterized in that... Includes the following steps: (1) Dissolve the crude oil of microalgae in ethanol, add 5%~40% of Candida antarctica lipase B, and carry out a specific hydrolysis reaction at 35~65℃ for 5-16 hours. (2) After the reaction, centrifuge and filter to remove the immobilized enzyme and collect the ethanol phase; (3) Molecular distillation was carried out under conditions of vacuum less than 10 Pa and temperature of 110-150 °C, and the resulting heavy phase was collected; (4) After the obtained heavy phase is evaporated to remove ethanol, water is added and stirred. Then, the phase is allowed to stand and separate into layers. Water-soluble impurities are removed, and the oil phase is collected to obtain high-purity microalgae glycerol glycolipid.

3. The preparation method according to claim 2, characterized in that: The microalgae crude oil raw material in step (1) includes crude oil or decolorized refined oil obtained by extraction of one or more of the following: *Microcystis aeruginosa*, *Haematococcus pluvialis*, *Spirulina*, *Chlorella vulgaris*, *Euglena*, *Dunaliella salina*, *Chlamydomonas reinhardtii*, and *Scenedesmus*.

4. The preparation method according to claim 2, characterized in that: In step (1), the ratio of algal oil to ethanol is 1:3 to 1:

20.

5. The preparation method according to claim 2, characterized in that: In step (1), magnetic or mechanical stirring with a stirring rate of 100-600 r / min is used during the reaction.

6. The preparation method according to claim 2, characterized in that: In step (2), centrifugation is carried out at 1500-6000 rpm for 5-20 minutes; filtration is performed using a 0.45 μm microporous membrane.

7. The preparation method according to claim 2, characterized in that: Step (4) The conditions for water washing are a temperature of 40-60℃, the amount of water added is 20-40% of the amount of oil, the stirring time is 10-30 minutes, and then let it stand still to separate into layers to remove water-soluble impurities. The oil phase can be collected to obtain a high-purity microalgae glycerol glycolipid product.

Images