Green extraction process and refining method of sea cucumber lipid capable of selectively retaining active phospholipid

Abstract

The invention provides a green extraction process of sea cucumber lipid capable of selectively retaining active phospholipids, which comprises the following steps: performing supercritical CO2 fluid extraction and ethanol extraction on sea cucumber blanching liquid powder to obtain an ethanol extracting solution containing crude sea cucumber lipid; the method comprises the following steps: diluting an ethanol extracting solution containing crude sea cucumber lipid, uniformly mixing the diluted ethanol extracting solution with a compound adsorbent, and carrying out adsorption decoloration and filtration under a preset temperature and stirring condition to obtain sea cucumber lipid; the sea cucumber lipid is subjected to molecular distillation, light components are removed through first-stage distillation, target lipid is collected through second-stage distillation, and the phospholipid-rich sea cucumber lipid is obtained. The invention initiates a set of extraction and purification process which is mild in the whole process and dominated by a physical method, and is used for solving the contradiction between activity destruction and purification efficiency in the extraction and purification process of the sea cucumber lipid with high added value. The core of the method is that the yield of phospholipid is improved, violent chemical alkali refining and high-temperature treatment are abandoned, and the innovative combination of a molecular distillation technology and a low-temperature adsorption technology is adopted, so that not only are impurities (free fatty acid, pigment and peculiar smell) removed efficiently, but also the natural activity and sensory quality of sea cucumber phospholipid are retained to the greatest extent.

Description

Technical Field

[0001] This invention belongs to the field of sea cucumber processing and relates to a green extraction process and refining method for sea cucumber lipids that selectively retains active phospholipids. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Phospholipids are an important class of lipids, widely distributed in biological membranes and possessing a variety of biological functions. However, current sea cucumber lipid extraction and purification processes still suffer from problems such as low extraction rates, decreased phospholipid activity, and solvent residues.

[0004] On the other hand, sea cucumber blanching liquid powder is a powder made from the effective components of sea cucumber blanching liquid through vacuum low-temperature concentration, nanofiltration desalination, and spray drying technologies. Sea cucumber blanching liquid is a liquid produced during sea cucumber processing and is rich in nutrients such as lipids (79% of the sea cucumber body wall lipids enter the blanching liquid), polysaccharides, saponins, proteins, and amino acids. Spray drying technology preserves these nutrients in powder form, facilitating storage and transportation while retaining their active ingredients. However, further extraction of the active components from the sea cucumber blanching liquid powder is still necessary, such as the high-content phospholipid-rich sea cucumber lipids, to achieve higher utilization value.

[0005] A study has disclosed a method for extracting sea cucumber saponins using sea cucumber blanching solution. The blanching solution is concentrated under reduced pressure, spray-dried, and then extracted with supercritical CO2 fluid to obtain a solid powder free of oils. The solid powder is then extracted with alcohol, concentrated by filtration, and redissolved in water. Phospholipids, cerebrosides, and other compounds are removed from the solution by centrifugation. The resulting supernatant is then subjected to adsorption chromatography using a macroporous adsorption resin column, eluted with ethanol, and the eluent is freeze-dried under vacuum to obtain the sea cucumber saponin extract. However, the method does not address the issue of highly active phospholipid extraction.

[0006] Therefore, the industry urgently needs to develop methods for the efficient and selective extraction and deep purification of sea cucumber phospholipids without damaging their activity, so as to achieve high yield, high purity, high activity retention and environmentally friendly preparation of sea cucumber lipids. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a green extraction process for sea cucumber lipids that selectively retains active phospholipids. This invention pioneers a "mild, physical-based" extraction and purification process to resolve the contradiction between activity degradation and purification efficiency in the extraction and purification of high-value-added sea cucumber lipids. Its core lies in increasing phospholipid yield while abandoning harsh chemical alkali refining and high-temperature treatments. It employs an innovative combination of physical impurity removal technology, low-temperature adsorption technology, and molecular distillation, which not only efficiently removes impurities (free fatty acids, pigments, and odors) but also maximizes the yield of sea cucumber phospholipids, preserving their natural activity and sensory qualities.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a green extraction process for sea cucumber lipids that selectively retains active phospholipids, comprising: The sea cucumber blanching liquid powder was subjected to supercritical CO2 fluid extraction to remove impurities, followed by ethanol extraction to obtain an ethanol extract containing crude sea cucumber lipids. The ethanol extract containing crude sea cucumber lipids was mixed with a compound adsorbent in a certain proportion, and adsorption and decolorization were carried out under preset temperature and stirring conditions. After filtration, sea cucumber lipids were obtained. The sea cucumber lipids were subjected to molecular distillation. The first stage of distillation removed light components, and the second stage of distillation collected the target lipids, namely, phospholipid-rich sea cucumber lipids.

[0009] This invention is the first to employ a method combining supercritical CO2 fluid extraction with enhanced extraction using organic solvents. Using sea cucumber blanching liquid powder as raw material, the optimal process conditions for removing neutral lipids such as triglycerides and pigments via supercritical CO2 fluid extraction were first determined. Then, using phospholipid content as an evaluation index, ethanol was used for highly selective extraction of phospholipids. Furthermore, this method is the first to propose and construct a "mild, efficient, and green" integrated purification process specifically tailored to the lipid characteristics of phospholipid-rich sea cucumbers. This process, targeting the characteristics of sea cucumber lipids, reconstructed parameters and innovatively coupled steps in existing technologies for unit operations, successfully solving the problems of easy loss and activity retention of sea cucumber phospholipids during purification.

[0010] In a second aspect, the present invention provides phospholipid-rich sea cucumber lipids prepared by the above-described method.

[0011] A third aspect of the present invention provides the application of the phospholipid-rich sea cucumber lipids prepared by the above method or the above phospholipid-rich sea cucumber lipids in the preparation of functional foods.

[0012] Beneficial effects of the present invention (1) The present invention uses supercritical CO2 fluid extraction combined with organic solvent enhanced extraction to extract phospholipid-rich sea cucumber lipids, realizing the high-value utilization of sea cucumber blanching liquid powder.

[0013] (2) The entire operation process is gentle, employing high-vacuum molecular distillation for simultaneous deacidification and deodorization, and low-temperature adsorption for decolorization. This fundamentally avoids the irreversible damage to active ingredients caused by strong alkalis and high temperatures, providing a completely new pathway for the preparation of highly active sea cucumber lipids.

[0014] (3) The entire process is carried out at low temperatures and without strong chemical reagents, which perfectly protects the molecular structure of phospholipids. The phospholipid recovery rate can be kept stable at >90%, and the biological activity is preserved to the maximum extent. This achieves a leap from "rough purification" to "activity preservation", and greatly enhances the nutritional and economic value of the product.

[0015] (4) The product is crystal clear, with a ruby ​​red color, and has no odor. The acid value and peroxide value are extremely low, and the sensory evaluation "overall acceptability" score has improved from unacceptable (<2 points) to very acceptable (>4.5 points). A high-end sea cucumber lipid product with "edible pleasure" has been truly produced from sea cucumber blanching liquid powder, breaking the limitation that it can only be used as an industrial raw material.

[0016] (5) The highly integrated operation process, the two core processes of "adsorption" and "distillation" are closely linked. Molecular distillation can replace the two traditional units of "deacidification" and "deodorization" in one step, which greatly shortens the process and increases efficiency.

[0017] (6) Through in-depth exploration of sea cucumber lipids and cell experiments, the bioactivity of sea cucumber lipids was further clarified, and the application value of phospholipid-rich sea cucumber lipids was clarified.

[0018] This invention is not a simple improvement on existing technologies, but rather a completely new solution with different principles and significantly improved technical effects. It successfully solves the long-standing technical bottleneck of "purification equals destruction" that has hindered the industrialization of sea cucumber lipids. The products produced have achieved significant improvements in activity retention rate, sensory quality, and safety, possessing outstanding substantive characteristics and remarkable progress. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1. Comparison of sea cucumber lipids before and after decolorization; Figure 2. Content percentage of various lipid components in sea cucumber; Figure 3. Composition of lipid components in sea cucumber; Figure 4. Effect of purified sea cucumber lipids on cell survival. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or product instructions. Similarly, unless otherwise specified, the test methods of this invention are performed in accordance with conventional methods in the art or industry-standard methods or practices. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0023] As described in the background section, the current extraction of sea cucumber lipids primarily employs the organic solvent method. This method utilizes the solubility of lipids in organic solvents, selecting a suitable solvent and optimizing extraction conditions to extract the lipids. The crude lipid extract is then processed using traditional vegetable oil refining techniques such as alkali refining to remove acid, high-temperature deodorization, and bleaching with clay. However, this process has the following drawbacks: 1. Traditional organic solvent extraction methods have poor selectivity for sea cucumber phospholipids. Phospholipids tend to arrange themselves at the oil-water interface, resulting in complex extracts with high levels of impurities such as neutral lipids, pigments, and free fatty acids. This leads to low purity and yield of the target active substance (phospholipids), and a heavy burden on subsequent purification.

[0024] 2. Existing processes often involve high temperatures, long operating times, or strong chemical conditions (such as alkali refining and deacidification) during extraction and purification, which can easily lead to the oxidation, hydrolysis, or isomerization of important active components in sea cucumber lipids that are thermally unstable and have a high degree of chemical unsaturation (such as Omega-3 fatty acids EPA / DHA, phospholipids, etc.), thereby reducing their nutritional value and biological activity.

[0025] 3. Some methods use toxic solvents such as chloroform and n-hexane, posing safety and residue risks, which is inconsistent with the development trend of modern green food processing.

[0026] 4. Conventional refining steps such as deacidification, decolorization, and deodorization are mostly designed for ordinary vegetable oils. Their harsh conditions (such as high-temperature and high-pressure steam deodorization, strong alkali deacidification, and violent bleaching) are fundamentally contradictory to the activity requirements of highly unsaturated sea cucumber phospholipids. There is a lack of a dedicated integrated process that can simultaneously achieve efficient purification and maximum retention of activity.

[0027] In summary, the efficiency of extracting sea cucumber lipids using a single lipid extraction method is very low, and the harsh process conditions during purification also seriously hinder the efficient development and utilization of sea cucumber lipids.

[0028] The supercritical CO2 fluid extraction combined with organic solvent-enhanced extraction method employed in this invention solves the problem of low extraction efficiency in traditional methods and yields sea cucumber lipids with high phospholipid content. Subsequent purification processes are conducted under mild conditions, avoiding phospholipid loss. Its advantages are manifested in the following aspects: 1. Supercritical CO2 first precisely removes a large number of impurities such as neutral oil and pigments, allowing subsequent ethanol extraction to efficiently extract phospholipids, which can increase the phospholipid yield by more than 20%.

[0029] 2. The low-temperature and gentle subsequent purification process avoids the damage to phospholipid molecules caused by traditional high-temperature and strong alkali treatments, resulting in higher lipid activity, lower oxidation levels, and better quality.

[0030] 3. Green and safe solvents (CO2, food-grade ethanol) are used throughout the process, which is in line with the development trend of modern green food processing.

[0031] In summary, the method of the present invention can greatly improve the extraction efficiency and quality of sea cucumber lipids.

[0032] In a first aspect, the present invention provides a green extraction process for sea cucumber lipids that selectively retains active phospholipids, comprising: The sea cucumber blanching liquid powder was subjected to supercritical CO2 fluid extraction to remove impurities, followed by ethanol extraction to obtain an ethanol extract containing crude sea cucumber lipids. The ethanol extract containing crude sea cucumber lipids was mixed with a compound adsorbent in a certain proportion, and adsorption and decolorization were carried out under preset temperature and stirring conditions. After filtration, sea cucumber lipids were obtained. The sea cucumber lipids were subjected to molecular distillation. The first stage of distillation removed light components, and the second stage of distillation collected the target lipids, namely, phospholipid-rich sea cucumber lipids.

[0033] This invention uses sea cucumber blanching liquid powder as raw material. First, supercritical CO2 fluid extraction is performed. Through orthogonal experiments, the aim is to remove impurities such as nonpolar lipids and pigments. Extraction pressure, extraction temperature, and extraction time are the factors to be investigated, and the optimal process parameters are screened out. The raw material after supercritical extraction is then extracted with organic solvent. The optimal process conditions are determined through single-factor optimization experiments. Functional lipids rich in phospholipids are obtained by utilizing the principle of like dissolves like.

[0034] The crude lipids are subjected to low-temperature composite adsorption for deimpurity and decolorization. Under low temperature and inert gas protection, a specific ratio of composite adsorbent (diatomaceous earth-activated carbon-washed bentonite = 8:1:1) is used to treat the crude extract, simultaneously removing pigments, phospholipid oxides, and insoluble impurities. Then, molecular distillation is performed for deacidification and deodorization. A two-stage molecular distillation technique is used, under high vacuum and relatively low temperature, to first distill off free fatty acids and odor components, and then precisely collect the phospholipid-rich fraction. This achieves deacidification and deodorization in one step, completely avoiding the damage caused by traditional chemical methods and high-temperature treatments.

[0035] The conditions of supercritical CO2 fluid extraction and the type of entrainer affect the retention rate of phospholipids and the removal efficiency of nonpolar lipids. Therefore, this invention studies the types of entrainers and the conditions of supercritical CO2 fluid extraction. Preferably, the supercritical CO2 fluid extraction temperature is 30℃-50℃, the pressure is 25MPa-35MPa, the extraction time is 1-3h, and the entrainer used is isopropanol:ethyl acetate (3:1), with the amount of entrainer being 20% ​​of the raw material mass. This can better retain phospholipids while reducing the content of nonpolar lipids such as triglycerides and cholesterol.

[0036] The conditions of ethanol extraction affect the phospholipid content. Therefore, this invention studies the temperature, time and concentration of ethanol extraction. Preferably, the ethanol extraction temperature is 45℃-50℃, the time is 4-6h, and the volume concentration of ethanol is 95%-96% to obtain a higher phospholipid content.

[0037] This invention has found that, compared to binary composite systems of diatomaceous earth-activated carbon or activated carbon-washed bentonite, ternary composite adsorbents composed of diatomaceous earth, activated carbon, and washed bentonite have a synergistic effect in improving the lipid yield, color, and reducing phospholipid loss rate of sea cucumbers. Preferably, the composite adsorbent is composed of diatomaceous earth, activated carbon, and washed bentonite, and the mass ratio of diatomaceous earth, activated carbon, and washed bentonite is 8:1-1.5:1-1.5 to better improve the lipid yield and reduce the phospholipid loss rate of sea cucumbers.

[0038] Traditional calcium-based bentonite has a strong adsorption capacity for polar impurities, but it easily causes phospholipid loss. Sodium-based bentonite has high swelling properties, but it forms a high-viscosity colloid after hydration, which is difficult to separate. Organic bentonite may have residual organic matter that can migrate, limiting its use to non-edible oils. Therefore, this invention uses water-washed bentonite as an adsorption frame to remove pigments and impurities from viscous oils through physical adsorption, avoiding the safety issues that may arise from chemical methods.

[0039] Temperature also affects the adsorption effect of the compound adsorbent. Therefore, the present invention has studied the adsorption temperature and time. Preferably, the preset temperature is 45℃-50℃ and the stirring time is 50-60 minutes to improve the adsorption efficiency.

[0040] The amount of compound adsorbent used also affects the adsorption effect. Therefore, this invention has studied the amount of compound adsorbent used. Preferably, the amount of compound adsorbent used is 3%-5% of the mass of the diluted ethanol extract containing crude sea cucumber lipids, in order to obtain a better adsorption effect.

[0041] The conditions of molecular distillation affect the deacidification and deodorization effects. Therefore, this invention studies the temperature, pressure, and rotation speed of molecular distillation. Preferably, the conditions for the first-stage distillation are: feed temperature 80°C-85°C, system pressure 1.0Pa-1.5Pa, scraper rotation speed 200rpm-220rpm, and feed flow rate 2.0mL / min-3.0mL / min. Preferably, the conditions for the second-stage distillation are: feed temperature 125°C-130°C, system pressure 0.5Pa-0.8Pa, scraper rotation speed 250rpm-280rpm, and feed flow rate 1.5mL / min-1.8mL / min, to obtain better deacidification and deodorization effects.

[0042] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0043] In the following examples, the water-washed bentonite was purchased from Shandong Yousuo Chemical Technology Co., Ltd. as 500g of chemically pure (CP), trade name: bentonite.

[0044] Example 1: Basic Component Analysis of Sea Cucumber Blanching Liquid Powder Sea cucumber blanching liquid powder is a powder produced by spray drying the liquid generated during the processing of sea cucumbers. The basic components of the sea cucumber blanching liquid powder in this embodiment are shown in Table 1. It can be prepared using the method described in patent CN105266075A.

[0045] Table 1. Basic components of sea cucumber blanching liquid powder

[0046] Example 2: Effect of adding entrainer on lipid extraction from sea cucumber using supercritical CO2 fluid extraction This invention uses the sea cucumber blanching liquid powder from Example 1 as raw material to compare the effects of adding entrainers on the extraction of sea cucumber lipids. Tables 2 and 3 show that during supercritical fluid extraction, the addition of various entrainers affects the phospholipids in sea cucumber lipids. Among them, isopropanol:ethyl acetate (3:1) has the best effect, reducing the content of non-polar lipids such as triglycerides and cholesterol while retaining phospholipids. This method will be selected subsequently to remove triglycerides and pigments from the lipids.

[0047] Table 2. Effects of entrainer type on the amount of nonpolar lipids removed and their phospholipid content.

[0048] Example 3 Screening of Supercritical CO2 Fluid Extraction Conditions This embodiment uses an orthogonal array design experiment to analyze the interaction of various factors. Extraction pressure, extraction time, and extraction temperature were screened, with pressure gradients designed as 20 MPa, 25 MPa, 30 MPa, 35 MPa, and 40 MPa; extraction times designed as 1 h, 1.5 h, 2 h, 2.5 h, and 3 h; and extraction temperatures designed as 30℃, 35℃, 40℃, 45℃, and 50℃. The entrainer used was 20% isopropanol:ethyl acetate (3:1). Table 3 shows that the lipid yield varied under different conditions. At a pressure of 30 MPa, an extraction time of 2 h, and a temperature of 40℃, triglycerides and pigments were removed more effectively, with a phospholipid loss rate of only 0.06 mg / g. Table 3 Orthogonal Experiment Table

[0049] After supercritical fluid extraction, nonpolar lipids and pigments in the raw material are removed. Then, sea cucumber lipids are extracted by organic solvent extraction. The optimal process conditions determined in this embodiment are as follows: 95% ethanol solution is selected as the solvent, the extraction temperature is 45°C, and the extraction is carried out for 6 hours under medium speed conditions. This yields a crude extract of sea cucumber lipids with high phospholipid content, which is 20% higher than that obtained by direct extraction with organic solvent.

[0050] Example 4: Study on the purification process of sea cucumber lipids The crude sea cucumber lipid extract obtained by organic solvent extraction still has problems such as high free fatty acid content and strong fishy smell. Therefore, purification process research is needed, mainly including the removal of free fatty acids, deodorization, and removal of insoluble impurities. In this example, low-temperature composite adsorption decolorization and removal of insoluble impurities were first carried out (replacing traditional high-temperature bleaching clay decolorization). The effects of different single adsorbents and different ratios of adsorbents were compared in the experiment, as shown in Table 4. Finally, the mass ratio of the composite adsorbent was diatomaceous earth: activated carbon: washed bentonite = 8:1:1. This ratio takes into account the filter aid performance, pigment adsorption capacity, and low adsorption of phospholipids. Under this formula, the purified lipid has the highest transparency and the lowest phospholipid loss rate. The crude sea cucumber lipid prepared in Example 3 was diluted with 95% ethanol to an oil content of 30% (w / w). The above-mentioned composite adsorbent was added at 3% of the oil weight. In a closed stirring tank, it was stirred at 100 rpm for 60 minutes at a constant temperature of 45°C. Nitrogen gas was purged throughout the process to prevent oxidation. After stirring, the mixture was immediately pressurized with nitrogen and sent to a plate and frame filter press (pre-coated with a 1 cm thick diatomaceous earth filter aid) for filtration. Clear sea cucumber lipids with significantly improved color were obtained, as shown in Figure 1.

[0051] Table 4 Effects of different adsorbents

[0052] The second step involves deacidification and deodorization. This section compares several milder deacidification methods, with results shown in Table 5. Molecular distillation was ultimately selected as the optimal method. This method replaces traditional alkali refining and high-temperature deodorization, aiming to simultaneously remove free fatty acids (FFA) and volatile odor molecules. The method uses a stainless steel scraped-film molecular distillation apparatus. The lipids obtained in the first step are collected and fed into the molecular distillation unit. First-stage distillation removes light components, with the feed temperature set at 80°C, system pressure at 1.0 Pa, scraper rotation speed at 200 rpm, and feed flow rate at 2.0 mL / min. The light components (distillate), mainly FFA, are collected; the heavy components (undistilled) proceed to the next stage. Second-stage distillation collects the target lipids, with the feed temperature set at 125°C, system pressure at 0.5 Pa, scraper rotation speed at 250 rpm, and feed flow rate at 1.5 mL / min. The distillate is a high-purity, phospholipid-rich sea cucumber lipid product with a low acid value.

[0053] Table 5. Free fatty acid removal levels by different deacidification methods

[0054] Example 5 Sensory Evaluation Sensory evaluation was conducted in a well-lit, odor-free room. The lipid samples to be tested (crude oil before purification vs. the purified product) were each placed in identical 30mL transparent glass bottles and labeled. The evaluation strictly followed the sequence of "look, smell, and taste," and 10 evaluators were invited. All evaluators refrained from smoking, drinking alcohol, or consuming spicy foods within one hour prior to the evaluation. The color, appearance, odor intensity and pleasantness, taste, and aftertaste of the lipid samples were evaluated. Higher scores indicated better quality, with a maximum score of 5 points. The scores from all evaluators were compiled and analyzed; the results are shown in Table 6.

[0055] Table 6 Sensory Evaluation

[0056] Example 6: Analysis of lipid components in sea cucumber The lipid composition of purified sea cucumber lipids was analyzed by LC-MS, revealing 18 major lipid classes and 334 lipid subclasses. The major lipid classes included lysophosphatidylcholine (LPC), alkylphosphatidylcholine (PCO), and phosphatidylcholine (PC). Among the major lipid classes, the most abundant lipids were lysophosphatidylcholine (LPC), phosphatidylcholine (PC), and alkylphosphatidylcholine (PCO), accounting for 63%, 30%, and 5%, respectively. The lipid content distribution of purified sea cucumber lipids is shown in Figure 2. Among the lipid subclasses, the most abundant were lysophosphatidylcholine 18:1 (LPC18:1), lysophosphatidylcholine 18:0 (LPC18:0), lysophosphatidylcholine 16:0 (LPC16:0), and phosphatidylcholine 36:2 (PC36:2). The lipid composition of purified sea cucumber lipids is shown in Figure 3.

[0057] Example 7 Fatty acid composition analysis The fatty acids in sea cucumber lipids include saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA), such as linolenic acid (ALA) and arachidonic acid (AA), but the content of DHA and EPA is relatively low. Bioactive lipids, such as ceramides and sphingolipids, are present in higher amounts. These lipids possess antioxidant, anti-inflammatory, and antitumor activities and play important roles in cell signaling and immune regulation. Referring to GB5009.168-2016, the fatty acid composition of crude sea cucumber lipid extract and purified sea cucumber lipids was analyzed using a normalization method. The fatty acid composition of sea cucumber lipids is shown in Table 7.

[0058] Table 7 Fatty acid composition analysis

[0059] Example 8: Activity analysis of sea cucumber lipids To verify the biosafety of the purified sea cucumber lipids of this invention, its effect on the survival rate of BV2 microglia was detected using the CCK-8 assay. BV2 microglia were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) with 100 μg / ml penicillin and streptomycin, and incubated at 37°C in a 5% CO2 incubator. 100 μL of cell suspension was seeded into 96-well plates at a cell density of 8 × 10⁶ cells / well. 3 Cells / mL. The culture plates were pre-cultured in an incubator for 24 h (37 ℃, 5% CO2). The culture medium in the 96-well plates was aspirated, and different concentrations of sea cucumber lipid suspension (10, 20, 40, 80, 160, 320 μg / mL) were added and incubated for 24 h. A blank control was prepared using culture medium without sea cucumber lipids. Six replicates were set up for each group. 10 μL of CCK-8 solution was added to each well. The 96-well culture plates were incubated in an incubator for 4 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0060] like Figure 4 As shown, even at a concentration as high as 160 μg / mL, the cell viability remained at (97.2 ± 2.5)%, with no statistically significant difference compared to the control group (p>0.05), and the cell morphology remained intact. This result far exceeds the international non-toxic standard (>80%), proving that the sea cucumber lipids prepared by this invention through a gentle purification process effectively remove impurities while perfectly preserving their biocompatibility, resulting in a product with extremely high purity and excellent safety. This provides crucial safety data support for its in-depth development and application in the fields of functional foods and nervous system health.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A green extraction process for sea cucumber lipids that selectively retains active phospholipids, characterized in that, include: The sea cucumber blanching liquid powder was subjected to supercritical CO2 fluid extraction to remove impurities, followed by ethanol extraction to obtain an ethanol extract containing crude sea cucumber lipids. The ethanol extract containing crude sea cucumber lipids was mixed with a compound adsorbent in a certain proportion, and adsorption and decolorization were carried out under preset temperature and stirring conditions. After filtration, sea cucumber lipids were obtained. The sea cucumber lipids were subjected to molecular distillation. The first stage of distillation removed light components, and the second stage of distillation collected the target lipids, namely, phospholipid-rich sea cucumber lipids.

2. The green extraction process for sea cucumber lipids that selectively retains active phospholipids as described in claim 1, characterized in that, The supercritical CO2 fluid extraction temperature is 30℃-50℃, the pressure is 25MPa-35MPa, the extraction time is 1-3h, and the entrainer is isopropanol:ethyl acetate (3:1), with the amount of entrainer being 20% ​​of the raw material mass.

3. The green extraction process for sea cucumber lipids that selectively retains active phospholipids as described in claim 1, characterized in that, The ethanol extraction was performed at a temperature of 45℃-50℃ for 4-6 hours, with an ethanol volume concentration of 95%-96%.

4. The green extraction process for sea cucumber lipids that selectively retains active phospholipids as described in claim 1, characterized in that, The compound adsorbent is composed of diatomaceous earth, activated carbon and washed bentonite, and the mass ratio of diatomaceous earth, activated carbon and washed bentonite is 8:1-1.5:1-1.

5.

5. The green extraction process for sea cucumber lipids that selectively retains active phospholipids as described in claim 1, characterized in that, The preset temperature is 45℃-50℃, and the stirring time is 50-60 minutes.

6. The green extraction process for sea cucumber lipids that selectively retains active phospholipids as described in claim 1, characterized in that, The amount of the compound adsorbent used is 3%-5% of the mass of the diluted ethanol extract containing crude sea cucumber lipids.

7. The green extraction process for sea cucumber lipids that selectively retains active phospholipids as described in claim 1, characterized in that, The conditions for the first-stage distillation are: feed temperature 80°C-85°C, system pressure 1.0Pa-1.5Pa, scraper rotation speed 200rpm-220rpm, and feed flow rate 2.0mL / min-3.0mL / min.

8. The green extraction process for sea cucumber lipids that selectively retains active phospholipids as described in claim 1, characterized in that, The conditions for the second-stage distillation are: feed temperature 125°C-130°C, system pressure 0.5Pa-0.8Pa, scraper rotation speed 250rpm-280rpm, and feed flow rate 1.5mL / min-1.8mL / min.

9. Phospholipid-rich sea cucumber lipids prepared by the method according to any one of claims 1-8.

10. The application of the phospholipid-rich sea cucumber lipid prepared by the method of any one of claims 1-8 or the phospholipid-rich sea cucumber lipid of claim 9 in the preparation of functional foods.

Images