A method for enriching and purifying high-purity phospholipid krill oil from antarctic krill oil and application

Abstract

The application belongs to the technical field of oil extraction of euphausia superba, and particularly relates to a method for enriching and purifying high-purity phospholipid phosphorus shrimp oil in Antarctic krill oil and application, wherein a low eutectic solvent prepared by mixing L-menthol and octanoic acid is used to efficiently extract and separate Antarctic krill astaxanthin, and then ethanol is used to extract polar crude phospholipid, so as to effectively reduce the content of triglyceride in the krill oil. For the extracted krill oil crude product, a multi-stage supercritical CO2 extraction process is used for gradient deep purification, so as to prepare high-purity phospholipid phosphorus shrimp oil containing high-activity astaxanthin and fatty acids. By introducing saponin and beta-cyclodextrin for shear microemulsification, soft capsules are prepared to realize the production and application of high-activity krill oil, so as to effectively improve the in-vitro stability and bioavailability of the krill oil, and the krill oil is used for regulating lipid metabolism.

Description

Technical Field

[0001] This invention belongs to the field of krill oil extraction technology, specifically relating to a method and application for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. Background Technology

[0002] Antarctic krill oil is rich in phospholipid-type polyunsaturated fatty acids (EPA, DHA) and the natural powerful antioxidant astaxanthin. Its unique molecular structure gives it extremely high bioavailability, showing great application potential in areas such as lipid metabolism regulation, cardiovascular health, and neuroprotection. Traditional krill oil extraction methods mostly employ organic solvent extraction or single supercritical CO2 extraction technology. With the increasing demands for raw material purity in high-value-added health products, how to efficiently obtain high-purity phospholipids while maximizing the preservation of the activity of free astaxanthin has become a research hotspot in the field of marine biolipid engineering. Existing extraction and refining processes face the typical polarity paradox. The target product, phospholipids, is highly polar, while astaxanthin and non-target byproducts, triglycerides, are both nonpolar. It is extremely difficult for a single solvent to selectively remove triglycerides while retaining astaxanthin. To improve the purity of phospholipids, multiple solvent precipitation or high-temperature refining is usually used. This not only leads to a large loss or thermal degradation of astaxanthin, but also makes high-purity phospholipids prone to oil phase precipitation and stratification during long-term storage due to thermodynamic instability, which seriously affects the uniformity of the product and its clinical efficacy. Summary of the Invention

[0003] To address the above issues, this invention provides a method and application for enriching and purifying high-purity phospholipids in Antarctic krill oil. The method employs a low-eutectic solvent extraction to separate neutral lipids, combined with multi-stage supercritical CO2 elution for phospholipid purification. Furthermore, it introduces plant saponins and cyclodextrin for self-assembly microemulsion, achieving steady-state fusion of polar and non-polar active components in an anhydrous system. This results in the synergistic effect of triglyceride removal, efficient astaxanthin extraction, and phospholipid purification and separation.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The enrichment and purification method involves subjecting Antarctic krill oil to multiple supercritical CO2 extractions, enrichment, and microemulsifications to obtain high-purity phospholipid krill oil. The specific steps are as follows: Step 1: Place Antarctic krill oil in a supercritical CO2 extraction vessel, introduce CO2 for static extraction for 30 min, open the separation valve of the extraction vessel for dynamic extraction for 1 h, and collect extract 1. Extract 1 contains free fatty acids, esterified astaxanthin and some phospholipids that are similarly miscible with supercritical CO2. Step 2: Introduce CO2 and an entrainer into the extraction vessel. The extraction time is 1-3 h, the extraction pressure is 15-25 MPa, the extraction temperature is 30-50℃, the CO2 flow rate is 10-20 L / h, and the entrainer flow rate is 0.5 L / h. At this time, the fluid polarity increases, and the remaining polar phospholipids in the extraction vessel are purified efficiently. Extract A is collected. CO2 and an entrainer are introduced again for extraction, and extract B is collected. Extract A and extract B are combined to obtain extract 2. Step 3: Combine extract 1 and extract 2, homogenize and emulsify, and remove entrainers by rotary evaporation to obtain high-purity phospholipid krill oil.

[0005] Furthermore, the entrainer is prepared from citric acid and 95% ethanol by volume, wherein the mass fraction of citric acid in the entrainer is 0.5%.

[0006] Furthermore, the preparation method of the Antarctic krill oil is as follows: S1: Antarctic krill is freeze-dried and pulverized to obtain freeze-dried powder. The freeze-dried powder is added to a eutectic solvent, stirred and extracted, and filtered to obtain extract and filter residue 1. Eutectic solvent is beneficial for dissolving astaxanthin and fatty acid components with strong hydrogen bonding ability, while non-polar triglycerides cannot be extracted and dissolved. S2: Add 95% ethanol (by volume) to filter residue 1 for extraction and filtration to obtain extract 1 and filter residue 2. Filter residue 2 is extracted and filtered again to obtain extract 2. Extract 1, extract 2 and extract are combined and rotary evaporated to obtain Antarctic krill oil.

[0007] Furthermore, the eutectic solvent is prepared from L-menthol and octanoic acid in a molar ratio of 1:2.

[0008] The present invention also provides an application of high-purity phospholipid krill oil, which is used to prepare soft capsules.

[0009] Furthermore, the soft capsule is used to regulate and improve lipid metabolism.

[0010] Furthermore, the soft capsule comprises the following raw materials in parts by weight: 50 parts of high-purity phospholipid krill oil, 1 part of soybean saponins, 5 parts of β-cyclodextrin, 12 parts of glycerin, 30 parts of gelatin, and 24 parts of purified water.

[0011] Furthermore, the preparation method of the soft capsule is as follows: X1: Soybean saponins and β-cyclodextrin are mixed and dissolved to obtain an embedding matrix solution. High-purity phospholipid krill oil is slowly added to the embedding matrix solution, and the mixture is sheared and homogenized to obtain an embedding solution. The solvent in the embedding solution is removed by rotary evaporation under reduced pressure to obtain the contents. X2: Add glycerin, gelatin and purified water to a gelling tank, heat and stir to obtain a gel solution. Pump the contents and gel solution separately into a soft capsule machine for capsule compression and injection. After cooling and shaping, drying in a rotating drum, washing and collecting the capsules, soft capsules are obtained.

[0012] The beneficial effects achieved by this invention are as follows: The enrichment and purification method for high-purity phospholipid krill oil provided by this invention breaks through the limitations of traditional single extraction processes. It utilizes the hydrogen bond recognition ability of eutectic solvents to target and extract astaxanthin, and supplements it with ethanol to extract crude phospholipids, effectively reducing the content of non-polar triglycerides in Antarctic krill oil and significantly improving the purity of the target product. Citric acid and ethanol are introduced as a composite entrainer for supercritical extraction. The trace amounts of organic acids not only regulate the polar environment to improve the extraction efficiency of phospholipids, but also have antioxidant effects, protecting the heat-sensitive active substances during the extraction process. In the application of high-purity phospholipid krill oil, natural saponins are used to reduce interfacial tension for astaxanthin and phospholipid components that are easily oxidized and have unstable activity. Combined with the hydrophobic cavity of β-cyclodextrin, functional components are targeted for recognition and microemulsion encapsulation, effectively isolating oxygen and avoiding oxidative degradation. This solves the problem of easy layering and clumping of high-purity phospholipid oil in soft capsules. The krill oil soft capsules prepared by this method have good effects in assisting to lower blood lipids, regulate metabolism, protect the liver, and improve sleep quality. It also greatly improves the shelf-life stability and bioavailability of the product. Attached Figure Description

[0013] Figure 1 The yield of Antarctic krill oil extracted in Example 1 and Comparative Example 1; Figure 2 The results of phospholipid content detection for the enriched and purified high-purity phospholipid krill oil in Examples 2-10 are shown. Figure 3 The results of the response surface methodology investigation on the effects of extraction time, extraction pressure, extraction temperature and CO2 flow rate on the phospholipid content in high-purity phospholipid krill oil are presented. Figure 4 The results of the study on the changes in triglyceride content in subjects before and after administration of the soft capsules prepared in Example 11; Figure 5 The results of the study on the changes in total cholesterol content before and after subjects took the soft capsules prepared in Example 11. Detailed Implementation

[0014] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0016] Unless otherwise specified, all methods described in the following embodiments are conventional. Unless otherwise specified, all materials used in the following embodiments are new materials purchased from the market. All parts are by weight. Specifically, the Antarctic krill is frozen Antarctic krill with a moisture content of 81.4%; the β-cyclodextrin used has the product number Y256133 and CAS number 7585-39-9; the soybean saponins used have a purity of 95%; the 95% ethanol used is food grade; and the gelatin used is type A gelatin.

[0017] Example 1: This example provides a method for extracting Antarctic krill oil from Antarctic krill. The specific method is as follows: S1: Antarctic krill was freeze-dried and pulverized to obtain freeze-dried powder. The moisture content of the freeze-dried powder was 2.86%. 500g of freeze-dried powder was added to 5 L of eutectic solvent and stirred at 45℃ and 300 rpm for 1.5 h. The mixture was then filtered to obtain extract and filter residue 1. S2: Add 2.5 L of 95% ethanol to filter residue 1 for extraction at 60℃ for 2 h with stirring speed of 150 rpm. Filter the mixture to obtain extract 1 and filter residue 2. Add 2.5 L of 95% ethanol to filter residue 2 again for extraction for 1 h and filter the mixture to obtain extract 2. Combine extract 1, extract 2 and extract and rotary evaporate at 35℃ until solvent-free condensation occurs to obtain Antarctic krill oil.

[0018] The Antarctic krill oil used in the following examples was extracted according to the method described in this example.

[0019] Example 2: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The specific steps are as follows: Step 1: Place 10 parts of Antarctic krill oil in a supercritical CO2 extraction vessel, introduce N2 and CO2 for static extraction for 30 min, open the separation valve of the extraction vessel for dynamic extraction for 1 h, with an extraction pressure of 15 MPa, an extraction temperature of 30℃, and a CO2 flow rate of 10 L / h, and collect the extract 1. Step 2: N2 and CO2 are introduced into the extraction vessel, along with an entrainer. The extraction time is 1 h, the extraction pressure is 15 MPa, the extraction temperature is 30℃, the CO2 flow rate is 10 L / h, and the entrainer flow rate is 0.5 L / h. Extract A is collected. CO2 and entrainer are introduced again for extraction with the same parameters as above. Extract B is collected. Extract A and extract B are combined to obtain extract 2. Step 3: Combine extract 1 and extract 2, homogenize and emulsify at 12000 rpm for 15 min, and remove entrainers by rotary evaporation to obtain high-purity phospholipid krill oil.

[0020] Example 3: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The difference from Example 2 is that the extraction process parameters in step 2 are set as follows: extraction time 2 h, extraction pressure 15 MPa, extraction temperature 30℃, CO2 flow rate 10 L / h. The remaining processes are the same as in Example 2, and high-purity phospholipid krill oil is obtained.

[0021] Example 4: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The difference from Example 2 is that the extraction process parameters in step 2 are set as follows: extraction time 3 h, extraction pressure 15 MPa, extraction temperature 30℃, CO2 flow rate 10 L / h. The remaining processes are the same as in Example 2, and high-purity phospholipid krill oil is obtained.

[0022] Example 5: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The difference from Example 3 is that the extraction process parameters in step 2 are set as follows: extraction time 2 h, extraction pressure 15 MPa, extraction temperature 40℃, CO2 flow rate 10 L / h. The remaining processes are the same as in Example 3, and high-purity phospholipid krill oil is obtained.

[0023] Example 6: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The difference from Example 3 is that the extraction process parameters in step 2 are set as follows: extraction time 2 h, extraction pressure 15 MPa, extraction temperature 50℃, CO2 flow rate 10 L / h. The remaining processes are the same as in Example 3, and high-purity phospholipid krill oil is obtained.

[0024] Example 7: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The difference from Example 5 is that the extraction process parameters in step 2 are set as follows: extraction time 2 h, extraction pressure 20 MPa, extraction temperature 40℃, CO2 flow rate 10 L / h. The remaining processes are the same as in Example 5, and high-purity phospholipid krill oil is obtained.

[0025] Example 8: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The difference from Example 5 is that the extraction process parameters in step 2 are set as follows: extraction time 2 h, extraction pressure 25 MPa, extraction temperature 40℃, CO2 flow rate 10 L / h. The remaining processes are the same as in Example 5, and high-purity phospholipid krill oil is obtained.

[0026] Example 9: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The difference from Example 7 is that the extraction process parameters in step 2 are set as follows: extraction time 2 h, extraction pressure 20 MPa, extraction temperature 40℃, CO2 flow rate 15 L / h. The remaining processes are the same as in Example 7, and high-purity phospholipid krill oil is obtained.

[0027] Example 10: This example provides a method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil. The difference from Example 7 is that the extraction process parameters in step 2 are set as follows: extraction time 2 h, extraction pressure 20 MPa, extraction temperature 40℃, CO2 flow rate 20 L / h. The remaining processes are the same as in Example 7, and high-purity phospholipid krill oil is obtained.

[0028] Example 11: This example provides an application of high-purity phospholipid krill oil from Antarctic krill oil. The high-purity phospholipid krill oil is used to prepare soft capsules with lipid metabolism regulation function. The high-purity phospholipid krill oil is prepared according to the parameters and methods described in Example 9. The soft capsules comprise the following raw materials in parts by weight: 50 parts high-purity phospholipid krill oil, 1 part soybean saponin, 5 parts β-cyclodextrin, 12 parts glycerol, 30 parts gelatin, and 24 parts purified water. The preparation method is as follows: X1: Mix 1 part of soybean saponins and 5 parts of β-cyclodextrin and dissolve them in 60 parts of 95% ethanol at 45℃ to obtain an embedding matrix solution. Take 50 parts of high-purity phospholipid krill oil and slowly add it to the embedding matrix solution. Shear and homogenize at 10,000 rpm for 10 min to obtain an embedding solution. Remove the solvent from the embedding solution by rotary evaporation under reduced pressure to obtain the contents. X2: Add 12 parts of glycerin, 30 parts of gelatin and 24 parts of purified water to a gelling tank, heat and stir at 70℃ and 30 rpm for 1 hour to melt and mix, and obtain a gel solution. After degassing, pump the contents and gel solution into a soft capsule machine for capsule compression and injection. After cooling and shaping, drying in a rotating drum, washing and picking up the capsules, soft capsules are obtained.

[0029] Comparative Example 1: Extraction of Antarctic krill oil from freeze-dried krill powder using organic solvent method: 2.5 L of acetone was added to 500 g of Antarctic krill powder for extraction for 2 h at a stirring speed of 150 rpm. After filtration, 2.5 L of acetone was added to the filter residue for extraction for another 1 h and then filtered again. The two filtrates were collected and combined, and then rotary evaporated at 35 °C until no solvent was released and condensed to obtain Antarctic krill oil.

[0030] Antarctic krill oil yield investigation: The yield of Antarctic krill oil extracted according to the methods described in Example 1 and Comparative Example 1 was investigated respectively. Yield % = Antarctic krill oil mass / freeze-dried powder mass × 100%. The results are shown in the figure. Figure 1 The contents of phospholipids, astaxanthin, tocopherols, EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid) and triglycerides in Antarctic krill oil extracted in Example 1 and Comparative Example 1 were investigated respectively. The results are shown in Table 1.

[0031] Determination of high-purity phospholipid krill oil content: The phospholipid content in the high-purity phospholipid krill oil extracted in Examples 2-10 was determined by thin-layer chromatography. The results are shown in the figure. Figure 2 .

[0032] Response surface methodology for process optimization: Extraction time, extraction pressure, extraction temperature, and CO2 flow rate were used as influencing factors, and phospholipid content was used as the detection index. Response surface curves were plotted to determine the optimal extraction process. The results are shown below. Figure 3 .

[0033] Efficacy study of krill oil soft capsules in lowering blood lipids: Changes in triglyceride and total cholesterol levels in blood samples from 10 subjects were recorded one month before and after taking the krill oil soft capsules prepared in Example 11. Subject numbers were 1-10. Each soft capsule weighed 0.5 g, and the dosage was 1 g / day / person. Results are shown below. Figure 4 and Figure 5 .

[0034] The health benefits of krill oil soft capsules were investigated: The levels of systemic inflammatory markers hs-CRP (high-sensitivity C-reactive protein), liver damage markers ALT (alanine aminotransferase), and AST (aspartate aminotransferase) in the blood of 10 subjects were recorded one month before and after taking the krill oil soft capsules prepared in Example 11. The eye protection and sleep improvement effects of the krill oil soft capsules before and after taking the subjects were evaluated by OSDI (Ocular Surface Disease Index) and PSQI (Pittsburgh Sleep Quality Index). The results are shown in Table 2.

[0035] Note: The OSDI (Outcome Detection and Diagnosis) aims to screen and diagnose patients with dry eye syndrome. It assesses the severity of dry eye and includes three dimensions: "Ocular Symptoms," "Visual Function," and "Environmental Triggers," totaling 12 items. Each item is scored from 0 to 4, with a minimum score of 0 for none, 1 for a small portion of the time, 2 for half the time, 3 for most of the time, and 4 for all of the time. The "Ocular Symptoms" dimension has 3 items, the "Visual Function" dimension has 6 items, and the "Environmental Triggers" dimension has 3 items. The final score is calculated by multiplying the sum of all scores by the number of items answered, then multiplying by 25. The final score ranges from 0 to 100, with scores ≤20 indicating mild symptoms, 21-45 indicating moderate symptoms, and ≥46 indicating severe symptoms.

[0036] Table 1. Determination of components in high-purity phospholipid krill oil extracted in Example 1 and Comparative Example 1.

[0037] Table 2. Evaluation of the efficacy of soft capsules

[0038] Table 1 and Figure 1 The results showed that the yields of Antarctic krill oil extracted in Example 1 and Comparative Example 1 were 18.4% and 11.2%, respectively. The phospholipid content in the Antarctic krill oil extracted in Example 1 was significantly higher than that in Comparative Example 1, while the triglyceride content was significantly lower. This indicates that the fractional extraction process in Example 1 can effectively remove triglycerides for further purification. Furthermore, the EPA and DHA contents showed a positive correlation with the phospholipid content, indicating that the EPA and DHA were in a phospholipid-bound form with stronger activity.

[0039] Figure 2 The results showed that, with extraction time, extraction pressure, extraction temperature and CO2 flow rate as process evaluation indicators, the extraction process parameters were optimized. Among them, the process parameters of Example 9 yielded the highest phospholipid content in the high-purity phospholipid krill oil. The purification effect was better under the conditions of extraction time of 2 h, pressure of 20 MPa, extraction temperature of 40℃ and CO2 flow rate of 15 L / h.

[0040] Figure 3 The results showed that the F-value of the model was 5.08 and the P-value was 0.0022, indicating that the process parameters had a significant impact on the purity of phospholipids with low error.

[0041] Figure 4 and Figure 5 The results showed that the soft capsules prepared based on the krill oil extracted in Example 2 could help reduce the levels of triglycerides and cholesterol in the subjects, demonstrating a good lipid-lowering ability.

[0042] Table 2 shows that the soft capsules prepared in Example 11 have good effects on reducing inflammatory response in the body, improving liver damage, promoting eye health and improving sleep quality. The abundant marine phospholipids, astaxanthin and highly active unsaturated fatty acids in the soft capsules have good effects on regulating human metabolism.

[0043] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0044] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil, characterized in that, The enrichment and purification method involves multiple supercritical CO2 extractions, enrichments, and microemulsifications of Antarctic krill oil to obtain high-purity phospholipid krill oil. Specific steps are as follows: Step 1: Place Antarctic krill oil in a supercritical CO2 extraction vessel and perform static extraction and dynamic extraction successively, and collect the extract 1. Step 2: Pass CO2 and entrainer into the extraction vessel for extraction, collect extract A, extract again and collect extract B, combine extract A and extract B to obtain extract 2; Step 3: Combine extract 1 and extract 2, homogenize and emulsify, and rotary evaporate to obtain high-purity phospholipid krill oil.

2. The method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil according to claim 1, characterized in that, In step 1, the static extraction time is 30 min; In step 2, the extraction process parameters are as follows: extraction time is 1-3 h, extraction pressure is 15-25 MPa, extraction temperature is 30-50℃, and CO2 flow rate is 10-20 L / h.

3. The method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil according to claim 2, characterized in that, The entrainer is prepared from citric acid and 95% ethanol by volume; The entrainer contains 0.5% citric acid by mass.

4. The method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil according to claim 1, characterized in that, The preparation method of the Antarctic krill oil is as follows: S1: Antarctic krill was freeze-dried and pulverized to obtain freeze-dried powder. The freeze-dried powder was added to a eutectic solvent, stirred and extracted, and filtered to obtain extract and filter residue 1, respectively. S2: Add 95% ethanol (by volume) to filter residue 1 for extraction and filtration to obtain extract 1 and filter residue 2. Filter residue 2 is extracted and filtered again to obtain extract 2. Extract 1, extract 2 and extract are combined and rotary evaporated to obtain Antarctic krill oil.

5. The method for enriching and purifying high-purity phospholipid krill oil from Antarctic krill oil according to claim 4, characterized in that, The eutectic solvent is prepared from L-menthol and octanoic acid in a molar ratio of 1:

2.

6. An application of high-purity phospholipid krill oil obtained by the method according to any one of claims 1-5, characterized in that, The high-purity phospholipid krill oil is used to prepare soft capsules; The soft capsule comprises the following raw materials in parts by weight: 50 parts high-purity phospholipid krill oil, 1 part soybean saponins, 5 parts β-cyclodextrin, 12 parts glycerin, 30 parts gelatin, and 24 parts purified water. The preparation method is as follows: X1: Mix and dissolve soybean saponins and β-cyclodextrin to obtain an encapsulation matrix solution. Add high-purity phospholipid krill oil to the encapsulation matrix solution, homogenize, and rotary evaporate to obtain the contents. X2: Mix glycerin, gelatin and purified water, heat and stir to obtain a gel solution. Press the contents and gel solution into pellets, cool and set, dry in a rotating drum, wash and collect the pellets to obtain soft capsules.

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