A method for extracting antidepression active euphausia superba oil by subcritical combined solvent extraction

Abstract

The application discloses a method for extracting antidepression active Euphausia superba oil by adopting subcritical combination solvent, and belongs to the technical field of deep processing of Euphausia superba. The method is a three-stage gradient extraction method of "subcritical butane-acetone-ethanol", and realizes differential separation and enrichment of lipid components by using solvent polarity difference. The first-stage subcritical butane extraction is carried out under low temperature, closed and oxygen isolation conditions, and high-efficiency desolventizing is realized by using reduced pressure gasification, so that the heat-sensitive active ingredients are effectively reserved, and meanwhile, oxidation and metamorphosis of raw materials are avoided, thereby laying a foundation for preparation of subsequent high-quality extracts. The method successfully prepares three Euphausia superba oil products with significant component differences, and especially, the third-stage extract and the enzymatic modification product thereof exhibit excellent antidepression activity. The application realizes functional separation of Euphausia superba lipid components, and provides strong support for development of personalized and diversified antidepression products.

Description

Technical Field

[0001] This invention belongs to the field of seafood processing technology, specifically relating to a method for extracting antidepressant active Antarctic krill oil using subcritical combined solvent extraction. Background Technology

[0002] Antarctic krill ( Euphausia superba Antarctic krill are among the most biodiverse organisms in the Antarctic Ocean. They are rich in polyunsaturated fatty acids (n-3 PUFAs), proteins and peptides, phospholipids (PL), astaxanthin (ASTA), flavonoids, and vitamins A and E, making them widely used in high-value-added health food products. Among these, Antarctic krill oil and astaxanthin, as natural, safe, and highly effective dietary supplements, show great promise in alleviating depression and improving central nervous system dysfunction.

[0003] Antarctic krill oil ( Antarctic krill Antarctic krill oil comprises 12-50% of the dry weight (dw) of krill, and its content is affected by species, season, age, harvest time, and processing and storage methods. Antarctic krill oil has a complex composition, including phospholipids (PL, 38.89%-80.69%), triacylglycerols (TAG, 0.59%-43.70%), diacylglycerols (DAG, ND~17.00%), monoacylglycerols (MAG, ND~72.00%), and free fatty acids (FFA, ND~16.10%). It is also rich in astaxanthin, vitamin A, vitamin E, and other active ingredients. Studies have shown that different lipid components exhibit significant differences in their physiological activities. For example, phospholipid-type polyunsaturated fatty acids (PL-PUFA) play an important role in brain development and the prevention of neurodegenerative diseases; triacylglycerol-type saturated fatty acids (TAG-SFA) play an important role in energy supply; and astaxanthin is considered a naturally occurring strong antioxidant. Therefore, the personalized separation and extraction of Antarctic krill oil is of significant scientific and practical value for in-depth research into the roles of various active ingredients in antidepressant effects. However, the coexistence of polar phospholipids, low- to medium-polarity trace components, and non-polar triglycerides in Antarctic krill oil leads to the limitations of single-solvent extraction methods, which suffer from high selectivity and incomplete extraction, making it difficult to effectively enrich and separate different lipid components. Furthermore, the oxidation of the oil restricts its high-value development and application.

[0004] Currently, common methods for krill oil extraction include organic solvent extraction (such as petroleum ether, n-hexane, and ethanol), enzymatic solvent extraction, supercritical CO2 extraction, and subcritical extraction. While each method has its advantages, none can efficiently extract total lipids while systematically separating phospholipids, neutral lipids, and trace active ingredients. Polar solvents (such as ethanol) are effective at extracting phospholipids, while non-polar solvents (such as n-hexane and subcritical butane) are more suitable for extracting non-polar components like TAG. Moderately polar solvents, such as acetone, are effective at extracting moderately polar trace components like astaxanthin and vitamins, but have low solubility for phospholipids. Mixed solvents, while helping to increase total lipid yield, lack the ability to target and enrich specific components. This specific solvent-component relationship provides a framework and theoretical basis for designing fractional extraction processes. Currently, although methods for fractional extraction of krill oil exist, there are still shortcomings in maximizing and refining lipid extraction while improving oil quality and adapting to large-scale industrial operations. Summary of the Invention

[0005] To address the shortcomings of existing technologies in Antarctic krill oil extraction, such as mixed components, insufficient substrate purity in enzymatic modification, and low separation efficiency of antidepressant functional components, this invention aims to provide a method for preparing Antarctic krill oil rich in different antidepressant functional components based on a three-stage extraction using a specific polarity-increasing gradient of "subcritical butane-acetone-ethanol," combined with enzymatic modification. This invention does not simply utilize solvent polarity differences, but creatively achieves "step-by-step impurity removal" and "differential enrichment" of antidepressant active ingredients through a specific solvent sequence. First, subcritical butane is used as the first-stage extraction solvent to specifically remove and recover triglycerides (neutral lipids), eliminating their interference with subsequent phospholipid extraction. Then, acetone is used for secondary extraction to separate trace components such as astaxanthin, further purifying the defatted meal. Finally, ethanol is used for tertiary extraction to obtain high-purity phospholipid components. Based on this high-purity phospholipid substrate, further targeted enzymatic modification was carried out. Compared with direct modification of crude oil extracted by a single solvent, this preparation method significantly reduced steric hindrance and interference from competing substrates, thereby more efficiently enriching DHA / EPA at specific sites on the phospholipids. This method not only prepared three antidepressant active products with significant compositional differences (triglyceride type, astaxanthin type, and functionalized phospholipid type), achieving functional separation of lipid components with different chain lengths and saturation levels, but also provided key technical support and structure-activity relationship basis for the precise intervention and personalized product development of Antarctic krill oil in the field of antidepressant treatment.

[0006] To achieve the above-mentioned objectives, the present invention is specifically implemented through the following technical solution: A method for preparing antidepressant active Antarctic krill oil using a subcritical butane-acetone-ethanol three-stage gradient extraction process includes the following steps: (1) Raw material pretreatment: After the frozen Antarctic krill is vacuum freeze-dried, the Antarctic krill is taken out, quickly ground and crushed to a particle size of 0.25 mm, packed into a self-sealing bag, and stored at -80℃ away from light.

[0007] (2) First-stage extraction: Subcritical butane is used to perform the first-stage extraction of Antarctic krill powder to obtain first-stage extracted Antarctic krill oil and first-stage extracted Antarctic krill meal. The first-stage extraction of Antarctic krill powder using subcritical butane involves mixing Antarctic krill powder and butane at a mass-volume ratio of 1:1 to 1:14 (g / mL), extracting at 40°C for 60 min, and then separating the target product in a separation tank. The solvent is removed by vacuum to obtain first-stage extracted Antarctic krill oil and first-stage extracted Antarctic krill meal. After the solvent evaporates, the residue is used as the raw material for the next stage of extraction.

[0008] (3) Secondary extraction: Acetone is used to perform secondary extraction on the Antarctic krill meal extracted in the first stage to obtain secondary extracted Antarctic krill oil and secondary extracted Antarctic krill meal. The secondary extraction of the Antarctic krill powder extracted in the first stage with acetone is carried out by mixing the Antarctic krill meal extracted in the first stage with acetone at a mass-volume ratio of 1:5 (g / mL), stirring at 750 rpm, extracting at 30°C, and extracting for 40 min. After stirring, the mixture is filtered immediately, and the solvent is removed from the filtrate to obtain secondary Antarctic krill oil and secondary Antarctic krill meal.

[0009] (4) Tertiary extraction: The secondary extracted Antarctic krill meal was subjected to tertiary extraction with ethanol to obtain extracted Antarctic krill oil. The tertiary extraction of the secondary extracted Antarctic krill meal with ethanol was carried out by mixing the secondary extracted Antarctic krill meal with ethanol at a mass-volume ratio of 1:4 (g / mL), stirring at 750 rpm, extracting at 30°C, and extracting for 40 min. After stirring, the mixture was filtered immediately, and the solvent was removed from the filtrate to obtain the tertiary extracted Antarctic krill oil.

[0010] (5) In step (4), the Antarctic krill oil was extracted in three stages and modified under the following conditions: 5-30% immobilized phospholipase A1 (accounting for % of total substrate mass), DHA / EPA: krill oil = 1:1-10:1 (mL / g), 5-36 h, and 35-55℃.

[0011] This invention provides Antarctic krill oil prepared by the method described above.

[0012] This invention provides the application of the Antarctic krill oil described above in the preparation of antidepressant drugs or food.

[0013] The beneficial effects of this invention are as follows: (1) Compared with the traditional single solvent extraction method, the total lipid yield of the three-stage gradient extraction process is increased by 9.34%~47.96%.

[0014] (2) The present invention improves the proportion of phospholipid DHA / EPA (25% / 50%) by 31.71% by enzymatic modification of Antarctic krill oil obtained by third-stage ethanol extraction.

[0015] (3) This invention employs subcritical extraction technology as the first-stage extraction process. This process is carried out under low-temperature, closed, and oxygen-deficient conditions. After extraction, the solvent liquid butane is efficiently removed by vacuum pump depressurization and vaporization. The vaporized butane is then liquefied by a compressor and condenser and returned to the solvent tank. This technology not only has the advantages of high solvent removal efficiency and low residue, but also effectively prevents the degradation of heat-sensitive bioactive components and avoids the oxidative deterioration of the remaining raw materials after the first-stage extraction, thus providing quality assurance for the products of subsequent second and third-stage gradient extractions. In addition, this process is simple to operate, has good solvent removal effect, and low equipment cost, making it suitable for large-scale industrial production.

[0016] (4) The subcritical butane-acetone-ethanol three-stage gradient extraction process systematically solves the problem of efficient separation and targeted enrichment of different polar lipid categories and fatty acids with different chain lengths and saturation in Antarctic krill oil. For example, the first-stage butane mainly extracts glycerol lipids (such as TAG 16:0-16:1-20:5); compared with the first-stage butane, the second-stage acetone has a stronger ability to extract trace active ingredients such as AST, VA, and VE; the third-stage ethanol mainly targets phospholipid components (such as PC 20:5-22:6).

[0017] (5) The subcritical butane-acetone-ethanol three-stage gradient extraction process of the present invention successfully prepared three krill oil products with significant differences in composition and different functional activities. This not only provides technical support for the efficient utilization of Antarctic krill oil lipid resources and the quality assurance of products, but also provides strong support for the research on the functional characteristics of krill oil and the development of personalized products by realizing the functional separation of lipid components.

[0018] (6) The three-stage extraction of Antarctic krill oil and its corresponding modified Antarctic krill oil in this invention showed significant antidepressant effects in three behavioral tests, and exhibited an antidepressant trend of PL-1>QY>PL>TAG. Attached Figure Description

[0019] Figure 1 The process flow diagram for the subcritical butane-acetone-ethanol three-stage gradient extraction process is shown below. Figure 2 Figure showing the results of three-stage gradient extraction of the main lipid composition of Antarctic krill oil; Figure 3 The graph shows the lipid extraction rate results of the three-stage gradient extraction process compared with traditional single solvent extraction. Figure 4Heat maps of key substances in Antarctic krill oil extracted using different processes; Figure 5 A graph showing the effect of modified Antarctic krill oil on the proportion of phospholipid-type DHA / EPA. Figure 6 This image shows the antidepressant effects of different active ingredients in Antarctic krill oil. Detailed Implementation

[0020] The following specific embodiments will illustrate the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or in the product manual.

[0021] The Antarctic krill used in this invention was purchased from Shanghai Kaichuang Ocean Fishery Co., Ltd.; phospholipase A1 (Lecitase® Ultra, hydrolytic activity unit 10.84 KLU / g) was purchased from Novozymes, Denmark; the immobilization carrier D380 ion exchange resin was from Zhengzhou Hecheng New Material Technology Co., Ltd.; and ethyl ester DHA / EPA was from Xi'an Yatu Biotechnology Co., Ltd.

[0022] The two total lipid extraction methods used in this invention are the Folch method and the Soxhlet extraction method: (1) Folch method: Weigh 3 g of freeze-dried Antarctic krill powder and add chloroform / methanol (2:1, v / v) at a material-to-liquid ratio of 1:12 (g:mL). Stir at 750 r / min for 2 h (30℃). After extraction, filter the solution and add 0.2 times the volume of 0.9% NaCl solution to the filtrate. Shake well and allow to stand for separation. Collect the lower organic phase and remove the solvent by vacuum rotary evaporation at 40~45℃ to obtain Antarctic krill oil.

[0023] (2) Soxhlet extraction: Refer to national standard GB / T5009.6-2016.

[0024] The testing methods used in this invention: (1) The fatty acid composition of Antarctic krill oil extracted by different methods was determined by gas chromatography (GC-FID) with a hydrogen flame ionization detector.

[0025] (2) Weigh Antarctic krill oil, add 1 mL of methyl tert-butyl ether / methanol (3:1, v / v), vortex for 15 min, and take 100 μL each of sample and internal standard (DG(12:0_12:0), PE(12:0_12:0), PC(13:0_13:0), PG(12:0_12:0), TG(17:0_17:0_17:0)), and dilute to 10 μg / mL with methyl tert-butyl ether / methanol (3:1, v / v). Take 1 mL of the sample solution containing internal standard, add 200 μL of water, vortex for 1 min, and incubate at 4℃ for 10000 hours. × g Centrifuge for 10 min; collect the supernatant and concentrate to dryness. Reconstitute the concentrate with 500 μL acetonitrile / isopropanol (1:1, v / v), vortex for 3 min, and then centrifuge at 10000 mL. × g Centrifuge for 3 min, take the supernatant and pass it through a 0.45 μm organic membrane, and use ACQUITY I-Class plus+Synapt XS ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry to analyze the lipid composition of krill oil.

[0026] Example 1: Three-stage extraction process and lipid composition of Antarctic krill oil (1) Subcritical butane primary extraction: Weigh freeze-dried Antarctic krill, grind it into powder, and add butane as the extraction solvent at a material-to-liquid ratio of 1:7 (g / mL). Extract at 40℃ for 60 min. After extraction, transfer the mixture to a separation tank. Evaporate the butane under reduced pressure (-0.1 MPa), collect the extracted oil at the bottom of the tank to obtain the first-stage Antarctic krill oil, weigh it, calculate the oil yield, and determine its chemical composition. At the same time, collect the residual Antarctic krill meal after extraction, weigh it, and use it as the raw material for the next stage of extraction.

[0027] (2) Acetone secondary extraction: Acetone was added to the primary extracted shrimp meal at a material-to-liquid ratio of 1:5 (g / mL), and the mixture was magnetically stirred at 30℃ for 40 min. After the extraction, the mixture was filtered and centrifuged at 8000 rpm for 20 min at 4℃. The supernatant was collected and desolventized by vacuum rotary evaporation at 45℃ to obtain the second-grade Antarctic krill oil. The yield of the oil was calculated and its chemical composition was determined after weighing. The second-grade Antarctic krill meal was recovered, weighed, and used as the raw material for the next extraction stage.

[0028] (3) Tertiary ethanol extraction: Anhydrous ethanol was added to the shrimp meal extracted in the secondary extraction at a material-to-liquid ratio of 1:4 (g / mL), and the mixture was magnetically stirred at 30℃ for 40 min. After the extraction, the mixture was filtered and centrifuged at 8000 rpm for 20 min at 4℃. The supernatant was collected and the solvent was removed by vacuum rotary evaporation at 45℃ to obtain the third-grade krill oil. The yield of the oil was calculated and its chemical composition was determined after weighing.

[0029] Table 1. Yield and lipid composition (%) of Antarctic krill oil obtained from the third-stage preparation in Example 1

[0030] Table 2 Fatty acid composition of Antarctic krill oil obtained in Example 1 (third-stage preparation)

[0031] Comparative Example 1: Extraction of Antarctic Krill Oil with Different Solvents (1) Weigh freeze-dried Antarctic krill powder and add chloroform:methanol (2:1, v / v), anhydrous ethanol, isopropanol, acetone and n-hexane at a material-to-liquid ratio of 1:12 (g / mL). Stir at 750 rpm for 2 h (30℃).

[0032] (2) Chloroform: The filtrate obtained from methanol extraction needs to be added with 0.2 times 0.9% NaCl solution, shaken and allowed to stand to separate into layers, the lower organic phase is collected and the solvent is removed by vacuum rotary evaporation at 40℃.

[0033] (3) After the extraction of other solvents is completed, filter and centrifuge at 8000 rpm for 20 min at 4℃, collect the supernatant and remove the solvent by rotary evaporation at 45℃.

[0034] Example 2: Enrichment of DHA / EPA by Antarctic krill oil enzyme modification Accurately weigh the raw material from the third-stage ethanol extraction of Antarctic krill oil in Example 1 into a 50 mL stoppered conical flask. Add ethyl ester DHA / EPA (1:2), Antarctic krill oil (DHA / EPA: krill oil = 6 mL: 1 g), and 1.00% water, and shake well. Then add 15% immobilized phospholipase A1 and place in a water bath shaker at 50°C and 200 r / min for 12 h. After the reaction is complete, immediately filter and collect the filtrate. Simultaneously, wash the conical flask and immobilized enzyme with n-hexane. Combine the filtrate and washing liquid and transfer to a round-bottom flask. Remove the solvent by rotary evaporation at 45°C to obtain the crude product. Use acetone (pre-cooled at 4°C) to deoil the crude product until the supernatant evaporates to dryness on a clean glass slide without any oil residue. Then centrifuge to collect the precipitate layer, and dry it with nitrogen to obtain modified Antarctic krill oil. Seal the product phospholipids in a brown bottle and store at 4°C.

[0035] Example 3: Antidepressant effects of different active ingredients in Antarctic krill oil Male C57 / 6J mice were acclimatized for 7 days and then randomly divided into 7 groups. The following gavage doses were set: Control group, Model group (CUMS), Positive control group (Fluoxetine), Whole oil group (oil sample obtained by mixing first, second, and third grade Antarctic krill oils in Example 1 at ratios of 24.16%, 33.11%, and 42.74%, respectively, QY), TAG group (Antarctic krill oil extracted with first-stage butane), PL group (Antarctic krill oil extracted with third-stage ethanol), and Modified PL group (PL-1). The Control and Model groups received isocaloric maltodextrin by gavage, the Positive control group received fluoxetine by gavage at a dose of 2.6 mg / (kg bw d), and the Sample group received fluoxetine by gavage at a dose of 200 mg / (kg bw d). Mice were administered gavage for 4 consecutive weeks. Except for the control group, the other mice were modeled using CUMS. After the experiment, all mice underwent behavioral tests including: sucrose preference test (SPT), elevated plus maze (EPM), and tail suspension test (TST).

[0036] (1) CUMS modeling procedure: A 4-week CUMS stimulation program was conducted. Except for the Control group, mice in other groups were subjected to a series of chronic, unpredictable, and mild stressors. The stressors included: 24-hour water / food fasting, 24-hour empty water bottle / empty cage, day / night reversal (12 / 12 h), 5-minute tail clamping, 6-hour restraint, 45° tilt (24 h), 5-minute shaking, and damp bedding. To ensure the unpredictability of the modeling, 2-3 different stressors were randomly applied each day, and the same stressor could not be reused within a week.

[0037] (2) Compared with the Control group, the CUMS group showed a significant decrease in sucrose skewness (≥20%) and a significant decrease in the number of times the open arms of the elevated cross maze were entered. p <0.01), the tail immobility time was significantly increased by ≥30% ( p <0.0001), behavioral results indicate that the CUMS model was successfully established. Compared with the CUMS group, the Antarctic krill oil group and the Fluoxetine group showed significantly improved recovery of sucrose preference and significantly increased number of open arm entries in the elevated cruciate maze, approaching control levels, while significantly reduced tail immobility time. p<0.0001). Experimental results showed that CUMS did not significantly affect the mood and behavior of mice in the presence of different components of krill oil. This indicates that different components of Antarctic krill oil all have certain effects in improving depression, and the antidepressant effect showed a trend of PL-1>QY>PL>TAG. Therefore, the three extracts obtained from the graded extraction of Antarctic krill oil all hold promise for extensive exploration and development for the prevention and treatment of depression, as well as providing raw materials for the subsequent development of personalized and diversified antidepressant products.

[0038] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for extracting Antarctic krill oil with antidepressant activity, characterized in that: Includes the following steps: (1) Antarctic krill is dried and pulverized to obtain Antarctic krill powder; (2) Mix Antarctic krill powder with butane for extraction. After extraction, the target product is separated in a separation tank. Solvent is removed by vacuum to obtain primary extracted Antarctic krill oil and primary extracted Antarctic krill meal. After the solvent evaporates, they are used as raw materials for the next extraction stage. (3) The primary extract of Antarctic krill meal is mixed evenly with acetone and extracted. After extraction, the mixture is filtered and the solvent is removed to obtain secondary extract of Antarctic krill oil and secondary extract of Antarctic krill meal. (4) Secondary extraction of Antarctic krill meal was performed by mixing and extracting with ethanol, followed by filtration and solvent removal from the filtrate to obtain tertiary extraction of Antarctic krill oil. (5) Immobilized phospholipase A1 was added to the three-stage extraction of Antarctic krill oil for modification.

2. The method according to claim 1, characterized in that, In step (2), the mass-to-volume ratio of Antarctic krill powder to butane is 1:1 to 1:14, g / mL.

3. The method according to claim 1, characterized in that, In step (2), the extraction temperature is 40℃ and the extraction time is 60~180 min.

4. The method according to claim 1, characterized in that, In step (3), the mass-to-volume ratio of primary extracted Antarctic krill meal to acetone is 1:2 to 1:6, g / mL.

5. The method according to claim 1, characterized in that, In step (3), the extraction temperature is 30℃ and the extraction time is 40min.

6. The method according to claim 1, characterized in that, In step (4), the mass-to-volume ratio of the secondary extracted Antarctic krill meal to ethanol is 1:2 to 1:6, g / mL.

7. The method according to claim 1, characterized in that, In step (4), the extraction temperature is 30℃ and the extraction time is 40min.

8. The method according to claim 1, characterized in that, In step (5), the amount of immobilized phospholipase A1 added is 5-30% of the mass of the three-stage extracted Antarctic krill oil; the modification is carried out at 35-55℃ for 5-36 h.

9. Antarctic krill oil prepared by any one of the methods described in claims 1 to 8.

10. The use of Antarctic krill oil as described in claim 9 in the preparation of antidepressant drugs or food.

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