Method for separating and extracting active components of microalgae by using a three-phase system and application thereof

Abstract

The application discloses a method for separating and extracting active components of microalgae by using a three-phase system and application thereof. The method comprises the following steps: preparing a hydrophobic deep eutectic solvent (DES) by taking lidocaine as a hydrogen bond acceptor and fatty acids (capric acid, lauric acid and myristic acid) as hydrogen bond donors; adding microalgae powder into a mixed system of the DES and a salt solution, adding an organic solvent after ultrasonic treatment, and then performing extraction by using a three-phase system, and finally collecting target active substances (lipids, astaxanthin, proteins and saccharides) in different zones. The method solves the technical problems that lipids and astaxanthin are easily co-dissolved and the separation cost is extremely high in the later stage, and the three-phase solvents can be recycled and utilized, and are suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the fields of biochemical engineering and natural product extraction and separation technology, and in particular to a method for separating and extracting active ingredients from microalgae using a three-phase system and its application. Background Technology

[0002] Microalgae (such as Haematococcus pluvialis, Chlorella, and Spirulina) are rich in various high-value-added active ingredients and are typical co-production biological resources. Taking Haematococcus pluvialis as an example, it can accumulate the super antioxidant astaxanthin under specific conditions; sugars account for about 30% to 40% of the cell dry weight and have antioxidant, anti-aging, anti-inflammatory, anti-tumor, lipid-lowering, and immune-enhancing biological activities; at the same time, its protein content is 16% to 23%, with a reasonable amino acid composition, making it a high-quality protein source with liver-protective and antioxidant effects; in addition, microalgal lipids, as important energy substances and nutritional supplements, also have extremely high development and utilization value. However, due to the dense three-layered cell wall structure of Haematococcus pluvialis and the small differences in polarity among its components (such as lipids and pigments, proteins and sugars), traditional extraction methods cannot separate components with similar polarities.

[0003] Chinese patent CN114377432B describes a method for separating astaxanthin with different configurations from Haematococcus pluvialis extract using supercritical fluid chromatography. Chinese patent application CN117384074A describes a method for extracting astaxanthin from Haematococcus pluvialis using a cellulase- and pectinase-assisted eutectic solvent. Chinese patent application CN117700572A describes a method for extracting crude polysaccharides, an intracellular active substance from Haematococcus pluvialis, using choline amino acid ionic liquid to disrupt cell walls. Chinese patent CN119701403B discloses a technique for enriching proteins in Haematococcus pluvialis using a three-phase partitioning system. However, these methods mostly focus on the separation of single components or the recovery of substances with similar polarities from Haematococcus pluvialis, failing to achieve in-situ regional enrichment of active substances with small polarity differences within a single operating unit, resulting in limitations such as limited extracted components and low separation selectivity. Therefore, researching a technique capable of simultaneously extracting, separating, and regionally enriching multiple active substances within Haematococcus pluvialis is of great significance. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for separating and extracting active ingredients from microalgae using a three-phase system.

[0005] Another object of the present invention is to provide an application of the method for separating and extracting active ingredients from microalgae using a three-phase system.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for separating and extracting active ingredients from microalgae using a three-phase system includes the following steps:

[0008] (1) Preparation of hydrophobic eutectic solvent:

[0009] Lidocaine (hydrogen bond acceptor) and fatty acid (hydrogen bond donor) are mixed and stirred at 70-90°C until the solution is clear and transparent. Then, the mixture is allowed to stand and cool to room temperature to obtain a hydrophobic eutectic solvent (DES). The fatty acid is at least one of decanoic acid, lauric acid, and tetradecanoic acid.

[0010] (2) Extraction in a three-phase system:

[0011] Microalgae powder was added to a mixture of hydrophobic eutectic solvent (DES) and salt solution, and ultrasonically treated at 30–60°C and 100–300W for 10–60 min (using ultrasound to disrupt cells and promote directional migration of components). Then, an organic solvent was added, and the mixture was thoroughly mixed and centrifuged to induce phase separation. The target active substances were then collected in separate layers (from top to bottom: L1 layer rich in lipids (organic solvent), L2 layer rich in astaxanthin (hydrophobic eutectic solvent), L3 layer rich in proteins, and L4 layer rich in carbohydrates (salt solution)). Finally, lipids, astaxanthin, proteins, and carbohydrates were obtained through separation and purification.

[0012] The molar ratio of lidocaine (hydrogen bond acceptor) and fatty acid (hydrogen bond donor) in step (1) is 1:(1-2); preferably 1:1.

[0013] The stirring time mentioned in step (1) is more than 1 hour.

[0014] The fatty acid mentioned in step (1) is preferably decanoic acid.

[0015] The microalgae mentioned in step (2) are at least one of Haematococcus pluvialis, Spirulina, Chlorella, Dunaliella salina, and diatoms; preferably Haematococcus pluvialis.

[0016] The algae mentioned include Dunaliella salina and others.

[0017] The salt solution mentioned in step (2) is an aqueous sodium sulfate solution with a concentration of 10% to 20% by mass (preferably 15% by mass).

[0018] In step (2), the extraction temperature is preferably 50°C.

[0019] The organic solvent mentioned in step (2) is a low-polarity organic solvent; preferably at least one of ethyl acetate and isooctane.

[0020] The preferred conditions for ultrasonic treatment in step (2) are: 200W ultrasonic treatment for 40 minutes.

[0021] The solid-liquid ratio of the microalgae powder and the three-phase system (hydrophobic eutectic solvent + salt solution + organic solvent) in step (2) is 1g: (10-50)mL; preferably 1g: (20-50)mL; more preferably 1g: 30mL.

[0022] The mass ratio of the hydrophobic eutectic solvent (DES), salt solution and organic solvent in the three-phase system described in step (2) is (0.40~0.55): (0.15~0.35): (0.15~0.35); preferably 0.44:0.28:0.28.

[0023] The centrifugation conditions described in step (2) are: rotation speed 8000-12000 rpm, centrifugation time 5-15 min; preferably: rotation speed 8000-10000 rpm, centrifugation time 10-15 min; more preferably: rotation speed 10000 rpm, centrifugation time 10 min.

[0024] The separation and purification described in step (2) can be carried out using conventional methods in the art. For example, the lower layer L4 can be removed using a separatory funnel, and the upper layers L1 and L2 can be removed sequentially using a dropper. Then, lipids, astaxanthin, proteins and carbohydrates can be separated from the L1, L2, L3 and L4 layers respectively.

[0025] The lipid separation and purification method is as follows: First, the L1 layer is rotary evaporated to obtain a concentrate; then, it is decolorized with activated carbon, followed by alkali washing, water washing, drying with anhydrous Na2SO4, filtration and evaporation to obtain the lipid.

[0026] The conditions for rotary evaporation are: 40–50°C, -0.08 MPa, until no more organic solvent can be evaporated (volume remains unchanged).

[0027] The decolorization is carried out using activated carbon; the amount of activated carbon used is 1 to 2% of the mass of the decolorization system.

[0028] The decolorization conditions are: 50±2℃, decolorization for 30±5min.

[0029] The alkaline washing is performed using a 0.1M NaOH solution with a pH of 8-9.

[0030] The astaxanthin was separated and purified using XAD-8 macroporous adsorption resin. The eluent used was an ethanol-water solution with a volume ratio of 50~65:35~50 and an ethanol-ethyl acetate solution with a volume ratio of 80~95:5~20.

[0031] The sugars were separated and purified using a Sepharose CL-6B gel column with distilled water as the eluent and a flow rate of 0.5–1 mL / min.

[0032] The protein was separated and purified using a 20%–70% ammonium sulfate solution. After standing at 4°C in the dark, it was centrifuged to obtain the final product.

[0033] The time for keeping the plant in the dark is 8-10 hours.

[0034] The centrifugation conditions are: 8000~10000 r / min for 20~30 min.

[0035] The method for separating and extracting active ingredients from microalgae using a three-phase system further includes a step of recovering the solvent after step (2); the solvent includes at least one of an organic solvent (L1 layer), a hydrophobic eutectic solvent (L2 layer), and a salt solution (L4 layer).

[0036] The method for separating and extracting active ingredients from microalgae using a three-phase system is applied in the separation and extraction of active ingredients from microalgae.

[0037] The microalgae active ingredients include at least one of lipids, astaxanthin, proteins, and sugars; preferably lipids, astaxanthin, proteins, and sugars.

[0038] The sugars include at least one of D-glucose, rhamnose, and xylose; preferably D-glucose.

[0039] The present invention has the following advantages and effects compared with the prior art:

[0040] 1. This invention provides a method for the simultaneous extraction and separation of multiple bioactive substances from microalgae using a three-phase system controlled by interfaces. This method constructs a three-phase system consisting of a phase-forming salt solution, a hydrophobic eutectic solvent (DES), and a low-polarity organic solvent to simultaneously enrich lipids, astaxanthin, proteins, and carbohydrates from Haematococcus pluvialis. Microalgae powder is added to the extraction system composed of the above three phases. Utilizing the effect of temperature on interfacial energy and partition coefficient, and with the assistance of ultrasonic force, bioactive substances of different polarities are driven to migrate directionally to specific phases. After centrifugation, the system spontaneously forms a clear four-layer structure, achieving in-situ extraction and physical isolation of complex biological components within a single unit operation. Furthermore, all three phase solvents can be recovered and recycled, demonstrating significant economic efficiency and versatility.

[0041] 2. The method of the present invention solves the following technical problems: (1) Simultaneous refining of complex biomass matrix: For multi-component co-production resources such as microalgae, it solves the problem that existing technologies cannot simultaneously achieve efficient extraction of components with huge polarity spans such as lipids, pigments, proteins, and sugars in one operation unit; (2) Refined separation of components with similar polarity: It solves the technical problem that lipids and carotenoids (such as astaxanthin) are easily co-soluble in traditional organic solvent extraction, and the separation cost in the later stage is extremely high; (3) Efficient cell wall disruption and in-situ enrichment of dense cell walls: It solves the problem that microalgae with a three-layer dense cell wall structure, such as Haematococcus pluvialis, are not completely disrupted under mild conditions, and cross-contamination is very likely to occur after component extraction; (4) Sustainability and safety of solvent system: It solves the environmental and cost problems of traditional extraction processes using a large amount of toxic organic solvents, which are difficult to recycle and cannot be recycled; (5) Universality and scalability of process: It solves the problem that the extraction technology has poor compatibility among different types of microalgae (Chlorella, Spirulina, Diatoms, etc.) and the problem of decreasing efficiency in large-scale production.

[0042] 3. The three-phase system of this invention exhibits a synergistic effect: it simultaneously achieves high extraction rates of astaxanthin (42.55–73.38%), lipids (55.35–91.83%), proteins (29.02–77.58%), and carbohydrates (64.39–84.85%), with all indicators exceeding those of other solvent systems. This three-phase system can also be used for the extraction of other plants with high protein, polysaccharide, fat, and pigment content. Attached Figure Description

[0043] Figure 1 This is a flowchart of the process for separating and extracting active ingredients from microalgae using a three-phase system, as described in this invention.

[0044] Figure 2 This is an extraction effect diagram of the active ingredients extracted from microalgae according to the extraction conditions of Example 5; wherein, (a) is the content of fatty acid components; (b) is the high performance liquid chromatogram of astaxanthin; (c) is the SDS-PAGE electrophoresis band of Haematococcus pluvialis and the interface layer complex; (d) is the determination of monosaccharide components in the extract (from left to right, the peaks appearing on the standard curve are xylose, arabinose, mannose, glucose, galactose, rhamnose and trehalose, respectively).

[0045] Figure 3 These are microscopic images and scanning electron microscope images of Haematococcus pluvialis powder before and after extraction (under the extraction conditions of Example 5); wherein, (a) is untreated Haematococcus pluvialis powder, optical microscope image with oil immersion objective 1000x; (b) is extracted Haematococcus pluvialis powder, optical microscope image with oil immersion objective 1000x; (c) is untreated Haematococcus pluvialis powder, electron microscope image with 1000x magnification; (d) is extracted Haematococcus pluvialis powder, electron microscope image with 1000x magnification.

[0046] Figure 4 This is a diagram showing the extraction effect of the three-phase separation system of the present invention (under the extraction conditions of Example 5) on other microalgae (spirulina, chlorella, Dunaliella salina, and diatoms). Detailed Implementation

[0047] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0048] In this invention, the ambient temperature (room temperature) is 25°C.

[0049] Example 1

[0050] An extraction system and method for simultaneous and efficient extraction and separation of multiple active components from microalgae, the process is as follows: Figure 1 As shown in Table 1, the solvent system and extraction conditions are as follows: The specific steps of the extraction method are as follows:

[0051] (1) Preparation of hydrophobic eutectic solvent

[0052] Lidocaine (hydrogen bond acceptor) and fatty acid (lauric acid, hydrogen bond donor) were mixed in a molar ratio of 1:1 and stirred at 70°C for 1 h until the solution was clear and transparent. Then, the mixture was allowed to stand and cool to room temperature to obtain a hydrophobic eutectic solvent (DES).

[0053] (2) Construction of a three-phase extraction system and temperature-controlled ultrasound-assisted extraction

[0054] ①Preparation of salt solution: Add sodium sulfate (Na2SO4) to deionized water to prepare a sodium sulfate aqueous solution with a concentration of 15% by mass;

[0055] ② Add Haematococcus pluvialis algal powder (source: Beijing Zhuoyuan Hangcheng Technology Development Co., Ltd.) (CASMA Procurement Platform Product No.: NFSW-079) to a mixture of hydrophobic DES (lidocaine / lauric acid) and salt solution (sodium sulfate aqueous solution), and sonicate at 50 ℃ and 200 W for 30 minutes. The system was thoroughly mixed for 1 minute (using ultrasound to break up cells and promote the directional migration of components), then an organic solvent (ethyl acetate) was added and vortexed (1 minute) to ensure thorough mixing. The solid-liquid ratio of the algal powder to the three-phase extraction system (hydrophobic DES + salt solution + organic solvent, mass ratio = 0.44:0.28:0.28) was controlled at 1:20 (g / mL) (i.e., the mass ratio of algal powder to the total of all solvents is 1:15.6, g / g). Finally, solid-liquid centrifugation (8000 rpm for 15 minutes) was used to induce phase separation (the system spontaneously formed a four-layer heterogeneous structure), and then the target active substances were collected in sections (from top to bottom, the L1 layer rich in lipids (organic solvent), the L2 layer rich in astaxanthin (hydrophobic DES), the L3 layer rich in interfacial proteins, and the L4 layer rich in sugars (salt solution) were collected).

[0056] ③ Separation and purification

[0057] Use a separatory funnel to remove the lower layer L4, and use a plastic dropper to assist in phase separation, then sequentially remove the upper layers L1 and L2.

[0058] Lipid purification: First, the L1 layer was rotary evaporated (40-50℃, -0.08 MPa, until no more organic solvent could be evaporated) to obtain a concentrate; then, it was decolorized with activated carbon (1-2% of the concentrate by mass, 50℃, 30 min), followed by alkaline washing (0.1M NaOH solution, pH 8-9), water washing, drying with anhydrous Na2SO4, and finally filtration and evaporation to dryness.

[0059] Astaxanthin purification: XAD-8 macroporous adsorption resin was used as the eluent. The eluents were ethanol-water solution (volume ratio 50~65:35~50) and ethanol-ethyl acetate solution (volume ratio 80~95:5~20).

[0060] Sugar purification: Sepharose CL-6B gel column; eluent: distilled water; flow rate: 0.5~1 mL / min.

[0061] Protein purification: Ammonium sulfate solution (mass concentration 20%~70%) was used; the protein was allowed to stand at 4 ℃ in the dark for 8~10 h; and then centrifuged at 8000~10000 r / min for 20~30 min.

[0062] (3) Solvent recovery and recycling extraction

[0063] The recovered L1, L2, and L4 three-phase solvents can be directly used for the simultaneous extraction of the next batch of microalgae.

[0064] Examples 2-6

[0065] Following the method in Example 1, the solvent system and extraction conditions (Table 1) were adjusted to extract Haematococcus pluvialis powder, and then the target active substances were collected in separate sections.

[0066] Comparative Examples 1-12

[0067] Following the method in Example 1, the solvent system and extraction conditions (Table 1) were replaced to extract Haematococcus pluvialis powder, and then the target active substances were collected.

[0068] Table 1

[0069]

[0070] Example 1

[0071] I. The active substances extracted from Examples 1-6 and Comparative Examples 1-12 were determined, as follows:

[0072] 1. Extraction rates of lipids, astaxanthin, proteins, and carbohydrates

[0073] E a (mg / g)=m a / M formula (4-1)

[0074] E in the formula a Indicates the extraction yield of lipids, astaxanthin, proteins, and carbohydrates, m ɑ The values ​​represent the mass (mg) of lipids, astaxanthin, protein, and carbohydrates, while M represents the mass (g) of Haematococcus pluvialis.

[0075] 2. Distribution coefficient

[0076] The distribution coefficient (K) can be calculated according to the following equations (4-2), (4-3), (4-4), and (4-5):

[0077] K1 =C L1 / C L2 Equation (4-2)

[0078] K2 =C L1 / C L2 Equation (4-3)

[0079] K3 =C L3 / C L4 Equation (4-4)

[0080] K4 =C L3 / CL4 Equation (4-5)

[0081] In the formula, K1 represents the partition coefficient of lipids (Fat), K2 represents the partition coefficient of astaxanthin (Ast), K3 represents the partition coefficient of proteins (Prot), and K4 represents the partition coefficient of carbohydrates (Sac); C L1 C L2 C L1 C L2 These represent the concentrations of the active substances in the extract in the respective layers (L1, L2, L3, L4).

[0082] 3. Methods for determining active substance components

[0083] (1) Determination of astaxanthin content and its components

[0084] The total Astaxanthin (Ast) content in *Haematococcus pluvialis* was determined by repeated extraction with ethanol. The Astaxanthin concentration was detected by high-performance liquid chromatography (HPLC, UltiMate 3000, Thermo Fisher, USA) (Reference: GAO J, FANG C, LIN Y, et al. Enhanced extraction of astaxanthin using aqueous biphasic systems composed of ionic liquids and potassium phosphate [J]. Food chemistry, 2020, 309: 125672.). The mobile phase was dichloromethane / methanol / acetonitrile / water (5 / 85 / 5.5 / 4.5, v / v), and the flow rate was 1.0 mL·min⁻¹. -1 HPLC analysis was performed using a commercial C18 column (4.6 mm × 250 mm, 5 µm) at a column temperature of 298.15 K. UV detection was performed at 474 nm, with an injection volume of 10.0 µL. The Ast content in each solvent was calculated based on the standard curve. Three replicates were performed.

[0085] (2) Determination of lipid and fatty acid components

[0086] The fatty acid content in Haematococcus pluvialis was determined using Soxhlet extraction. The lipid content was determined according to GB / T5009.6-2016, "National Food Safety Standard - Determination of Fat in Food". First, fatty acids were extracted from Haematococcus pluvialis according to the method described in the reference (SOMPACH G, RODKLONGTAN A, NITISINPRASERT S, et al. Microencapsulating role of wheyprotein isolate and sucrose in protecting the cell membrane and enhancing survival of probiotic lactobacilli strains during spray drying, storage, and simulated gastrointestinal passage [J]. Food research international, 2022, 159: 111651.). The specific steps are as follows:

[0087] Weigh approximately 0.4 g of algal powder into a centrifuge tube, recording the precise mass. Resuspend in 10 mL of ice-cold 0.85% (w / v) NaCl solution. Vortex to completely suspend and hydrate the algal powder, then incubate on ice for 10-15 minutes. Disrupt the cells using an ultrasonic disruptor at 300 W for 20 cycles. Mix the cell suspension thoroughly with 15 mL of a 1:2 (v / v) dichloromethane-methanol mixture at room temperature for 2 minutes. Then add 10 mL of a 1:1 (v / v) dichloromethane-distilled water mixture to the cell suspension and mix for 2 minutes. Incubate at room temperature for 10 minutes, then filter the lower phase containing fatty acids through a 0.22 μm nylon syringe filter and dry under vacuum.

[0088] The esterification of fatty acids was performed according to the method described in the literature (RODRIGUES RO, COSTA H, LIMA R, et al. Simple methodology for the quantitative analysis of fatty acids in human red blood cells [J]. Chromatographia, 2015, 78(19): 1271-81.). Specifically, the fatty acids were hydrolyzed in a water bath at 90 ± 1 °C with 0.25 mL of methanol-potassium hydroxide solution (0.25 M) for 10 min. Then, 2 mL of methanol-boron trifluoride solution (14%) was added, and the reaction mixture was incubated at 90 ± 1 °C for another 60 min. Liquid-liquid extraction was performed using a hexane (6 mL) / distilled water (2 mL) system to obtain fatty acid methyl esters. The upper hexane phase was filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a vial for subsequent GC analysis (the extract sample was from the start of the esterification reaction).

[0089] Fatty acid methyl esters were analyzed using a gas chromatograph (6890 N, Agilent, USA) equipped with a flame ionization detector. The column used was an SPTM-2560 capillary column (100 m, 0.25 mm, 0.20 µm). The analytical conditions were as follows: injector temperature 250 °C; carrier gas: hydrogen; detector temperature 250 °C; injection volume: 1 µL. The column temperature program was: 140 °C, hold for 5 min; ramp to 175 °C at 10 °C / min, then ramp to 210 °C at 2 °C / min, hold for 5 min; ramp to 218 °C at 1 °C / min, hold for 3 min; ramp to 240 °C at 30 °C / min, hold for 17 min.

[0090] (3) Determination of carbohydrates and their monosaccharide components

[0091] The total sugar content in Haematococcus pluvialis was determined using the dinitrosalicylic acid method. Following the method described in the reference (Xiao Jinyan, Song Ruiping, Wang Baobei, et al. Physicochemical properties and in vitro antioxidant activity of polysaccharides from Haematococcus pluvialis residue precipitated by gradient alcohols [J]. Food Industry Technology, 2025, 46(06): 75-84.), high-performance liquid chromatography (HPLC) was used to detect the monosaccharide types in the extract. The chromatographic conditions were: Eclipse XDB-C18 column (4.6 mm x 150 mm, 5 pm); mobile phase: a mixture of phosphate buffer and acetonitrile (83:17, V / V); flow rate: 1.0 mL / min; column temperature: 25 ℃; injection volume: 20 Ul; detection at 245 nm.

[0092] (4) Determination of crude protein content and molecular weight

[0093] The protein content was determined according to GB / T6432-2018 "Determination of Crude Protein in Feed - Kjeldahl Method". The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was used as described in the literature (Li Qin, Wang Meng, Yuan Shuo, et al. Study on the structural and functional characteristics of proteins in Haematococcus pluvialis [J]. Grain and Oil Food Science and Technology, 2024, 32(04): 201-9.): SDS-PAGE pre-prepared gel (12.5% ​​separating gel, 3% stacking gel), voltage 80 V / 120 V. The gel plates were fixed at room temperature, stained with Coomassie Brilliant Blue R-250 with shaking for 30 min, and then destained overnight. Algal powder and the interfacial complex were separately taken and added to 10 ml of lysis buffer (UREA, 50 mM Tris-HCl, 100 mM NaCl, 2% (w / v) SDS (sodium dodecyl sulfate), 10 mM DTT (dithiothreitol), 1× Protease inhibitor, pH 7.6), and thoroughly ground for lysis. The mixture was then centrifuged at 14800 rpm for 30 min, and the supernatant was collected and transferred to a tube again.

[0094] (5) Results

[0095] The results are shown in Table 2. Figure 2 and Figure 3 As shown.

[0096] Table 2

[0097]

[0098] As can be seen from the results in Table 2, the three-phase system of the present invention simultaneously achieved high extraction rates of astaxanthin, lipids, proteins, and carbohydrates. Among them, Example 5 showed the best extraction effect (73.38% astaxanthin, 91.83% lipids, 77.58% protein, and 84.85% carbohydrates), and all indicators exceeded the corresponding indicators of other solvent systems (Comparative Examples 1-6), single solvent systems (Comparative Examples 7-9), and any two-phase combination (Comparative Examples 10-12).

[0099] like Figure 2As shown in (a), the proportions of major fatty acids in algal powder and L1 / L2 are basically consistent: C18:2n6t (linoleic acid, trans): algal powder 51.91%, L1 49.39%, L2 49.60%; C18:1n9t (oleic acid, trans): algal powder 13.63%, L1 14.87%, L2 14.83%, slightly increased after extraction; C18:1n9c (oleic acid, cis): algal powder 13.50%, L1 14.74%, L2 14.70%, consistent with the trans trend; C16:0 (palmitic acid): algal powder 6.99%, L1 6.52%, L2 6.47%, slightly decreased; C18:0 (stearic acid): algal powder 5.18%, L1 5.53%, L2 5.46%, slightly increased. The ratio of saturated to unsaturated fatty acids remained essentially constant: the total of major saturated fatty acids (C16:0, C18:0) was 12.17% in algal powder, 12.05% in L1, and 11.93% in L2; the total of major unsaturated fatty acids (C18:1, C18:2) was 78.95% in algal powder, 79.74% in L1, and 79.93% in L2. From the proportions, the three-phase system constructed in this study showed no obvious selective trend in the extraction of fatty acids from Haematococcus pluvialis, meaning that both L1 and L2 phases could completely capture all types of fatty acids in the algal powder without component divergence due to polarity differences. This characteristic is of great significance for maintaining the natural composition of oil products, avoiding the loss of functional fatty acids (such as linoleic acid and oleic acid), and ensuring the nutritional value of the extracted fats.

[0100] The extracted L1 / L2 was diluted with methanol and the chromatogram of Ast was obtained by high-performance liquid chromatography (HPLC) at 474 nm. Figure 2 As shown in (b) above. Similar proportions of the Ast component were detected in both L1 and L2 layers. The three characteristic peaks represent the monoesters of C18 linolenic acid (Pack 2, Ast-E1) with three double bonds, C18 linoleic acid (Pack 3, Ast-E2) with two double bonds, oleic acid (Ast-E3) with only one double bond, and the saturated fatty acid C16 palmitic acid (Pack 4, Ast-E4). Palmitic acid and oleic acid astaxanthin monoesters had the highest yields, followed by linolenic acid. The proportions of the three main fatty acids were approximately: linolenic acid (C18:3, 14.52%), linoleic acid (C18:2, 28.39%), and palmitic acid (C18:1, 39.10%).

[0101] The molecular weight of proteins in the L3 interface layer and Haematococcus pluvialis powder was determined. SDS-PAGE electrophoresis showed that the major subunits of Haematococcus pluvialis proteins were concentrated around 14.4 kDa, 18.4 kDa, and 38 kDa. Figure 2As shown in (c), the richer bands (38, 30, 27, 23, 19, 17 kDa) in the L3 sample indicate the presence of a more complex protein composition in the extract. The 38 kDa band may correspond to the γ-subunit of phycoerythrin or other high-molecular-weight proteins.

[0102] The L4 extract after extraction and centrifugation was diluted 1000 times and analyzed by high performance liquid chromatography (HPLC). The HPLC chromatogram of the monosaccharide is shown below. Figure 2 As shown in (d) in the figure. Studies have demonstrated that Haematococcus pluvialis has a good monosaccharide conversion rate. Therefore, different types of monosaccharide standards and L4 extract were analyzed by HPLC to study the monosaccharide conversion, composition, hydrolysis and extraction effects of the three-phase system on sugars in Haematococcus pluvialis. In this study, the L4 extract and the spectra obtained by the monosaccharide standards (mixed standards) showed similar peak curves, and the retention times of the characteristic peaks of the monosaccharides detected in the extract were consistent with those of their corresponding standards. The results showed that the most abundant monosaccharide in Haematococcus pluvialis was D-glucose (3523.20 mg / g, 79.36%), followed by rhamnose (516.40 mg / g, 11.63%) and xylose (399.80 mg / g, 9.01%), while mannose, trehalose, galactose and arabinose were not detected.

[0103] like Figure 3 As shown, untreated Haematococcus pluvialis cells appear round and bright red under both microscopic and scanning electron microscopy, possessing an intact cell wall structure and significant pigment accumulation. Microscopic observation reveals that the extracted Haematococcus pluvialis cells become significantly lighter in color, indicating the dissolution of pigments and other active substances. The cells exhibit barrier rupture and cell wall disruption, with cytoplasmic contents flowing out and dispersing in the solution. The fragmented Haematococcus pluvialis (L3 layer complex) after extraction displays a complex, clumped structure under scanning electron microscopy.

[0104] Example 2

[0105] Following the parameters and conditions in Example 5, Haematococcus pluvialis was replaced with Spirulina, Chlorella, Dunaliella salina, and diatoms (Table 3), with other conditions remaining the same. After extraction, the partition coefficient (K) was calculated according to the method in Example 1. The results are as follows: Figure 4 As shown, the method of the present invention is applicable to the extraction of other algal species.

[0106] Table 3

[0107]

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for separating and extracting active ingredients from microalgae using a three-phase system, characterized in that, Includes the following steps: (1) Preparation of hydrophobic eutectic solvent: Lidocaine and fatty acids are mixed and stirred at 70–90°C until the solution is clear and transparent. Then, the mixture is allowed to stand and cool to room temperature to obtain a hydrophobic eutectic solvent. The fatty acid is at least one of decanoic acid, lauric acid, and tetradecanoic acid. (2) Extraction in a three-phase system: Microalgae powder was added to a mixture of hydrophobic eutectic solvent and salt solution, and ultrasonically treated at 30–60°C and 100–300 W for 10–60 min. Then, an organic solvent was added, and the mixture was thoroughly mixed and centrifuged to induce phase separation. The target active substances were then collected in separate layers: from top to bottom, the L1 layer rich in lipids, the L2 layer rich in astaxanthin, the L3 layer rich in proteins, and the L4 layer rich in carbohydrates were collected. Finally, lipids, astaxanthin, proteins, and carbohydrates were obtained through separation and purification.

2. The method according to claim 1, characterized in that: The microalgae mentioned in step (2) are at least one of Haematococcus pluvialis, Spirulina, Chlorella, Dunaliella salina, and diatoms.

3. The method according to claim 1, characterized in that: The molar ratio of lidocaine to fatty acid mentioned in step (1) is 1:1 to 2; The salt solution mentioned in step (2) is an aqueous solution of sodium sulfate with a concentration of 10% to 20% by mass. The organic solvent mentioned in step (2) is a low-polarity organic solvent; The mass ratio of the hydrophobic eutectic solvent, salt solution and organic solvent in the three-phase system described in step (2) is 0.40-0.55: 0.15-0.35: 0.15-0.

35.

4. The method according to claim 3, characterized in that: The molar ratio of lidocaine to fatty acid mentioned in step (1) is 1:1; The salt solution mentioned in step (2) is an aqueous solution of sodium sulfate with a concentration of 15% by mass. The organic solvent mentioned in step (2) is at least one of ethyl acetate and isooctane; The mass ratio of the hydrophobic eutectic solvent, salt solution and organic solvent in the three-phase system described in step (2) is 0.44:0.28:0.

28.

5. The method according to claim 1, characterized in that: The solid-liquid ratio of the microalgae powder to the three-phase system in step (2) is 1g:10-50mL.

6. The method according to claim 5, characterized in that: The solid-liquid ratio of the microalgae powder to the three-phase system in step (2) is 1g: 20-50mL.

7. The method according to claim 1, characterized in that: The stirring time mentioned in step (1) is more than 1 hour; In step (2), the extraction temperature is 50℃; The conditions for ultrasonic treatment in step (2) are: 200W ultrasonic treatment for 40 minutes; The centrifugation conditions described in step (2) are: rotation speed 8000-12000 rpm, centrifugation time 5-15 min.

8. The method according to claim 1, characterized in that: The step (2) is followed by a further step of recovering the solvent; wherein the solvent includes at least one of an organic solvent, a hydrophobic eutectic solvent, and a salt solution.

9. The application of the method for separating and extracting microalgal active ingredients using a three-phase system as described in any one of claims 1 to 8 in the separation and extraction of microalgal active ingredients.

10. The application according to claim 9, characterized in that: The microalgae active ingredients include at least one of lipids, astaxanthin, proteins, and sugars; The sugars mentioned include at least one of D-glucose, rhamnose, and xylose.

Images