Extracting agent for synchronously separating active substances of haematococcus pluvialis, preparation method and separation method

Abstract

The invention provides an extraction agent for synchronously separating haematococcus pluvialis active substances, a preparation method and a separation method, and relates to the technical field of natural product extraction, the extraction agent comprises 20-50% by mass of an extraction phase and the balance of water; the extraction phase is quaternary phosphonium ionic liquid or eutectic solvent; the quaternary phosphonium ionic liquid comprises tetrabutyl phosphine trifluoroacetate or tributyl octyl phosphonium bromide; the eutectic solvent comprises quaternary phosphonium ionic liquid and fatty acid in a molar ratio of (1: 1)-(5: 3). The Haematococcus pluvialis polysaccharide is faintly acid, has the capacity of forming Van der Waals force and hydrogen bond interaction, and has the wall breaking effect and high solubility of Haematococcus pluvialis. Liquid-liquid phase separation can be formed, wall breaking of haematococcus pluvialis and extraction of astaxanthin, carbohydrate and protein can be synchronously carried out, no organic solvent is used, operation is easy and convenient, the extraction time is short, and the extraction efficiency is high. The technical problem that in the prior art, an extracting agent cannot effectively separate multiple active substances of haematococcus pluvialis is solved.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction, and in particular to an extractant for synchronously separating active substances of Haematococcus pluvialis, a preparation method and a separation method. Background Art

[0002] Astaxanthin, as one of the main components of Haematococcus pluvialis, has biological activities such as anti-oxidation, enhancing the body's immunity, anti-aging and anti-aging; carbohydrates in Haematococcus pluvialis account for about 30% to 40% of the cell dry mass, and the polysaccharide content is as high as 6% to 10% of the dry mass, mainly with biological activities such as anti-oxidation, anti-aging, anti-inflammatory, anti-tumor, lowering blood lipids, and enhancing immunity; at the same time, the protein content in Haematococcus pluvialis is 16% to 23%, with a high amino acid content and a reasonable composition. It is a good source of protein with antioxidant effects and the effect of improving acute alcoholic liver damage.

[0003] In the prior art, the separation of active substances in Haematococcus pluvialis is mostly the separation of a single substance. For example, Chinese patent application number 202111570709.1 designs a method for separating different configurations of astaxanthin in Haematococcus pluvialis extract by supercritical fluid chromatography. Chinese patent application number 202311552352.3 designs a method for extracting astaxanthin from Haematococcus pluvialis with the assistance of cellulase and pectinase in a low eutectic solvent. Chinese patent CN117700572A with application number 202310873866.2 designs a method for breaking the cell wall of choline amino acid ionic liquid and extracting crude polysaccharides, which are active substances in Haematococcus pluvialis cells. The above methods only separate single substances in Haematococcus pluvialis, and cannot effectively separate multiple active substances in Haematococcus pluvialis, and have great limitations.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide an extractant for simultaneously separating active substances of Haematococcus pluvialis, so as to solve the technical problem that the extractant in the prior art cannot effectively separate multiple active substances of Haematococcus pluvialis.

[0006] The second object of the present invention is to provide a method for preparing an extractant for synchronously separating active substances from Haematococcus pluvialis.

[0007] The third object of the present invention is to provide a method for separating active substances from Haematococcus pluvialis synchronously.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides an extractant for synchronously separating active substances of Haematococcus pluvialis, comprising an extraction phase having a mass ratio of 20% to 50%, and the remainder being water;

[0010] The extraction phase is a quaternary phosphine ionic liquid or a deep eutectic solvent;

[0011] The quaternary phosphine ionic liquid includes tetrabutylphosphine trifluoroacetate or tributyloctylphosphine bromide;

[0012] The low eutectic solvent comprises a quaternary phosphine ionic liquid and a fatty acid in a molar ratio of 1:1 to 5:3.

[0013] Further, the ionic liquid includes tetrabutylphosphine trifluoroacetate or tributyloctylphosphine bromide;

[0014] Preferably, the fatty acid includes any one of lauric acid, capric acid or D,L-lactic acid.

[0015] In a second aspect, the present invention provides a method for preparing the above-mentioned extractant. When the extraction phase is a quaternary phosphine ionic liquid, the quaternary phosphine ionic liquid is mixed with water according to the formula amount to obtain the extractant;

[0016] When the extraction phase is a low eutectic solvent, the quaternary phosphine ionic liquid and fatty acid are stirred and heated at 50-80° C. for at least 2 hours according to the formula amount to obtain a low eutectic solvent, and the low eutectic solvent is mixed with water to obtain an extractant.

[0017] Furthermore, the stirring speed is 100 to 500 rpm.

[0018] In a third aspect, the present invention provides a method for synchronously separating active substances from Haematococcus pluvialis, comprising the following steps:

[0019] A. adding Haematococcus pluvialis to the above-mentioned extractant or the extractant prepared by the above-mentioned preparation method, extracting, and centrifuging to obtain a homogeneous extract of active substances of Haematococcus pluvialis;

[0020] B. Phase separation is performed on the homogeneous extract of active substances from Haematococcus pluvialis to obtain an extraction phase containing astaxanthin, an interface layer containing protein and a water-enriched phase containing carbohydrates, and the phases are purified to obtain astaxanthin, protein and carbohydrates respectively.

[0021] Furthermore, the mass volume ratio of the Haematococcus pluvialis to the extractant is 1:20 to 1:50.

[0022] Further, the extraction includes ultrasonic extraction or stirring extraction;

[0023] The extraction temperature is 30-50°C;

[0024] Preferably, the extraction time is 30 to 60 minutes;

[0025] Preferably, the power of the ultrasound is 100-200W;

[0026] Preferably, the stirring rate is 400-600 rpm.

[0027] Furthermore, the rotation speed of the centrifugal separation is 8000-10000 rpm;

[0028] Preferably, the centrifugal separation time is 10 to 15 minutes;

[0029] Preferably, the centrifugal separation temperature is 20-30°C.

[0030] Further, the phase separation includes standing and heating;

[0031] Preferably, the standing time is 10 to 30 minutes;

[0032] Preferably, raising the temperature includes raising the temperature to 30-50°C.

[0033] Further, the purification includes purification of astaxanthin by adsorption, purification of carbohydrates by molecular separation and purification of proteins by salting out;

[0034] Preferably, the adsorbent for purifying astaxanthin by adsorption method comprises XAD-8 macroporous adsorption resin;

[0035] Preferably, the eluent for purifying astaxanthin by adsorption method comprises ethanol-water solution and ethanol-ethyl acetate solution;

[0036] Preferably, the volume ratio of the ethanol-water solution is 50-65:35-50;

[0037] Preferably, the volume ratio of the ethanol-ethyl acetate solution is 80-95:5-20;

[0038] Preferably, the separating agent for molecular separation and purification of carbohydrates comprises a Sepharose CL-6B gel column;

[0039] Preferably, the eluent for molecular separation and purification of carbohydrates comprises distilled water;

[0040] Preferably, the flow rate of the eluent for molecular separation and purification of carbohydrates is 0.5 to 1 mL / min;

[0041] Preferably, the salting-out reagent for purifying protein by the salting-out method comprises an ammonium sulfate solution with a concentration of 20% to 70%;

[0042] Preferably, the salting-out method for purifying protein comprises static centrifugation;

[0043] Preferably, the standing includes standing at 4°C in the dark for 8 to 10 hours;

[0044] Preferably, the centrifugation comprises centrifugation at 8000-10000 r / min for 20-30 min.

[0045] The invention provides an extractant for synchronously separating active substances of Haematococcus pluvialis, which uses quaternary phosphine ionic liquids to replace traditional imidazole ionic liquids, and synthesizes a new type of low eutectic solvent with fatty acids; the low eutectic solvent is weakly acidic, and has the ability to form van der Waals forces and hydrogen bond interactions, and has both Haematococcus pluvialis wall breaking effect and high solubility. Both the ionic liquid aqueous solution and the low eutectic solvent aqueous solution have the phase change characteristics of low critical eutectic temperature, that is, increasing the temperature is conducive to phase separation, and liquid-liquid phase separation can be formed. Temperature regulation can promote the phase equilibrium transformation of the solvent system and the distribution of active substances of Haematococcus pluvialis. Astaxanthin with strong hydrophobicity mainly migrates to the extraction phase, and carbohydrates mainly migrate to the water-enriched phase. The liquid-liquid interface has the function of adsorbing proteins. The extractant has low viscosity, strong biocompatibility and high safety at room temperature; it can realize the simultaneous wall breaking of Haematococcus pluvialis and the extraction of astaxanthin, carbohydrates and proteins, and no organic solvent is used, the operation is simple, the extraction time is short, and the extraction efficiency is high. The invention solves the technical problem that the extractant in the prior art cannot effectively separate the multiple active substances of Haematococcus pluvialis. DETAILED DESCRIPTION

[0046] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0047] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0048] On one hand, the present invention provides an extractant for synchronously separating active substances of Haematococcus pluvialis, comprising an extraction phase with a mass ratio of 20% to 50%, and the remainder being water;

[0049] The extraction phase is a quaternary phosphine ionic liquid or a deep eutectic solvent;

[0050] The quaternary phosphine ionic liquid includes tetrabutylphosphine trifluoroacetate or tributyloctylphosphine bromide; the low eutectic solvent includes the quaternary phosphine ionic liquid and fatty acid in a molar ratio of 1:1 to 5:3.

[0051] Quaternary phosphine ionic liquids are used to replace traditional imidazole ionic liquids as the extraction phase, or a new low eutectic solvent is synthesized with fatty acids as the extraction phase; the low eutectic solvent is weakly acidic and has the ability to form van der Waals forces and hydrogen bond interactions, and has both the wall-breaking effect of Haematococcus pluvialis and high solubility. The extractant has low viscosity at room temperature, strong biocompatibility, and high safety; it can achieve the simultaneous wall-breaking of Haematococcus pluvialis and the extraction of astaxanthin, carbohydrates, and proteins, and no organic solvent is used, the operation is simple, the extraction time is short, and the extraction efficiency is high. The technical problem that the extractant in the prior art cannot effectively separate the various active substances of Haematococcus pluvialis is solved.

[0052] The mass concentration of the low eutectic solvent may be, but is not limited to, 20%, 23%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, 43%, 45%, 48% or 50%, or any value between 20% and 50%.

[0053] The molar ratio of the quaternary phosphine ionic liquid to the fatty acid may be, but is not limited to, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1 or 5:3, and may be any ratio between 1:1 and 5:3.

[0054] In some specific embodiments, the ionic liquid includes tetrabutylphosphine trifluoroacetate or tributyloctylphosphine bromide.

[0055] In some specific embodiments, the fatty acid includes any one of lauric acid (LU), capric acid (DA) or D,L-lactic acid (LA).

[0056] When the deep eutectic solvent reaches equilibrium, the fatty acid and the quaternary phosphine ionic liquid form [P 4448 ]Br / LU, [P 4448 ]Br / DA, [P 4448 ]Br / LA, [P 4444 ]CF3COO / LU,[P 4444 ]CF3COO / DA or [P 4444 ]CF3COO / LA.

[0057] According to another aspect of the present invention, a method for preparing the above-mentioned extractant is also provided. When the extraction phase is a quaternary phosphine ionic liquid, the quaternary phosphine ionic liquid is mixed with water according to the formula amount to obtain the extractant;

[0058] When the extraction phase is a low eutectic solvent, the quaternary phosphine ionic liquid and fatty acid are stirred and heated at 50-80° C. for at least 2 hours according to the formula amount to obtain a low eutectic solvent, and the low eutectic solvent is mixed with water to obtain an extractant.

[0059] In some specific embodiments, the stirring speed is 100-500 rpm.

[0060] According to another aspect of the present invention, there is also provided a method for synchronously separating active substances from Haematococcus pluvialis, comprising the following steps:

[0061] A. adding Haematococcus pluvialis to the above-mentioned extractant or the extractant prepared by the above-mentioned preparation method, extracting, and centrifuging to obtain a homogeneous extract of active substances of Haematococcus pluvialis;

[0062] B. Phase separation is performed on the homogeneous extract of active substances from Haematococcus pluvialis to obtain an extraction phase containing astaxanthin, an interface layer containing protein and a water-enriched phase containing carbohydrates, and the phases are purified to obtain astaxanthin, protein and carbohydrates respectively.

[0063] The above-mentioned extractant can produce the wall-breaking effect of Haematococcus pluvialis and high solubility performance, which can improve the extraction efficiency; at the same time, both the ionic liquid aqueous solution and the low eutectic solvent aqueous solution have the phase change characteristics of low critical eutectic temperature, and can form liquid-liquid phase separation through phase separation. The highly hydrophobic astaxanthin mainly migrates to the extraction phase, and the carbohydrates mainly migrate to the water-rich phase. The liquid-liquid interface has the function of adsorbing proteins.

[0064] When the extraction phase is a quaternary phosphine ionic liquid, an ionic liquid-enriched phase containing astaxanthin is formed; when the extraction phase is a low eutectic solvent, a low eutectic solvent-enriched phase containing astaxanthin is formed.

[0065] In some specific embodiments, the mass volume ratio of the Haematococcus pluvialis to the extractant is 1:20 to 1:50.

[0066] The mass volume ratio of the Haematococcus pluvialis to the extractant may be, but is not limited to, 1:20, 1:23, 1:25, 1:28, 1:30, 1:33, 1:35, 1:38, 1:40, 1:43, 1:45, 1:48 or 1:50, or any value between 1:20 and 1:50.

[0067] In some specific embodiments, the extraction includes ultrasonic extraction or stirring extraction. In order to improve the extraction efficiency, in some specific embodiments, the extraction temperature is 30 to 50° C. In some specific embodiments, the extraction time is 30 to 60 min. In some specific embodiments, the ultrasonic power of the ultrasonic extraction is 100 to 200 W. In some specific embodiments, the stirring rate is 400 to 600 rpm.

[0068] In order to separate the active substances of Haematococcus pluvialis, in some specific embodiments, the rotation speed of the centrifugal separation is 8000-10000 rpm; in some specific embodiments, the time of the centrifugal separation is 10-15 min; in some specific embodiments, the temperature of the centrifugal separation is 20-30°C.

[0069] Increasing the temperature is conducive to phase separation. In some specific embodiments, the phase separation includes standing and heating. Temperature regulation can promote the phase equilibrium transformation of the solvent system and promote the distribution of active substances in Haematococcus pluvialis. In some specific embodiments, the standing time is 10 to 30 minutes. In some specific embodiments, heating includes raising the temperature to 30 to 50°C.

[0070] In some specific embodiments, the purification includes purification of astaxanthin by adsorption, purification of carbohydrates by molecular separation and purification of proteins by salting out.

[0071] In some specific embodiments, the adsorbent for purifying astaxanthin by adsorption method includes XAD-8 macroporous adsorption resin, and the eluent for purifying astaxanthin by adsorption method includes ethanol-water solution and ethanol-ethyl acetate solution; the volume ratio of the ethanol-water solution is 50-65:35-50; the volume ratio of the ethanol-ethyl acetate solution is 80-95:5-20.

[0072] In some specific embodiments, the separation agent for molecular separation and purification of carbohydrates includes a Sepharose CL-6B gel column; the eluent for molecular separation and purification of carbohydrates includes distilled water; and the flow rate of the eluent for molecular separation and purification of carbohydrates is 0.5 to 1 mL / min.

[0073] In some specific embodiments, the salting-out reagent for purifying protein by the salting-out method comprises an ammonium sulfate solution with a concentration of 20% to 70%; the salting-out method for purifying protein comprises static centrifugation; the static standing comprises static standing at 4°C in the dark for 8 to 10 hours; the centrifugation comprises centrifugation at 8000 to 10000 r / min for 20 to 30 minutes.

[0074] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0075] The Haematococcus pluvialis in the following examples and comparative examples is Haematococcus pluvialis powder from Yunnan Baoshan Zeyuan Algae Industry Co., Ltd.

[0076] Example 1

[0077] The invention discloses an extractant for synchronously separating active substances of Haematococcus pluvialis, comprising tetrabutylphosphine trifluoroacetate with a mass ratio of 50%, and the solvent is deionized water.

[0078] The preparation is carried out according to the following method: tetrabutylphosphine trifluoroacetate and deionized water are mixed with water to obtain an extractant.

[0079] Example 2

[0080] An extractant for synchronously separating active substances from Haematococcus pluvialis comprises a low eutectic solvent and deionized water in a mass ratio of 1:2, wherein the low eutectic solvent comprises tributyloctylphosphonium bromide and D,L-lactic acid in a molar ratio of 4:3.

[0081] The preparation is carried out according to the following method: tributyloctylphosphonium bromide and D,L-lactic acid are placed on a magnetic stirrer (rotation speed is 300 rpm) at 65° C. and heated for at least 2 h until the solution reaches a state of equilibrium, and then mixed with water to obtain an extractant.

[0082] Example 3

[0083] An extractant for synchronously separating active substances from Haematococcus pluvialis comprises a low eutectic solvent and deionized water in a mass ratio of 3:7, wherein the low eutectic solvent comprises tetrabutylphosphine trifluoroacetate and D,L-lactic acid in a molar ratio of 5:3.

[0084] The preparation is carried out according to the following method: tetrabutylphosphine trifluoroacetate and D,L-lactic acid are placed on a magnetic stirrer (rotation speed is 300 rpm) at 65° C. and heated for at least 2 h until the solution reaches a state of equilibrium, and then mixed with water to obtain an extractant.

[0085] Example 4

[0086] An extractant for synchronously separating active substances from Haematococcus pluvialis comprises a low eutectic solvent and deionized water in a mass ratio of 1:4, wherein the low eutectic solvent comprises tributyloctylphosphonium bromide and lauric acid in a molar ratio of 1:1.

[0087] The preparation is carried out according to the following method: tributyloctylphosphonium bromide and lauric acid are placed on a magnetic stirrer (rotation speed is 300 rpm) and heated at 65° C. for at least 2 h until the solution reaches a state of equilibrium, and then mixed with water to obtain an extractant.

[0088] Example 5

[0089] An extractant for synchronously separating active substances from Haematococcus pluvialis comprises a low eutectic solvent and deionized water in a mass ratio of 2:3, wherein the low eutectic solvent comprises tributyloctylphosphonium bromide and decanoic acid in a molar ratio of 1:1.

[0090] The preparation is carried out according to the following method: tributyloctylphosphonium bromide and decanoic acid are placed on a magnetic stirrer (rotation speed is 300 rpm) at 65° C. and heated for at least 2 h until the solution reaches a state of equilibrium, and then mixed with water to obtain an extractant.

[0091] Example 6

[0092] An extractant for synchronously separating active substances from Haematococcus pluvialis comprises a low eutectic solvent and deionized water in a mass ratio of 1:1, wherein the low eutectic solvent comprises tetrabutylphosphine trifluoroacetate and lauric acid in a molar ratio of 1:1.

[0093] The preparation is carried out according to the following method: tetrabutylphosphine trifluoroacetate and lauric acid are heated at 65° C. on a magnetic stirrer (rotation speed is 300 rpm) for at least 2 h until the solution reaches a state of equilibrium, and then mixed with water to obtain an extractant.

[0094] Comparative Example 1

[0095] The extractant comprises tributyloctylphosphine chloride, glycerol-L-proline eutectic solvent and deionized water in a mass ratio of 7:5:8. In the raw material of the glycerol-L-proline eutectic solvent, the molar ratio of glycerol to L-proline is 1:3.

[0096] Embodiments 7 to 12

[0097] A method for synchronously separating active substances from Haematococcus pluvialis is provided, wherein the extractants provided in Examples 1 to 6 are respectively selected and operated according to the following steps:

[0098] (1) adding Haematococcus pluvialis powder to an extractant at a solid-liquid ratio of 1:20 g / ml, performing ultrasonic extraction at 200 W for 30 min at 50° C., and centrifuging at 8000 rpm for 15 min to obtain a homogeneous extract of active substances of Haematococcus pluvialis;

[0099] (2) heating to 50° C., standing the homogeneous extract of active substances from Haematococcus pluvialis for 30 minutes, and performing phase separation to obtain an extract phase containing astaxanthin, an interface layer containing protein, and a water-enriched phase containing carbohydrates;

[0100] (3) Astaxanthin purification: XAD-8 macroporous adsorption resin; eluent: ethanol-water solution (volume ratio 60:40), ethanol-ethyl acetate solution (volume ratio 95:5). Obtain astaxanthin extract. Carbohydrate purification: Sepharose CL-6B gel column; eluent: distilled water; flow rate: 0.5 mL / min. Obtain carbohydrate extract. Protein purification: ammonium sulfate solution (concentration of 20% to 70%); stand at 4°C in the dark for 8 hours; centrifuge at 9000 r / min for 20 minutes. Obtain protein extract.

[0101] Example 13

[0102] The difference from Example 8 is that (1) the Haematococcus pluvialis powder is added to the extractant at a solid-liquid ratio of 1:20 g / ml, ultrasonic extraction is performed at 200 W for 30 min at 50° C., and centrifugation is performed at 10,000 rpm for 10 min to obtain a homogeneous extract of active substances of Haematococcus pluvialis.

[0103] Embodiment 14

[0104] The difference from Example 9 is that (1) the Haematococcus pluvialis powder is added to the extractant at a solid-liquid ratio of 1:30 g / ml, ultrasonic extraction is performed at 200 W for 40 min at 40° C., and centrifugation is performed at 10,000 rpm for 10 min to obtain a homogeneous extract of active substances of Haematococcus pluvialis.

[0105] Embodiment 15

[0106] The difference from Example 10 is that (1) the Haematococcus pluvialis powder is added to the extractant at a solid-liquid ratio of 1:50 g / ml, ultrasonic extraction is performed at 200 W for 50 min at 30° C., and centrifugation is performed at 10,000 rpm for 10 min to obtain a homogeneous extract of active substances of Haematococcus pluvialis.

[0107] Example 16

[0108] The difference from Example 11 is that (1) the Haematococcus pluvialis powder is added to the extractant at a solid-liquid ratio of 1:40 g / ml, and ultrasonic extraction is performed at 100 W for 50 min at 50° C. and centrifuged at 9000 rpm for 10 min to obtain a homogeneous extract of active substances of Haematococcus pluvialis.

[0109] Embodiment 17

[0110] The difference from Example 12 is that (1) the Haematococcus pluvialis powder is added to the extractant at a solid-liquid ratio of 1:30 g / ml, and ultrasonic extraction is performed at 100 W for 40 min at 40° C. and centrifuged at 8000 rpm for 15 min to obtain a homogeneous extract of active substances of Haematococcus pluvialis.

[0111] Embodiment 18

[0112] The difference from Example 12 is that (1) the Haematococcus pluvialis powder is added to the extractant at a solid-liquid ratio of 1:20 g / ml, and the extraction is carried out at 50° C. with magnetic stirring at 500 rpm for 30 min, and centrifuged at 10000 rpm for 10 min to obtain a homogeneous extract of active substances of Haematococcus pluvialis;

[0113] Comparative Example 2

[0114] The difference from Example 7 is that the extractant provided in Comparative Example 1 is selected, and the rest is the same as Example 7.

[0115] Comparative Example 3

[0116] Acetone is selected as the extractant. The specific method includes: mechanically treating the Haematococcus pluvialis powder with a ball mill for 5 minutes, adding the Haematococcus pluvialis powder to the extractant according to a solid-liquid ratio of 1:20g / ml, extracting at 100bar and 40℃ for 20 minutes to obtain astaxanthin extract.

[0117] Comparative Example 4

[0118] Ethanol is selected as the extractant. The specific method includes: adding deionized water to the Haematococcus pluvialis powder according to a solid-liquid ratio of 1:25 g / ml; adjusting the pH value to 10.0; extracting at 90°C for 3 hours; centrifuging at 7000 r / min for 20 minutes; adding anhydrous ethanol (ethanol volume concentration is 40%) to the concentrated supernatant; precipitating at 4°C overnight; centrifuging at 7000 r / min for 20 minutes; removing proteins by Sevag method; freeze drying to obtain a carbohydrate extract.

[0119] Comparative Example 5

[0120] Sodium hydroxide was selected as the extractant. The specific method includes: resuspending the Haematococcus pluvialis powder in 0.05M phosphate buffer solution at a solid-liquid ratio of 1:15g / mL, adjusting the pH to 11.0 with 0.5M NaOH solution at 25°C, and continuously stirring at 200rpm for 30min. Then, centrifuge the mixture at 6000g for 15 minutes. Take the protein-containing supernatant, adjust the pH to 5.0 with 0.5MHCl, precipitate the protein, and centrifuge at 7000g for 15min. The protein was recovered. After dialysis at 4°C for 24h, freeze-drying was performed to obtain protein powder.

[0121] Comparative Example 6

[0122] Dimethylammonium dimethylcarbamate (DIMCARB) was selected as the extractant. The specific method includes: adding Haematococcus pluvialis powder to the extractant at a solid-liquid ratio of 1:20 g / mL, and extracting at 45°C for 75 minutes to obtain astaxanthin extract.

[0123] Comparative Example 7

[0124] Thymol and oleic acid were mixed in a mass ratio of 3:1 as an extractant. The specific method includes: adding Haematococcus pluvialis powder to the extractant in a solid-liquid ratio of 1:20 g / mL, extracting for 6 hours at 100 rpm magnetic stirring, and centrifuging at 2550×g for 10 minutes to obtain astaxanthin extract.

[0125] The extraction amount and distribution coefficient of each extract of Examples 7 to 18 and Comparative Examples 2 to 7 are shown in Tables 1 to 3.

[0126] Table 1

[0127]

[0128]

[0129] Table 2

[0130]

[0131] Table 3

[0132]

[0133] As shown in Tables 1 to 3, Examples 7 to 18 can achieve synchronous separation of astaxanthin, carbohydrates and proteins. Compared with Example 7, Comparative Example 2 uses other ionic liquid deep eutectic solvent water systems and cannot achieve synchronous separation; Comparative Examples 3 to 7 are commonly used extractants in the prior art and cannot achieve synchronous extraction.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An extractant for synchronously separating active substances from Haematococcus pluvialis, characterized in that: The invention comprises an extraction phase having a mass ratio of 20% to 50%, and the balance being water; The extraction phase is a quaternary phosphine ionic liquid or a deep eutectic solvent; The quaternary phosphine ionic liquid includes tetrabutylphosphine trifluoroacetate or tributyloctylphosphine bromide; The low eutectic solvent comprises a quaternary phosphine ionic liquid and a fatty acid in a molar ratio of 1:1 to 5:

3.

2. The extractant according to claim 1, characterized in that The fatty acid includes any one of lauric acid, capric acid or D,L-lactic acid.

3. The method for preparing the extractant according to claim 1 or 2, characterized in that: When the extraction phase is a quaternary phosphine ionic liquid, the quaternary phosphine ionic liquid is mixed with water according to the formula amount to obtain an extractant; When the extraction phase is a low eutectic solvent, the quaternary phosphine ionic liquid and the fatty acid are stirred and heated at 50-80° C. for at least 2 hours according to the formula amount to obtain a low eutectic solvent, and the low eutectic solvent is mixed with water to obtain an extractant.

4. The preparation method according to claim 3, characterized in that: The stirring speed is 100-500 rpm.

5. A method for synchronously separating active substances from Haematococcus pluvialis, characterized in that: The following steps are involved: A. adding Haematococcus pluvialis to the extractant according to claim 1 or 2 or the extractant prepared by the preparation method according to claim 3 or 4, extracting, and centrifuging to obtain a homogeneous extract of active substances of Haematococcus pluvialis; B. Phase separation is performed on the homogeneous extract of active substances from Haematococcus pluvialis to obtain an extraction phase containing astaxanthin, an interface layer containing protein and a water-enriched phase containing carbohydrates, and the phases are purified to obtain astaxanthin, protein and carbohydrates respectively.

6. The separation method according to claim 5, characterized in that The mass volume ratio of the Haematococcus pluvialis to the extractant is 1:20 to 1:

50.

7. The separation method according to claim 5, characterized in that The extraction includes ultrasonic extraction or stirring extraction; The extraction temperature is 30-50°C; Preferably, the extraction time is 30 to 60 minutes; Preferably, the power of the ultrasound is 100-200W; Preferably, the stirring rate is 400-600 rpm.

8. The separation method according to claim 5, characterized in that The rotation speed of the centrifugal separation is 8000-10000 rpm; Preferably, the centrifugal separation time is 10 to 15 minutes; Preferably, the centrifugal separation temperature is 20-30°C.

9. The separation method according to claim 5, characterized in that The phase separation includes standing and heating; Preferably, the standing time is 10 to 30 minutes; Preferably, raising the temperature includes raising the temperature to 30-50°C.

10. The separation method according to claim 5, characterized in that The purification includes purification of astaxanthin by adsorption, purification of carbohydrates by molecular separation and purification of proteins by salting out; Preferably, the adsorbent for purifying astaxanthin by adsorption method comprises XAD-8 macroporous adsorption resin; Preferably, the eluent for purifying astaxanthin by adsorption method comprises ethanol-water solution and ethanol-ethyl acetate solution; Preferably, the volume ratio of the ethanol-water solution is 50-65:35-50; Preferably, the volume ratio of the ethanol-ethyl acetate solution is 80-95:5-20; Preferably, the separating agent for molecular separation and purification of carbohydrates comprises a Sepharose CL-6B gel column; Preferably, the eluent for molecular separation and purification of carbohydrates comprises distilled water; Preferably, the flow rate of the eluent for molecular separation and purification of carbohydrates is 0.5 to 1 mL / min; Preferably, the salting-out reagent for purifying protein by the salting-out method comprises an ammonium sulfate solution with a concentration of 20% to 70%; Preferably, the salting-out method for purifying protein comprises static centrifugation; Preferably, the standing includes standing at 4°C in the dark for 8 to 10 hours; Preferably, the centrifugation comprises centrifugation at 8000-10000 r / min for 20-30 min.

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