A method for synchronously separating active substances of haematococcus pluvialis

Abstract

The application provides a method for synchronously separating active substances of Haematococcus pluvialis, and relates to the technical field of natural product extraction, and comprises an extraction phase with a mass ratio of 20%-50%, and the balance is water; the extraction phase is a quaternary phosphonium ionic liquid or a eutectic solvent; the quaternary phosphonium ionic liquid comprises tetrabutylphosphonium trifluoroacetate or tributyl octyl phosphonium bromide; and the eutectic solvent comprises a quaternary phosphonium ionic liquid and a fatty acid with a molar ratio of 1:1-5:3. The extraction phase has weak acidity, the ability to form van der Waals forces and hydrogen bond interactions, the Haematococcus pluvialis cell wall breaking effect and high solubility. The extraction phase can form liquid-liquid phase separation, can realize the synchronous extraction of Haematococcus pluvialis cell wall breaking, astaxanthin, carbohydrates and proteins, and has no organic solvent, is simple to operate, has short extraction time and high extraction efficiency. The method solves the technical problem that the extraction agent in the prior art cannot effectively separate multiple active substances of Haematococcus pluvialis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product extraction, and in particular to a separation method for simultaneously separating active substances of Haematococcus pluvialis. BACKGROUND

[0002] Astaxanthin, as one of the main components of Haematococcus pluvialis, has biological activities such as antioxidant, immune enhancement, anti-aging, and anti-aging. The carbohydrate content in Haematococcus pluvialis accounts for about 30% to 40% of the dry weight of the cell, and the polysaccharide content is as high as 6% to 10% of the dry weight, which has biological activities such as antioxidant, anti-aging, anti-inflammatory, anti-tumor, blood lipid-lowering, and immune enhancement. At the same time, the protein content in Haematococcus pluvialis is 16% to 23%, the amino acid content is high, and the composition is reasonable, which is a good source of protein and has antioxidant and acute alcohol-induced liver injury improvement effects.

[0003] In the prior art, the separation of active substances in Haematococcus pluvialis is mostly the separation of single substances. For example, a method for separating different configurations of astaxanthin in Haematococcus pluvialis extract by supercritical fluid chromatography is designed in Chinese Patent No. 202111570709.1. A method for extracting astaxanthin in Haematococcus pluvialis by low eutectic solvent assisted by cellulase and pectinase is designed in Chinese Patent No. 202311552352.3. A method for extracting crude polysaccharide of intracellular active substances in Haematococcus pluvialis by breaking the cell wall with choline amino acid ionic liquid is designed in Chinese Patent No. 202310873866.2 CN117700572A. The above methods only separate single substances in Haematococcus pluvialis, and cannot effectively separate multiple active substances in Haematococcus pluvialis, which has great limitations.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] One of the purposes of the present application 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 purpose of the present application is to provide a preparation method of the extractant for simultaneously separating active substances of Haematococcus pluvialis.

[0007] The third purpose of the present application is to provide a separation method for simultaneously separating active substances of Haematococcus pluvialis.

[0008] In order to achieve the above purposes of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides an extractant for simultaneously separating active substances of Haematococcus pluvialis, which comprises an extractant phase with a mass ratio of 20% to 50%, and the balance is water.

[0010] the extraction phase is a quaternary phosphonium ionic liquid or a deep eutectic solvent;

[0011] the quaternary phosphonium ionic liquid comprises tetrabutylphosphonium trifluoroacetate or tributyl octyl phosphonium bromide;

[0012] the deep eutectic solvent comprises a quaternary phosphonium ionic liquid and a fatty acid in a molar ratio of 1:1 to 5:3.

[0013] Further, the ionic liquid comprises tetrabutylphosphonium trifluoroacetate or tributyl octyl phosphonium bromide;

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

[0015] In a second aspect, the present application provides a preparation method of the above-mentioned extractant, when the extraction phase is a quaternary phosphonium ionic liquid, mixing the quaternary phosphonium ionic liquid with water according to the formula amount to obtain the extractant;

[0016] When the extraction phase is a deep eutectic solvent, stirring and heating the quaternary phosphonium ionic liquid and the fatty acid at 50-80°C for at least 2h to obtain the deep eutectic solvent, and mixing the deep eutectic solvent with water to obtain the extractant.

[0017] Further, the stirring speed is 100-500rpm.

[0018] In a third aspect, the present application provides a separation method for simultaneously separating Haematococcus pluvialis active substances, 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 Haematococcus pluvialis active substance homogeneous extract;

[0020] B. phase separating the Haematococcus pluvialis active substance homogeneous extract to obtain an extraction phase containing astaxanthin, an interface layer containing protein, and a water-rich phase containing carbohydrates, purifying, and obtaining astaxanthin, protein and carbohydrates, respectively.

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

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

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

[0024] Preferably, the extraction time is 30-60min;

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

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

[0027] Further, the centrifugal separation speed is 8000-10000 rpm.

[0028] Preferably, the centrifugal separation time is 10-15 min.

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

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

[0031] Preferably, the standing time is 10-30 min.

[0032] Preferably, the heating includes heating to 30-50℃.

[0033] Further, the purification includes adsorption method for purifying astaxanthin, molecular separation for purifying carbohydrates, and salting-out method for purifying proteins.

[0034] Preferably, the adsorbent for adsorption method for purifying astaxanthin includes XAD-8 macroporous adsorption resin.

[0035] Preferably, the eluent for adsorption method for purifying astaxanthin includes 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 separation agent for molecular separation for purifying carbohydrates includes Sepharose CL-6B gel column.

[0039] Preferably, the eluent for molecular separation for purifying carbohydrates includes distilled water.

[0040] Preferably, the flow rate of the eluent for molecular separation for purifying carbohydrates is 0.5-1 mL / min.

[0041] Preferably, the salting-out reagent for salting-out method for purifying proteins includes ammonium sulfate solution with a concentration of 20%-70%.

[0042] Preferably, the salting-out method for purifying proteins includes standing and centrifugation.

[0043] Preferably, the standing includes standing in the dark at 4℃ for 8-10 h.

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

[0045] The application provides an extractant for synchronously separating active substances of Haematococcus pluvialis, which comprises a quaternary phosphonium ionic liquid or a low eutectic solvent. The low eutectic solvent is weakly acidic and has the ability to form van der Waals forces and hydrogen bond interactions, and has the effects of Haematococcus pluvialis cell wall breaking and high solubility. The ionic liquid aqueous solution and the low eutectic solvent aqueous solution both have the phase transition characteristics of low critical solution temperature, that is, the phase separation is facilitated by increasing the temperature, liquid-liquid phase separation can be formed, temperature regulation can promote the phase equilibrium of the solvent system, and the active substances of Haematococcus pluvialis are distributed, the hydrophobic astaxanthin mainly migrates to the extraction phase, the carbohydrates mainly migrate to the water-rich phase, and the liquid-liquid interface has the function of adsorbing proteins. The extractant has low viscosity, high biocompatibility and high safety at room temperature, can realize the synchronous extraction of Haematococcus pluvialis cell wall breaking, astaxanthin, carbohydrates and proteins, and does not use organic solvents, and has the advantages of simple operation, short extraction time and high extraction efficiency. The technical problem that the extractant in the prior art cannot effectively separate multiple active substances of Haematococcus pluvialis is solved. DETAILED DESCRIPTION

[0046] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by a person of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including", as well as other forms such as "include", "includes" and "included", is not limiting.

[0047] Unless otherwise indicated, the methods and techniques of the present application 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.

[0048] In one aspect, the application provides an extractant for synchronously separating active substances of Haematococcus pluvialis, comprising an extraction phase in an amount of 20%-50% by mass, and the balance being water.

[0049] The extraction phase is a quaternary phosphonium ionic liquid or a low eutectic solvent.

[0050] The quaternary phosphonium ionic liquid comprises tetrabutylphosphonium trifluoroacetate or tributyl octyl phosphonium bromide; and the low eutectic solvent comprises a quaternary phosphonium ionic liquid and a fatty acid in a molar ratio of 1:1-5:3.

[0051] The traditional imidazole-based ionic liquid is replaced by a quaternary phosphonium-based ionic liquid as an extraction phase, or a new eutectic solvent is synthesized by combining a fatty acid as an extraction phase; the eutectic solvent is weakly acidic and has the ability to form van der Waals forces and hydrogen bond interactions, and has the breaking wall effect of Haematococcus pluvialis and high solubility. The extraction agent has low viscosity at room temperature, strong biocompatibility, and high safety; the breaking wall of Haematococcus pluvialis, the extraction of astaxanthin, carbohydrates and proteins can be carried out simultaneously, 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 extraction agent in the prior art cannot effectively separate the multiple active substances of Haematococcus pluvialis is solved.

[0052] The mass concentration of the eutectic solvent can 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 phosphonium-based ionic liquid and the fatty acid can 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, or any ratio between 1:1 and 5:3.

[0054] In some specific embodiments, the ionic liquid includes tetrabutylphosphonium trifluoroacetate or tributyl octyl phosphonium bromide.

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

[0056] When the eutectic solvent reaches an equilibrium state, the fatty acid and the quaternary phosphonium-based 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 application, a preparation method of the above-mentioned extraction agent is also provided. When the extraction phase is a quaternary phosphonium-based ionic liquid, the quaternary phosphonium-based ionic liquid is mixed with water according to the formula amount to obtain the extraction agent.

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

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

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

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

[0062] B. phase separating the Haematococcus pluvialis active substance homogeneous extract to obtain an extract phase containing astaxanthin, an interface layer containing protein, and a water-rich phase containing carbohydrates, purifying, and obtaining astaxanthin, protein, and carbohydrates, respectively.

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

[0064] When the extract phase is a quaternary phosphonium ionic liquid, an ionic liquid-rich phase containing astaxanthin is formed, and when the extract phase is a eutectic solvent, a eutectic solvent-rich phase containing astaxanthin is formed.

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

[0066] In some embodiments, the mass-volume ratio of the Haematococcus pluvialis to the extractant can 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, and can also be any value between 1:20 and 1:50.

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

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

[0069] The phase separation is facilitated by increasing the temperature, and in some specific embodiments, the phase separation comprises standing and heating. Temperature regulation can promote the phase equilibrium transition of the solvent system and facilitate the distribution of the active substance of Haematococcus pluvialis. In some specific embodiments, the standing time is 10-30 min. In some specific embodiments, the heating comprises increasing the temperature to 30-50℃.

[0070] In some specific embodiments, the purification comprises adsorption method for purifying astaxanthin, molecular separation for purifying carbohydrates, and salting-out method for purifying proteins.

[0071] In some specific embodiments, the adsorbent for adsorption method for purifying astaxanthin comprises XAD-8 macroporous adsorption resin, and the eluent for adsorption method for purifying astaxanthin comprises 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 for purifying carbohydrates comprises Sepharose CL-6B gel column; the eluent for molecular separation for purifying carbohydrates comprises distilled water; and the flow rate of the eluent for molecular separation for purifying carbohydrates is 0.5-1 mL / min.

[0073] In some specific embodiments, the salting-out reagent for salting-out method for purifying proteins comprises ammonium sulfate solution with a concentration of 20%-70%; the salting-out method for purifying proteins comprises standing and centrifugation; the standing comprises standing in the dark at 4℃ for 8-10 h; and the centrifugation comprises centrifugation at 8000-10000 r / min for 20-30 min.

[0074] The technical solutions of the present application will be described clearly and completely in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

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

[0076] Example 1

[0077] An extractant for simultaneous separation of Haematococcus pluvialis active substances, comprising 50% of tetrabutylphosphonium trifluoroacetate by mass, and deionized water as a solvent.

[0078] Prepared by mixing tetrabutylphosphonium trifluoroacetate and deionized water to obtain the extractant.

[0079] Example 2

[0080] An extractant for simultaneous separation of Haematococcus pluvialis active substances, comprising a eutectic solvent and deionized water in a mass ratio of 1:2, wherein the eutectic solvent comprises tributyl octyl phosphonium bromide and D, L-lactic acid in a molar ratio of 4:3.

[0081] Prepared by mixing tributyl octyl phosphonium bromide and D, L-lactic acid at 65°C on a magnetic stirrer (300 rpm) for at least 2 h until the solution reaches an equilibrium state, and mixing with water to obtain the extractant.

[0082] Example 3

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

[0084] Prepared by mixing tetrabutylphosphonium trifluoroacetate and D, L-lactic acid at 65°C on a magnetic stirrer (300 rpm) for at least 2 h until the solution reaches an equilibrium state, and mixing with water to obtain the extractant.

[0085] Example 4

[0086] An extractant for simultaneous separation of Haematococcus pluvialis active substances, comprising a eutectic solvent and deionized water in a mass ratio of 1:4, wherein the eutectic solvent comprises tributyl octyl phosphonium bromide and lauric acid in a molar ratio of 1:1.

[0087] Prepared by mixing tributyl octyl phosphonium bromide and lauric acid at 65°C on a magnetic stirrer (300 rpm) for at least 2 h until the solution reaches an equilibrium state, and mixing with water to obtain the extractant.

[0088] Example 5

[0089] An extractant for simultaneous separation of Haematococcus pluvialis active substances, comprising a eutectic solvent and deionized water in a mass ratio of 2:3, wherein the eutectic solvent comprises tributyl octyl phosphonium bromide and decanoic acid in a molar ratio of 1:1.

[0090] Preparation is carried out according to the following method: tributyl octyl phosphonium bromide and decanoic acid are heated at 65℃ on a magnetic stirrer (300 rpm) for at least 2h until the solution reaches equilibrium, mixed with water to obtain the extractant.

[0091] Example 6

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

[0093] Preparation is carried out according to the following method: tetrabutylphosphonium trifluoroacetate and lauric acid are heated at 65℃ on a magnetic stirrer (300 rpm) for at least 2h until the solution reaches equilibrium, mixed with water to obtain the extractant.

[0094] Comparative Example 1

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

[0096] Examples 7-12

[0097] A separation method for synchronously separating Haematococcus pluvialis active substances, using the extractants provided in Examples 1-6 respectively, is operated according to the following steps:

[0098] (1) Haematococcus pluvialis powder is added to the extractant at a solid-liquid ratio of 1:20 g / ml, 50℃, 200W ultrasonic extraction for 30min, 8000rpm centrifugal separation for 15min, to obtain Haematococcus pluvialis active substance homogeneous phase extract;

[0099] (2) The temperature is raised to 50℃, and the Haematococcus pluvialis active substance homogeneous phase extract is allowed to stand for 30min, and phase separation is performed, to obtain an extract phase containing astaxanthin, an interface layer containing protein, and a water-rich phase containing carbohydrates;

[0100] ​(3) Purification of astaxanthin: XAD-8 macroporous adsorption resin; eluent: ethanol-water solution (volume ratio 60:40), ethanol-ethyl acetate solution (volume ratio 95:5). Astaxanthin extract was obtained. Carbohydrate purification: Sepharose CL-6B gel column; eluent: distilled water; flow rate: 0.5 mL / min. Carbohydrate extract was obtained. Protein purification: ammonium sulfate solution (concentration of 20% to 70%); 4 ℃, dark, standing for 8 h; 9000 r / min centrifugal separation for 20 min. Protein extract was obtained.

[0101] Example 13

[0102] Different from Example 8, (1) Haematococcus pluvialis powder was added to the extraction agent at a solid-liquid ratio of 1:20 g / ml, 50 ℃, 200W ultrasonic extraction for 30 min, 10000 rpm centrifugal separation for 10 min, and Haematococcus pluvialis active substance homogeneous extract was obtained.

[0103] Example 14

[0104] Different from Example 9, (1) Haematococcus pluvialis powder was added to the extraction agent at a solid-liquid ratio of 1:30 g / ml, 40 ℃, 200W ultrasonic extraction for 40 min, 10000 rpm centrifugal separation for 10 min, and Haematococcus pluvialis active substance homogeneous extract was obtained.

[0105] Example 15

[0106] Different from Example 10, (1) Haematococcus pluvialis powder was added to the extraction agent at a solid-liquid ratio of 1:50 g / ml, 30 ℃, 200W ultrasonic extraction for 50 min, 10000 rpm centrifugal separation for 10 min, and Haematococcus pluvialis active substance homogeneous extract was obtained.

[0107] Example 16

[0108] Different from Example 11, (1) Haematococcus pluvialis powder was added to the extraction agent at a solid-liquid ratio of 1:40 g / ml, 50 ℃, 100W ultrasonic extraction for 50 min, 9000 rpm centrifugal separation for 10 min, and Haematococcus pluvialis active substance homogeneous extract was obtained.

[0109] Example 17

[0110] Different from Example 12, (1) Haematococcus pluvialis powder was added to the extraction agent at a solid-liquid ratio of 1:30 g / ml, 40 ℃, 100W ultrasonic extraction for 40 min, 8000 rpm centrifugal separation for 15 min, and Haematococcus pluvialis active substance homogeneous extract was obtained.

[0111] Example 18

[0112] Different from Example 12, (1) Haematococcus pluvialis powder was added to the extractant at a solid-liquid ratio of 1:20 g / ml, extracted at 50°C with magnetic stirring at 500 rpm for 30 min, and centrifuged at 10,000 rpm for 10 min to obtain a homogeneous extract of Haematococcus pluvialis active substances;

[0113] Comparative Example 2

[0114] Different from Example 7, the extractant provided in Comparative Example 1 was selected, and the rest was the same as in Example 7.

[0115] Comparative Example 3

[0116] Acetone was selected as the extractant. The specific method included: Haematococcus pluvialis powder was mechanically treated with a ball mill for 5 min, and Haematococcus pluvialis powder was added to the extractant at a solid-liquid ratio of 1:20 g / ml, extracted at 100 bar and 40°C for 20 min to obtain astaxanthin extract.

[0117] Comparative Example 4

[0118] Ethanol was selected as the extractant. The specific method included: Haematococcus pluvialis powder was added to deionized water at a solid-liquid ratio of 1:25 g / ml; the pH value was adjusted to 10.0; extraction was carried out at 90°C for 3 h; centrifugation was carried out at 7000 r / min for 20 min; anhydrous ethanol (ethanol volume concentration of 40%) was added to the concentrated supernatant; precipitation was carried out at 4°C overnight; centrifugation was carried out at 7000 r / min for 20 min; protein was removed by the Sevag method; freeze-drying was carried out to obtain a carbohydrate extract.

[0119] Comparative Example 5

[0120] Sodium hydroxide was selected as the extractant. The specific method included: Haematococcus pluvialis powder was resuspended in 0.05 M phosphate buffer solution at a solid-liquid ratio of 1:15 g / mL, the pH was adjusted to 11.0 with 0.5 M NaOH solution at 25°C, and continuous stirring was carried out at a speed of 200 rpm within 30 min. Then, the mixture was centrifuged at 6000g for 15 minutes. The supernatant containing protein was adjusted to pH 5.0 with 0.5 M HCl to precipitate the protein, and centrifuged at 7000g for 15 min. The protein was recovered. After dialysis at 4°C for 24 h, the protein powder was freeze-dried.

[0121] Comparative Example 6

[0122] Dimethylaminoformic acid dimethylammonium (DIMCARB) was selected as the extractant. The specific method included: Haematococcus pluvialis powder was added to the extractant at a solid-liquid ratio of 1:20 g / mL, and extracted at 45°C for 75 min to obtain astaxanthin extract.

[0123] Comparative Example 7

[0124] Thymol and oleic acid were selected as the extractant, and the mass ratio was 3:1. The specific method included: Haematococcus pluvialis powder was added into the extractant according to the solid-liquid ratio of 1:20 g / mL, and was extracted for 6 h under the condition of 100 rpm magnetic stirring; 2550xg centrifugation for 10 min. The astaxanthin extract was obtained.

[0125] The extraction amount and distribution coefficient of each extract of examples 7~18 and comparative examples 2~7 are shown in tables 1~3.

[0126] Table 1

[0127]

[0128] Table 2

[0129]

[0130] Table 3

[0131]

[0132] From tables 1~3, it can be seen that examples 7~18 can realize the simultaneous separation of astaxanthin, carbohydrates and proteins. Compared with example 7, comparative example 2 selected other ionic liquid Deep eutectic solvent The aqueous system cannot realize the simultaneous separation; comparative examples 3~7 are the extractants commonly used in the prior art, and cannot realize the simultaneous extraction.

[0133] Finally, it should be pointed out that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A separation method for synchronously separating a Haematococcus pluvialis active substance, characterized by, The method comprises the following steps: A. adding Haematococcus pluvialis to an extractant, extracting, and centrifuging to obtain a Haematococcus pluvialis active substance homogeneous extract; B. phase separating the Haematococcus pluvialis active substance homogeneous extract to obtain an extract phase containing astaxanthin, an interface layer containing protein, and a water-rich phase containing carbohydrates, purifying, and obtaining astaxanthin, protein, and carbohydrates, respectively; The extractant comprises an extract phase in a mass ratio of 20-50% and water in the rest; The extract phase is a quaternary phosphonium ionic liquid or a deep eutectic solvent; The quaternary phosphonium ionic liquid comprises tetrabutylphosphonium trifluoroacetate or tributyl octyl phosphonium bromide; The deep eutectic solvent comprises a quaternary phosphonium ionic liquid and a fatty acid in a molar ratio of 1:1-5:

3.

2. The separation method of claim 1, wherein, The fatty acid comprises any one of lauric acid, capric acid, or D, L-lactic acid.

3. The separation method of claim 2, wherein, The preparation method of the extractant comprises: When the extract phase is a quaternary phosphonium ionic liquid, the quaternary phosphonium ionic liquid is mixed with water according to the formula amount to obtain the extractant; When the extract phase is a deep eutectic solvent, the quaternary phosphonium ionic liquid and the fatty acid are stirred and heated at 50-80°C for at least 2 hours to obtain the deep eutectic solvent, and the deep eutectic solvent is mixed with water to obtain the extractant.

4. The separation method of claim 3, wherein, In the preparation method of the extractant, the stirring speed is 100-500 rpm.

5. The separation method of claim 1, wherein, The mass-volume ratio of the Haematococcus pluvialis to the extractant is 1:20-1:

50.

6. The separation method of claim 1, wherein, The extraction comprises ultrasonic extraction or stirring extraction; The extraction temperature is 30-50°C; The extraction time is 30-60 minutes.

7. The separation method of claim 6, wherein, The ultrasonic power is 100-200 W.

8. The separation method of claim 6, wherein, The stirring rate is 400-600 rpm.

9. The separation method of claim 1, wherein, The centrifugation speed is 8000-10000 rpm; The centrifugation time is 10-15 minutes; The centrifugation temperature is 20-30°C.

10. The separation method of claim 1, wherein, The phase separation comprises standing and heating; The standing time is 10-30 minutes; The heating comprises increasing the temperature to 30-50°C.

11. The separation method of claim 1, wherein, The purification comprises adsorption method for purifying astaxanthin, molecular separation for purifying carbohydrates, and salting-out method for purifying protein.

12. The separation method of claim 11, wherein, The adsorbent for purifying astaxanthin by the adsorption method comprises XAD-8 macroporous adsorption resin.

13. The separation method of claim 12, wherein, The eluent for purifying astaxanthin by the adsorption method comprises an ethanol-water solution and an ethanol-ethyl acetate solution.

14. The separation method of claim 13, wherein, The volume ratio of the ethanol-water solution is 50-65:35-50.

15. The separation method of claim 13, wherein, The volume ratio of the ethanol-ethyl acetate solution is 80-95:5-20.

16. The separation method of claim 11, wherein, The separation agent for purifying carbohydrates by the molecular separation comprises a Sepharose CL-6B gel column.

17. The separation method of claim 16, wherein, The eluent for purifying carbohydrates by the molecular separation comprises distilled water; The flow rate of the eluent for purifying carbohydrates by the molecular separation is 0.5-1 mL / min.

18. The separation method of claim 11, wherein, The salting-out reagent for purifying protein by the salting-out method comprises an ammonium sulfate solution with a concentration of 20-70%.

19. The separation method of claim 18, wherein, The salting-out method for purifying protein comprises standing and centrifugation; The standing comprises standing in the dark at 4°C for 8-10 hours; The centrifugation comprises centrifugation at 8000-10000 r / min for 20-30 minutes.

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