Separation method of phycocyanin of spirulina platensis through eutectic two-aqueous-phase extraction

Abstract

The embodiment of the invention discloses a separation method for extracting phycocyanin from spirulina platensis through eutectic aqueous two-phase extraction, relates to the technical field of extraction and purification of phycocyanin, and aims at solving the problems that in the prior art, when phycocyanin is extracted through a eutectic aqueous two-phase system, the cost is high, the raw material handling capacity is low, and the salt concentration is high. The separation method comprises the following steps: extracting spirulina platensis powder by adopting a CaCl2 solution, and carrying out freeze thawing wall breaking treatment and centrifugal treatment on the extracted spirulina platensis powder to obtain a crude extract containing phycocyanin; the method comprises the following steps: carrying out ultrafiltration desalting treatment on a crude extract containing phycocyanin, adding a deep eutectic solvent and a sodium potassium tartrate tetrahydrate solution, uniformly mixing to obtain an aqueous two-phase system, adjusting the pH value, uniformly mixing, vortex, standing for phase separation, centrifuging for promoting phase separation, separating an upper phase, and carrying out ultrafiltration treatment on the upper phase to obtain filtrate of the deep eutectic solvent component and a concentrated phycocyanin solution; the concentrated phycocyanin solution is subjected to freeze drying treatment, and phycocyanin powder is obtained.

Description

Technical Field

[0001] The present application relates to the technical field of extraction and purification of phycocyanin, and in particular to a method for separating phycocyanin from Spirulina platensis by eutectic double aqueous phase extraction. Background Art

[0002] Phycocyanin is made from Spirulina cultured in freshwater or seawater, and is produced through water extraction and purification processes. It is also known as C-phycocyanin. As a high-value natural product for biotechnology applications, phycocyanin has the potential for antioxidant, anti-cancer, antibacterial and anti-inflammatory effects, and has attracted much attention in health products, pharmaceuticals, food, feed and cosmetics. The economic value of phycocyanin varies depending on its purity, ranging from US$130 to US$15,000 per gram. The extraction and purification of phycocyanin is mainly divided into two steps. The first step is to break the cell wall of Spirulina to allow the intracellular protein phycocyanin to flow out. The second step is to remove impurities from the crude phycocyanin solution through purification methods. Among them, the methods for breaking the cell wall of Spirulina mainly include physical methods such as ultrasound, freeze-thaw, high-pressure homogenization, bead milling, ball milling, pulsed electric field, etc., chemical methods such as CaCl2, etc., and biological methods such as enzymatic hydrolysis, etc.; the current methods for purifying phycocyanin mainly include precipitation methods such as ammonium sulfate precipitation method, chromatography methods such as ion exchange chromatography, expanded bed chromatography, hydrophobic interaction chromatography, etc., membrane methods such as ultrafiltration, dialysis, etc., two-phase aqueous methods such as polymer-inorganic salt two-phase aqueous method, low eutectic solvent-inorganic salt two-phase aqueous method, etc., but the precipitation method has a long cycle and high salt easily denatures the protein. The chromatography method has the defects of high cost, cumbersome operation, and difficulty in industrial production. Aqueous two-phase extraction, as a liquid-liquid separation technology, can effectively extract, separate, purify and concentrate various biomolecules, such as proteins, viruses, enzymes and nucleic acids, and has the advantages of simple operation, mild conditions, high efficiency, cost-effectiveness, and easy industrial scale-up.

[0003] However, the existing technology uses a polymer (such as polyethylene glycol, dextran, methylcellulose) / salt two-phase aqueous system to extract phycocyanin, but the overall process is time-consuming, prone to microbial growth, and accumulation of toxins; in addition, there is also an existing technology that synthesizes chemical reagents with polyethylene glycol and organic solvents, and constructs a two-phase aqueous system with salt to extract phycocyanin, but the organic solvents used, such as dichloromethane, are harmful to the environment and human body; a study disclosed a method for extracting phycocyanin from Spirulina using a low eutectic solvent-salt two-phase aqueous system, but the low eutectic solvent consumed in the extraction is huge, and the secondary two-phase aqueous extraction process is complicated, the industrial production cost is too high, and it is not easy to scale up. Summary of the invention

[0004] The main purpose of the present application is to provide a method for separating phycocyanin from Spirulina platensis by eutectic two-phase extraction, aiming to solve the problems of high cost, low raw material processing capacity and high salt concentration in the prior art when using a low eutectic two-phase system to extract phycocyanin.

[0005] To achieve the above-mentioned purpose, the technical solution adopted in the embodiment of the present application is as follows: A method for separating phycocyanin from Spirulina platensis by eutectic double aqueous phase extraction, comprising the following steps:

[0006] The platensis powder is extracted with a CaCl2 solution, and the platensis powder is subjected to freeze-thaw cell wall breaking and centrifugation to obtain a crude extract containing phycocyanin.

[0007] The crude extract containing phycocyanin is subjected to ultrafiltration and desalination treatment to obtain an ultrafiltration crude extract containing phycocyanin;

[0008] Adding a low eutectic solvent and a potassium sodium tartrate solution to the ultrafiltration crude extract containing phycocyanin, mixing to obtain a two-phase aqueous system, adjusting the pH and mixing, vortexing, standing for phase separation, centrifuging to promote phase separation, separating the upper phase, and obtaining a low eutectic solvent upper phase rich in phycocyanin;

[0009] The upper phase of the deep eutectic solvent rich in phycocyanin is subjected to ultrafiltration to obtain a filtrate of the deep eutectic solvent component and a concentrated phycocyanin solution;

[0010] The concentrated phycocyanin solution is freeze-dried to obtain phycocyanin powder.

[0011] As some optional embodiments of the present application, the low eutectic solvent is composed of a hydrogen bond acceptor, a hydrogen bond donor and water in a mass ratio of 1:4:1; wherein the hydrogen bond acceptor is quaternary ammonium salts, amino acids, sugars, alcohols, and the hydrogen bond donor is polyethylene glycol.

[0012] As some optional embodiments of the present application, the quaternary ammonium salt is at least one of choline chloride and tetrapropylammonium bromide, the amino acid is proline, the sugar is at least one of glucose, fructose, and mannose, and the alcohol is at least one of sorbitol and xylitol; the polyethylene glycol is at least one of polyethylene glycol 300, polyethylene glycol 400, and polyethylene glycol 600.

[0013] As some optional embodiments of the present application, the mass ratio of the Spirulina platensis powder to the CaCl2 solution is 1:10 to 1:30.

[0014] As some optional embodiments of the present application, the concentration of the CaCl2 solution is 0.1 mol / L to 0.5 mol / L, the stirring time is 2 h to 6 h, and the stirring speed is 400 rpm to 600 rpm.

[0015] As some optional embodiments of the present application, the freeze-thaw conditions of the freeze-thaw cell wall breaking treatment are freezing at -20°C for 2h to 6h, thawing at 4°C for 4h to 10h, and the number of cycles is 1 to 4;

[0016] The centrifugal treatment is carried out under the centrifugal conditions of 8000 rpm / min to 10000 rpm / min, 10 min to 20 min, and a temperature of 4° C. to 26° C.

[0017] As some optional embodiments of the present application, the ultrafiltration conditions in the ultrafiltration desalination treatment and the ultrafiltration treatment are 2000r / min~4500r / min, 10min~20min, 3 times~6 times, and the ultrafiltration membrane is 3kDa~30kDa.

[0018] As some optional embodiments of the present application, the mass fraction of the low eutectic solvent in the two-phase aqueous system is 29% to 36%, the mass fraction of the tartrate is 29% to 32%, and the mass fraction of the ultrafiltration crude extract containing phycocyanin is 35% to 39%.

[0019] As some optional embodiments of the present application, the pH value of the two-phase aqueous system is adjusted to 6-8.

[0020] As some optional embodiments of the present application, the vortex time is 0.5min to 2min; the static phase separation time is 10min to 30min, and the static phase separation temperature is 25℃ to 26℃; the centrifugal conditions for centrifugal promotion of phase separation are 2000rpm to 3000rpm, 5min to 10min, and 25℃ to 26℃.

[0021] Compared with the prior art, the purity of phycocyanin separated by the technical solution described in the present application reaches pharmaceutical grade (Purity>3.0), and the yield reaches 84%; and the present application adopts a combination of CaCl2 and freeze-thaw method for extraction, which improves the dissolution rate of phycocyanin, and the purity of the crude extract alone reaches food grade (Purity>0.7), as high as 1.13, which is significantly better than the prior art; the present application also uses ultrafiltration to remove salt ions in the crude extract, so that the purity of phycocyanin after crude extraction is higher, the operation is simple, the time consumption is short, and it is conducive to the subsequent two-phase aqueous system to prepare high-purity and high-yield phycocyanin; and the low eutectic two-phase aqueous system is used to further purify phycocyanin, which is simple to operate, short cycle, mild conditions, high yield, and suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the separation process of the phycocyanin powder described in Example 1 of the present application. DETAILED DESCRIPTION

[0023] It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] Example 1

[0025] (1) Preparation of phycocyanin crude extract: 25 mL of 0.2 mol / L CaCl2 solution was added to 1 g of Spirulina platensis powder, and the stirring time was 3 h at a stirring speed of 600 rpm; the extracted Spirulina platensis powder was frozen at -20°C for 4 h, thawed at 4°C for 2 h, and freeze-thawed for 3 times; after the wall of Spirulina platensis powder was broken, the crude extract containing phycocyanin was obtained after centrifugation at 8000 rpm / min at 4°C for 10 min, and the purity was 1.13;

[0026] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 3500 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 3 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0027] (3) Extraction of phycocyanin by low eutectic aqueous two-phase system: add a low eutectic solvent synthesized by a mass ratio of 1:4:1 (sorbitol: polyethylene glycol 300: water) to the ultrafiltration crude extract obtained in step (2), and mix with potassium sodium tartrate solution to obtain a two-phase system, wherein the mass fraction of the low eutectic solvent is 35%, the mass fraction of tartrate is 30%, and the mass fraction of the ultrafiltration crude extract is 35%. Adjust the pH to 8 and mix, vortex for 2 minutes, stand at 25°C for 20 minutes, centrifuge at 25°C at 3000 rpm / min for 10 minutes to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0028] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 3500 r / min for 10 min on a 3 kDa ultrafiltration membrane (Millipore) for a total of 6 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0029] (5) Obtaining phycocyanin powder after freeze-drying: The phycocyanin solution obtained in step (4) is freeze-dried to obtain phycocyanin powder with a purity of 2.52 and a yield of 85%.

[0030] The separation process of the above phycocyanin powder is as follows Figure 1 shown.

[0031] Example 2

[0032] (1) Preparation of phycocyanin crude extract: 30 mL of 0.5 mol / L CaCl2 solution was added to 1 g of Spirulina platensis powder, and the stirring time was 6 h at a stirring speed of 600 rpm; the extracted Spirulina platensis powder was frozen at -20°C for 6 h, thawed at 4°C for 4 h, and freeze-thawed twice; after the wall of Spirulina platensis powder was broken, the crude extract containing phycocyanin was obtained after centrifugation at 10000 rpm / min at 4°C for 20 min, and the purity was 1.09;

[0033] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 2000 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 3 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0034] (3) Extraction of phycocyanin by low eutectic aqueous two-phase method: adding a low eutectic solvent synthesized by a mass ratio of 1:4:1 (proline: polyethylene glycol 600: water) to the ultrafiltration crude extract obtained in step (2), and mixing with a potassium sodium tartrate solution to obtain a two-phase aqueous system, wherein the mass fraction of the low eutectic solvent is 29%, the mass fraction of tartrate is 32%, and the mass fraction of the ultrafiltration crude extract is 39%. The pH value is adjusted to 7 and mixed, vortexed for 0.5 min, allowed to stand at 25° C. for 30 min, centrifuged at 25° C. at 3000 rpm / min for 10 min to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0035] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 2000 r / min for 10 min on a 3 kDa ultrafiltration membrane (Millipore) for a total of 6 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0036] (5) Obtaining phycocyanin powder after freeze drying: The phycocyanin solution obtained in step (4) is freeze dried to obtain phycocyanin powder with a purity of 2.57 and a yield of 85%.

[0037] Example 3

[0038] (1) Preparation of phycocyanin crude extract: add 20 mL of 0.2 mol / L CaCl2 solution to 1 g of Spirulina platensis powder, stir for 3 h, and stir at 400 rpm; freeze the extracted Spirulina platensis powder at -20°C for 4 h, thaw at 4°C for 4 h, and freeze-thaw for 3 times; after breaking the wall of Spirulina platensis powder, centrifuge at 10000 rpm / min at 4°C for 20 min to obtain a crude extract containing phycocyanin with a purity of 1.13;

[0039] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 3000 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 3 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0040] (3) Extracting phycocyanin through a low eutectic aqueous two-phase system: adding a low eutectic solvent synthesized by a mass ratio of 1:4:1 (mannose: polyethylene glycol 400: water) to the ultrafiltration crude extract obtained in step (2), and mixing with a potassium sodium tartrate solution to obtain a two-phase system, wherein the mass fraction of the low eutectic solvent is 30%, the mass fraction of tartrate is 31%, and the mass fraction of the ultrafiltration crude extract is 39%. The pH is adjusted to 7.5 and mixed, vortexed for 1 min, allowed to stand at 25° C. for 30 min, centrifuged at 25° C. at 3000 rpm / min for 10 min to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0041] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 2000 r / min for 10 min on a 3 kDa ultrafiltration membrane (Millipore) for a total of 6 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0042] (5) Obtaining phycocyanin powder after freeze-drying: The phycocyanin solution obtained in step (4) is freeze-dried to obtain phycocyanin powder with a purity of 2.84 and a yield of 87%.

[0043] Example 4

[0044] (1) Preparation of phycocyanin crude extract: add 25 mL of 0.1 mol / L CaCl2 solution to 1 g of Spirulina platensis powder, stir for 4 h, and stir at 500 rpm; freeze the extracted Spirulina platensis powder at -20°C for 4 h, thaw at 4°C for 6 h, and freeze-thaw twice; after breaking the wall of Spirulina platensis powder, centrifuge at 10000 rpm / min for 15 min at 4°C to obtain a crude extract containing phycocyanin with a purity of 1.11;

[0045] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 3000 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 3 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0046] (3) Extraction of phycocyanin by low eutectic aqueous two-phase system: add a low eutectic solvent synthesized by a mass ratio of 1:4:1 (tetrapropylammonium bromide: polyethylene glycol 400: water) to the ultrafiltration crude extract obtained in step (2), and mix with potassium sodium tartrate solution to obtain a two-phase system, wherein the mass fraction of the low eutectic solvent is 33%, the mass fraction of tartrate is 30%, and the mass fraction of the ultrafiltration crude extract is 37%. Adjust the pH to 7.5 and mix, vortex for 1 min, stand at 25° C. for 15 min, centrifuge at 25° C. at 3000 rpm / min for 10 min to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0047] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 3000 r / min for 10 min on a 30 kDa ultrafiltration membrane (Millipore) for a total of 6 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0048] (5) Obtaining phycocyanin powder after freeze-drying: The phycocyanin solution obtained in step (4) is freeze-dried to obtain phycocyanin powder with a purity of 2.50 and a yield of 83%.

[0049] Example 5

[0050] (1) Preparation of phycocyanin crude extract: add 20 mL of 0.2 mol / L CaCl2 solution to 1 g of Spirulina platensis powder, stir for 2 h, and stir at 400 rpm; freeze the extracted Spirulina platensis powder at -20°C for 2 h, thaw at 4°C for 3 h, and freeze-thaw for 3 times; after breaking the wall of Spirulina platensis powder, centrifuge at 10000 rpm / min at 4°C for 20 min to obtain a crude extract containing phycocyanin with a purity of 1.13;

[0051] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 4500 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 3 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0052] (3) Extraction of phycocyanin by low eutectic aqueous two-phase method: adding a low eutectic solvent synthesized by a mass ratio of 1:4:1 (proline: polyethylene glycol 400: water) to the ultrafiltration crude extract obtained in step (2), and mixing with a potassium sodium tartrate solution to obtain a two-phase aqueous system, wherein the mass fraction of the low eutectic solvent is 36%, the mass fraction of tartrate is 29%, and the mass fraction of the ultrafiltration crude extract is 35%. The pH value is adjusted to 6.5 and mixed, vortexed for 1.5 minutes, allowed to stand at 25° C. for 10 minutes, centrifuged at 25° C. at 3000 rpm / min for 10 minutes to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0053] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 4500 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 6 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0054] (5) Obtaining phycocyanin powder after freeze drying: The phycocyanin solution obtained in step (4) is freeze dried to obtain phycocyanin powder with a purity of 3.22 and a yield of 84%.

[0055] Comparative Example 1

[0056] (1) Preparation of phycocyanin crude extract: 20 mL of 0.2 mol / L CaCl2 solution was added to 1 g of Spirulina platensis powder, and the mixture was frozen at -20°C for 2 h, thawed at 4°C for 4 h, and freeze-thawed for 3 times; after the wall of Spirulina platensis powder was broken, the crude extract containing phycocyanin was obtained by centrifugation at 8000 rpm / min at 4°C for 15 min, and the purity was 0.72;

[0057] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 4500 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 3 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0058] (3) Extraction of phycocyanin by low eutectic aqueous two-phase method: adding a low eutectic solvent synthesized by a mass ratio of 1:4:1 (choline chloride: polyethylene glycol 400: water) to the ultrafiltration crude extract obtained in step (2), and mixing with a potassium sodium tartrate solution to obtain a two-phase aqueous system, wherein the mass fraction of the low eutectic solvent is 29%, the mass fraction of tartrate is 35%, and the mass fraction of the ultrafiltration crude extract is 36%. The pH is adjusted to 5.0 and mixed, vortexed for 2 minutes, allowed to stand at 25° C. for 30 minutes, centrifuged at 25° C. at 2000 rpm / min for 10 minutes to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0059] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 4500 r / min for 10 min on a 3 kDa ultrafiltration membrane (Millipore) for a total of 6 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0060] (5) Obtaining phycocyanin powder after freeze-drying: The phycocyanin solution obtained in step (4) is freeze-dried to obtain phycocyanin powder with a purity of 1.87 and a yield of 65%.

[0061] Comparative Example 2

[0062] (1) Preparation of phycocyanin crude extract: add 10 mL of 0.1 mol / L CaCl2 solution to 1 g of Spirulina platensis powder, stir for 4 h, and stir at 500 rpm; freeze the extracted Spirulina platensis powder at -20°C for 4 h, thaw at 4°C for 6 h, and freeze-thaw for 3 times; after breaking the wall of Spirulina platensis powder, centrifuge at 8000 rpm / min at 4°C for 20 min to obtain a crude extract containing phycocyanin with a purity of 1.09;

[0063] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 1500 r / min for 5 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 2 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0064] (3) Extraction of phycocyanin by low eutectic aqueous two-phase method: adding a low eutectic solvent synthesized by a mass ratio of 1:4:1 (tetrapropylammonium bromide: polyethylene glycol 400: water) to the ultrafiltration crude extract obtained in step (2), and mixing with a potassium sodium tartrate solution to obtain a two-phase aqueous system, wherein the mass fraction of the low eutectic solvent is 32%, the mass fraction of tartrate is 32%, and the mass fraction of the ultrafiltration crude extract is 36%. The pH value is adjusted to 6 and mixed, vortexed for 3 minutes, allowed to stand at 25° C. for 20 minutes, centrifuged at 25° C. at 3000 rpm / min for 10 minutes to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0065] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 3500 r / min for 10 min on a 3 kDa ultrafiltration membrane (Millipore) for a total of 3 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0066] (5) Obtaining phycocyanin powder after freeze-drying: The phycocyanin solution obtained in step (4) is freeze-dried to obtain phycocyanin powder with a purity of 1.64 and a yield of 58%.

[0067] Comparative Example 3

[0068] (1) Preparation of phycocyanin crude extract: add 15 mL of 0.4 mol / L CaCl2 solution to 1 g of Spirulina platensis powder, stir for 4 h, and stir at 500 rpm; freeze the extracted Spirulina platensis powder at -20°C for 3 h, thaw at 4°C for 2 h, and repeat the freeze-thaw cycle 4 times; after breaking the wall of Spirulina platensis powder, centrifuge at 10000 rpm / min at 4°C for 20 min to obtain a crude extract containing phycocyanin with a purity of 1.09;

[0069] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 3500 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 3 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0070] (3) Extracting phycocyanin by low eutectic aqueous two-phase method: adding a low eutectic solvent synthesized by a molar ratio of 1:2 (choline chloride: urea) to the ultrafiltration crude extract obtained in step (2), and mixing with potassium phosphate buffer to obtain a two-phase aqueous system, wherein the mass fraction of the low eutectic solvent is 36%, the mass fraction of phosphate is 29%, and the mass fraction of the ultrafiltration crude extract is 35%. The pH value is adjusted to 7.5 and mixed, vortexed for 0.5 min, allowed to stand at 25° C. for 30 min, centrifuged at 25° C. at 3000 rpm / min for 10 min to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0071] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 3500 r / min for 10 min on a 3 kDa ultrafiltration membrane (Millipore) for a total of 6 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0072] (5) Obtaining phycocyanin powder after freeze drying: The phycocyanin solution obtained in step (4) is freeze dried to obtain phycocyanin powder with a purity of 2.13 and a yield of 66%.

[0073] Comparative Example 4

[0074] (1) Preparation of phycocyanin crude extract: 30 mL of 0.5 mol / L CaCl2 solution was added to 1 g of Spirulina platensis powder, and the stirring time was 6 h at a stirring speed of 600 rpm; the extracted Spirulina platensis powder was frozen at -20°C for 6 h, thawed at 4°C for 4 h, and freeze-thawed twice; after the wall of Spirulina platensis powder was broken, the crude extract containing phycocyanin was obtained after centrifugation at 10000 rpm / min at 4°C for 20 min, and the purity was 1.06;

[0075] (2) Ultrafiltration desalination: The crude extract obtained in step (1) was ultrafiltered at 2000 r / min for 10 min using a 30 kDa ultrafiltration membrane (Millipore) for a total of 3 times to remove salt ions in the crude extract to obtain an ultrafiltered crude extract containing phycocyanin;

[0076] (3) Extraction of phycocyanin by low eutectic aqueous two-phase method: adding a low eutectic solvent synthesized by a mass ratio of 1:4:1 (glucose: polyethylene glycol 300: water) to the ultrafiltration crude extract obtained in step (2), and mixing with a potassium sodium tartrate solution to obtain a two-phase aqueous system, wherein the mass fraction of the low eutectic solvent is 32%, the mass fraction of tartrate is 31%, and the mass fraction of the ultrafiltration crude extract is 37%. The pH value is adjusted to 7 and mixed, vortexed for 0.5 min, allowed to stand at 25° C. for 30 min, centrifuged at 25° C. at 3000 rpm / min for 10 min to promote two-phase separation, separate the upper phase, and obtain a low eutectic solvent upper phase rich in phycocyanin;

[0077] (4) Ultrafiltration to recover the low eutectic solvent component and concentrate the phycocyanin solution: The upper phase obtained in step (3) was ultrafiltered at 2000 r / min for 10 min on a 50 kDa ultrafiltration membrane (Millipore) for a total of 6 times to obtain a filtrate of the low eutectic solvent component and a concentrated phycocyanin solution;

[0078] (5) Obtaining phycocyanin powder after freeze-drying: The phycocyanin solution obtained in step (4) is freeze-dried to obtain phycocyanin powder with a purity of 2.07 and a yield of 44%.

[0079] The following is a comparison of the raw material ratios, purities and yields of Examples 1-4 and Comparative Examples 1-4, as shown in Table 1:

[0080] Table 1

[0081]

[0082]

[0083] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction, characterized in that: The following steps are involved: The platensis powder is extracted with a CaCl2 solution, and the platensis powder is subjected to freeze-thaw cell wall breaking and centrifugation to obtain a crude extract containing phycocyanin. The crude extract containing phycocyanin is subjected to ultrafiltration and desalination treatment to obtain an ultrafiltration crude extract containing phycocyanin; Adding a low eutectic solvent and a potassium sodium tartrate solution to the ultrafiltration crude extract containing phycocyanin, mixing to obtain a two-phase aqueous system, adjusting the pH and mixing, vortexing, standing for phase separation, centrifuging to promote phase separation, separating the upper phase, and obtaining a low eutectic solvent upper phase rich in phycocyanin; The upper phase of the deep eutectic solvent rich in phycocyanin is subjected to ultrafiltration to obtain a filtrate of the deep eutectic solvent component and a concentrated phycocyanin solution; The concentrated phycocyanin solution is freeze-dried to obtain phycocyanin powder.

2. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 1, characterized in that: The low eutectic solvent is composed of a hydrogen bond acceptor, a hydrogen bond donor and water in a mass ratio of 1:4:1; the hydrogen bond acceptor is a quaternary ammonium salt, an amino acid, a sugar or an alcohol, and the hydrogen bond donor is polyethylene glycol.

3. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 2, characterized in that: The quaternary ammonium salt is at least one of choline chloride and tetrapropylammonium bromide, the amino acid is proline, the sugar is at least one of glucose, fructose and mannose, the alcohol is at least one of sorbitol and xylitol; the polyethylene glycol is at least one of polyethylene glycol 300, polyethylene glycol 400 and polyethylene glycol 600.

4. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 1, characterized in that: The mass ratio of the Spirulina platensis powder to the CaCl2 solution is 1:10 to 1:

30.

5. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 4, characterized in that: The concentration of the CaCl2 solution is 0.1 mol / L to 0.5 mol / L, the stirring time is 2 h to 6 h, and the stirring speed is 400 rpm to 600 rpm.

6. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 1, characterized in that: The freeze-thaw conditions of the freeze-thaw cell-breaking treatment are freezing at -20°C for 2h to 6h, thawing at 4°C for 4h to 10h, and the number of cycles is 1 to 4; The centrifugal treatment is carried out under the centrifugal conditions of 8000 rpm / min to 10000 rpm / min, 10 min to 20 min, and a temperature of 4° C. to 26° C.

7. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 1, characterized in that: The ultrafiltration conditions in the ultrafiltration desalination treatment and the ultrafiltration treatment are both 2000r / min to 4500r / min, 10min to 20min, 3 times to 6 times, and the ultrafiltration membrane is 3kDa to 30kDa.

8. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 1, characterized in that: The mass fraction of the low eutectic solvent in the two-phase aqueous system is 29% to 36%, the mass fraction of the tartrate is 29% to 32%, and the mass fraction of the ultrafiltration crude extract containing phycocyanin is 35% to 39%.

9. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 1, characterized in that: The pH value of the two-phase aqueous system is adjusted to 6-8.

10. The method for separating phycocyanin from Spirulina platensis by eutectic aqueous two-phase extraction according to claim 1, characterized in that: The vortexing time is 0.5min to 2min; the standing phase separation time is 10min to 30min, and the standing phase separation temperature is 25°C to 26°C; the centrifugal conditions for centrifugal promotion of phase separation are 2000rpm to 3000rpm, 5min to 10min, and 25°C to 26°C.

Images