WO2024108780A1 - Method for extracting and purifying phycocyanin from spirulina platensis - Google Patents

Abstract

A method for extracting and purifying phycocyanin from Spirulina platensis. Dried Spirulina platensis cells with polysaccharide enriched on the surface are obtained by means of spray drying with salt; by means of a salt solvent, the exudation of intracellular phycocyanin is promoted, improving the extraction rate of phycocyanin; by means of homogenizing the extraction solution carrying Spirulina platensis cells, appropriate cell fragments are obtained, so that the Spirulina platensis cell debris shows the characteristics of a polysaccharide micropolymer, that is, the Spirulina platensis cells migrate to the bottom phase with dextran while promoting the phase separation, so as to achieve the one-step separation and purification of phycocyanin; and finally, by means of an aqueous two-phase system formed by polyethylene glycol and the salt solvent, enrichment and purification are performed, so that finally the extraction rate and purity of phycocyanin are greatly improved. The method is simple to operate and suitable for large-scale extraction and purification of phycocyanin, reduces the production cost of phycocyanin and meets the requirements of large-scale application.

Description

A method for extracting and purifying phycocyanin from Spirulina platensis Technical Field

The invention belongs to the technical field of functional component extraction, and specifically relates to a method for extracting phycocyanin from dry biomass of Spirulina platensis .

Background technique

Phycocyanin is a natural pigment protein, mainly found in cyanobacteria and red algae. It is a color additive approved by the US Food and Drug Administration that does not require certification. Due to its functional activities such as anti-tumor, antioxidant, anti-inflammatory, and immune enhancement, it is increasingly being studied in the market, industry, and scientific fields. Phycocyanin has been extracted from various algae, and Spirulina platensis is considered to be a cheap and abundant source of phycocyanin and polysaccharides. However, the cell wall of Spirulina cells is difficult to destroy, and the dried Spirulina cell wall has stronger rigidity, which hinders the extraction of intracellular proteins. Therefore, there are many studies to improve the extraction rate of phycocyanin by improving the cell disruption process. At present, cell disruption mainly adopts: repeated freezing and thawing, ultrasound, microwave, chemical reagent treatment, enzymatic hydrolysis, swelling, homogenization, ultrafine shearing and grinding. These methods inevitably cause other impurities in the cell to dissolve and reduce the purity of phycocyanin, so there are also many methods for separating and purifying phycocyanin, such as salting out, chromatographic separation and extraction. The separation of cells in the early stage of purification will prolong the processing time and cause protein loss. It is not only costly and time-consuming but also affects the protein extraction rate. Therefore, a new process to reduce processing time and protein loss is necessary.

The two-phase aqueous extraction system is an extraction technology with mild conditions, large processing volume, easy continuous operation, environmental friendliness and the ability to maintain molecular biological activity. Researchers used a two-phase aqueous system formed by potassium phosphate and polyethylene glycol to separate and purify phycocyanin from Spirulina platensis through ten-stage countercurrent distribution, with a purity of 238% (Liu et al., Aqueous two-phase countercurrent distribution for the separation of c-phycocyanin and allophycocyanin from Spirulina platensis , 2012, 90: 111-117). In addition, the purity of phycocyanin from Spirulina platensis was increased by 2.1 times by using a two-phase aqueous system formed by trimethylamine and polyethylene glycol (Wang et al., Application of TMA-PEG to promote C-phycocyanin extraction from S.platensis in the PEG ATPS, 2017, 52: 283-294). The aqueous two-phase extraction system can achieve an ideal purification effect by purifying the phycocyanin extract. However, the traditional purification of phycocyanin requires the removal of the algae before purifying the extract. The aqueous two-phase purification can usually only perform liquid-liquid extraction and cannot achieve liquid-solid separation.

Summary of the invention

The present invention aims to solve the problem that the extraction of phycocyanin from Spirulina platensis requires the removal of the algae before purification of the extract, and that aqueous two-phase purification can usually only perform liquid-liquid extraction but cannot achieve liquid-solid separation. The invention provides a new process for one-step separation and purification of phycocyanin, and can reduce protein loss and processing costs during the processing.

The present invention introduces a two-phase aqueous system formed by polyethylene glycol and citrate, and utilizes the interaction between the glucan attached to the spirulina after drying and the polyethylene glycol to separate the algae from the phycocyanin while promoting phase separation. The method can improve the extraction rate and purity of phycocyanin, and the method is low in cost, simple to operate, and easy to scale up for production.

To achieve the purpose of the present invention, the present invention provides the following technical solutions:

A method for extracting and purifying phycocyanin from Spirulina platensis , screening a low-cost and efficient salt solvent to extract phycocyanin from Spirulina, and screening a phase-forming salt to achieve one-step algal separation and purification of phycocyanin through a two-phase aqueous system.

The specific steps include:

S1 Pretreatment: NaCl is added to the algae liquid before spray drying to promote the exudation of algae cell polysaccharides during the drying process and to adhere to the algae cells, which is beneficial for phase separation and cell separation in the later purification. The solid content of the algae liquid for spray drying is 50-150 g/L. 0.5-5% of metal chloride salts such as NaCl, KCl or _MgCl2 are added to the algae liquid. Fresh Spirulina is spray dried under the conditions of inlet temperature of 120-180 ^o C and outlet temperature of 60-100 ^o C.

S2 adds the spray-dried spirulina into a salt solvent, such as NaCl, KCl or MgCl ₂ , etc., and extracts phycocyanin at a liquid-solid ratio of 10-40 v/w, a salt concentration of 20-100 g/L, a pH of 5-10, an extraction temperature of 5-25 ^o C, a rotation speed of 50-200 rpm, and an extraction time of 6-30 h. The use of the salt solvent and its extraction conditions can ensure the maximum dissolution of phycocyanin, improve the stability of phycocyanin, and reduce the loss caused by the inactivation of phycocyanin during the extraction process.

S3 homogenizes the extract containing algae cells obtained in S2

Spirulina cells carry polysaccharides on their surface, and cell fragments of appropriate size exhibit the characteristics of polysaccharide micropolymers. In order to obtain appropriate Spirulina cell fragments, the extract carrying the algae cells is homogenized at a speed of 10,000-40,000 rpm for 3-15 seconds. Too large cell fragments cannot exhibit the characteristics of micro-polymers, which will make it difficult to form a two-phase in the subsequent separation. Too small cell fragments cannot exhibit the characteristics of polysaccharides, resulting in the cells existing in two phases in the subsequent separation, and the cell separation cannot be achieved.

S4: dilute the homogenized extract by 1-8 times and then add a phase-forming salt, preferably C ₆ H ₅ K ₃ O ₇ , C ₆ H ₅ Na ₃ O ₇ or C ₁₂ H ₁₀ Mg ₃ O ₁₄ , and polyethylene glycol with a molecular weight of 400-6000. Select the optimal phase-forming salt and polyethylene glycol, select a salt concentration of 8-40 wt%, a polyethylene glycol concentration of 15-40 wt% in the two-phase region of the phase diagram, and a total volume ratio of the algae liquid dilution to the two-phase water phase of 1:3-1:7. Purify phycocyanin and separate synchronous cells at a temperature of 5-25 ^o C, a pH of 5-9, and a time of 3-24 h, with phycocyanin in the upper phase and algae cells aggregated in the lower phase.

After S5 purification, the extraction rate and purity of phycocyanin in the upper phase were determined.

Further, the solid content of the algae liquid during the spray drying feed in S1 can be 50, 75, 100, 125, 150 g/L, preferably 75-125 g/L, and more preferably 100 g/L. The concentration of NaCl added to the concentrated algae liquid can be 0.5, 1, 2, 3, 4, 5%, preferably 2-4%, and more preferably 3%. Preferably, the inlet temperature can be 120, 130, 140, 150, 160, 170, 180 ° C, preferably 140-180 ^° C, and more preferably 175 ^° C. Preferably, the outlet temperature can ^be 60, 70, 80, 90, 100 ^° C, preferably 60-80 ^° C, and more preferably 75 ^° C.

Further, the salt solvent in S2 is preferably NaCl or KCl, more preferably NaCl. Preferably, the liquid-to-solid ratio is 10, 20, 30, 40 v/w, preferably 10-30 v/w, more preferably 20 v/w. Preferably, the salt concentration can be 20, 40, 60, 80, 100 g/L, preferably 20-80 g/L, more preferably 50 g/L. Preferably, the pH can be 5, 6, 7, 8, 9, 10, preferably 6-9, more preferably 7. Preferably, the leaching temperature can be 5, 10, 15, 20, 25 ^° C, preferably 10-20 ^° C, more preferably 15 ^° C. Preferably, the rotation speed can be 50, 75, 100, 125, 150, 200 rpm, preferably 100-150 rpm, more preferably 125 rpm. Preferably, the extraction time may be 6, 9, 12, 16, 20, 24, 26, 30 h, preferably 12-24 h, more preferably 24 h.

Further, the speed in S3 can be 10000, 20000, 30000, 40000, preferably 20000-30000 rpm, more preferably 30000 rpm. Preferably, the time can be 3, 5, 10, 15 s, preferably 5-10 s, more preferably 5 s.

Further, the dilution multiple of the algae liquid in S4 can be 1, 2, 4, 6, 8, preferably 1-4, more preferably 2. Preferably, the phase-forming salt can be C ₆ H ₅ K ₃ O ₇ , C ₆ H ₅ Na ₃ O ₇ or C ₁₂ H ₁₀ Mg ₃ O ₁₄ , preferably C ₆ H ₅ K ₃ O ₇ or C ₆ H ₅ Na ₃ O ₇ , more preferably C ₆ H ₅ K ₃ O _7. Preferably, the molecular weight of polyethylene glycol can be 400, 600, 800, 1000, 2000, 4000, 6000, preferably 2000-6000, more preferably 4000. Preferably, the salt concentration can be 8, 10, 20, 30, 40 wt%, preferably 8-20 wt%, more preferably 10 wt%. Preferably, the concentration of polyethylene glycol can be 15, 20, 25, 30, 35, 40 wt%, preferably 15-30 wt%, more preferably 20 wt%. Preferably, the total volume ratio of the algae liquid diluent to the two-phase aqueous solution can be 1:3, 1:4, 1:5, 1:6, 1:7, preferably, the total volume ratio of the algae liquid diluent to the two-phase aqueous solution is 1:4, 1:5, 1:6, more preferably 1:5. Preferably, the extraction temperature can be 5, 10, 15, 20, 25 ^° C, preferably 10-20 ^° C, more preferably 15 ^° C. Preferably, the pH can be 5, 6, 7, 8, 9, preferably 6-8, more preferably no pH adjustment (i.e. pH 8). Preferably, the time can be 3, 6, 12, 18, 24 h, preferably 6-18 h, more preferably 12 h.

First, dry spirulina cells enriched with polysaccharides on the surface are obtained by salt spray drying, and then a low-cost and efficient salt solvent is selected to promote the exudation of intracellular phycocyanin by destroying the integrity of spirulina cells; the salt solvent improves the extraction rate of phycocyanin by improving the water property of phycocyanin and maintaining the stability and functional activity of phycocyanin. Then, the extract carrying spirulina cells is homogenized to obtain suitable cell fragments, so that the spirulina cell fragments show the characteristics of polysaccharide micropolymers, and the spirulina cells migrate to the lower phase with glucan while promoting phase separation, thereby realizing the one-step separation and purification of phycocyanin; then, polyethylene glycol and the salt solvent are used to form a two-phase water system, and phycocyanin interacts with the phase-forming material through hydrophobic interaction, electrostatic interaction and other forces in the two-phase water system, and phycocyanin produces selective distribution behavior on the upper phase, forming a concentration difference, and achieving the purpose of enrichment and purification; in addition, the method of the present invention greatly improves the extraction rate and purity of phycocyanin.

The invention is simple to operate, suitable for large-scale extraction and purification of phycocyanin, reduces the cost of phycocyanin production, and meets the requirements of large-scale application.

Implementation

The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

Example

The total phycocyanin content was determined with reference to the Chinese national standard SNT1113-2002. The dried biomass of Spirulina was soaked in phosphate buffer (0.1 mol/L, pH 7.0) and then treated with ultrasound (100 W, 40 kHz) for 5 min in a fixed volume in a 250 mL volumetric flask. 250 mL of the mixture was transferred to a 300 mL plastic bottle and frozen at -20 ^° C for 12 h. Then, the mixture was thawed at 25 ^° C. The freeze-thaw procedure was repeated three times, and then centrifuged at 8000 rpm for 15 min, and all the supernatants were collected and mixed.

Fresh spirulina was filtered and concentrated to make the fixed matter content reach 75 g/L, and 0.5% NaCl was added. The fresh spirulina was dried at the inlet and outlet temperatures of 120 and 70 ^o C respectively to obtain dry spirulina. Spirulina dry biomass (3.3 g) and 100 mL of NaCl solution with a salt concentration of 60 g/L were added to a 250 mL flask (liquid-to-solid ratio of 30). The pH was adjusted to 7 and extracted at 125 rpm and 15 ^o C for 24 h. The extract carrying Spirulina cells was homogenized at 20000 rpm for 3 s, and the extract (without dilution) was added to 15 wt% polyethylene glycol 2000 and 8 wt% C ₆ H ₅ Na ₃ O ₇ solution. The total volume of the algae solution and the two-phase aqueous solution was 1:4, and the total volume of the mixed solution was 8 mL. The pH was adjusted to 8 and shaken at 15 ^o C and 125 rpm for 1 h. After settling at 20 ^° C for 24 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Fresh spirulina was filtered and concentrated to make the fixed matter content reach 50 g/L, and 2% NaCl was added. The fresh spirulina was dried at the inlet and outlet temperatures of 140 and 75 ^o C respectively to obtain dry spirulina. Spirulina dry biomass (5 g) and 100 mL of KCl solution with a salt concentration of 60 g/L were added to a 250 mL flask (liquid-solid ratio of 20). The pH was adjusted to 10 and extracted at 50 rpm and 25 ^o C for 16 h. The extract carrying spirulina cells was homogenized at 40000 rpm for 15 s, diluted 4 times, and added to 30 wt% polyethylene glycol 800 and 40 wt% C ₆ H ₅ K ₃ O ₇ solution. The total volume ratio of the algae liquid dilution to the two-phase aqueous solution was 1:3, and the total volume of the mixed solution was 9 mL. The pH was adjusted to 5 and shaken at 15 ^o C and 125 rpm for 1 h. After settling at 25 ^° C for 12 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Fresh spirulina was filtered and concentrated to make the fixed matter content reach 125 g/L, and 1% NaCl was added. The fresh spirulina was dried at the inlet and outlet temperatures of 160 and 90 ^o C respectively to obtain dry spirulina. Spirulina dry biomass (3.3 g) and 100 mL of MgCl ₂ solution with a salt concentration of 40 g/L were added to a 250 mL flask (liquid-to-solid ratio of 30). The pH was adjusted to 6 and extracted at 75 rpm and 5 ^o C for 6 h. The extract carrying Spirulina cells was homogenized at 30000 rpm for 5 s, diluted 8 times, and added to 25 wt% polyethylene glycol 1000 and 30 wt% C ₆ H ₅ K ₃ O ₇ solution. The total volume ratio of the algae liquid dilution to the two-phase aqueous solution was 1:5, and the total volume of the mixed solution was 10 mL. The pH was adjusted to 6 and shaken at 15 ^o C and 125 rpm for 1 h. After settling at 5 ^° C for 6 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Fresh spirulina was filtered and concentrated to make the fixed matter content reach 150 g/L, and 3% KCl was added. The fresh spirulina was dried at the inlet and outlet temperatures of 150 and 60 ^o C respectively to obtain dry spirulina. Spirulina dry biomass (2.5 g) and 100 mL of KCl solution with a salt concentration of 80 g/L were added to a 250 mL flask (liquid-solid ratio of 40). The pH was adjusted to 8 and extracted at 200 rpm and 20 ^o C for 26 h. The extract carrying Spirulina cells was homogenized at 10000 rpm for 10 s, diluted 6 times, and added to 35 wt% polyethylene glycol 400 and 10 wt% C ₆ H ₅ Na ₃ O ₇ solution. The total volume ratio of the algae liquid dilution to the two-phase aqueous solution was 1:7, and the total volume ratio of the mixed solution was 11 mL. The pH was adjusted to 9 and shaken at 10 ^o C and 125 rpm for 1 h. After settling at 10 ^° C for 18 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Fresh spirulina was filtered and concentrated to a fixed content of 100 g/L. 1% KCl was added and dried at inlet and outlet temperatures of 130 and 100 ^° C to obtain dry spirulina. Spirulina dry biomass (10 g) and 100 mL of MgCl ₂ solution with a salt concentration of 20 g/L were added to a 250 mL flask (liquid-solid ratio of 10). The pH was adjusted to 9 and extracted at 200 rpm and 25 ^° C for 12 h. The extract carrying Spirulina cells was homogenized at 40,000 rpm for 15 s, diluted 2 times, and added to 20 wt% polyethylene glycol 6000 and 20 wt% C ₁₂ H ₁₀ Mg ₃ O ₁₄ solutions. The total volume ratio of the algae liquid dilution to the two-phase aqueous solution was 1:6, and the total volume of the mixed solution was 12 mL. The pH was adjusted to 7 and the mixture was shaken at 15 ^° C and 125 rpm for 1 h. After settling at 15 ^° C for 3 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Fresh spirulina was filtered and concentrated to make the fixed matter content reach 150 g/L, and 5% MgCl ₂ was added. The fresh spirulina was dried at the inlet and outlet temperatures of 180 and 80 ^o C respectively to obtain dry spirulina. Spirulina dry biomass (10 g) and 100 mL of MgCl ₂ solution with a salt concentration of 20 g/L were added to a 250 mL flask (liquid-to-solid ratio of 10). The pH was adjusted to 5 and extracted at 100 rpm and 10 ^o C for 20 h. The extract carrying Spirulina cells was homogenized at 20000 rpm for 3 s, diluted 2 times, and added to 40 wt% polyethylene glycol 4000 and 20 wt% C ₁₂ H ₁₀ Mg ₃ O ₁₄ solution. The total volume ratio of the algae liquid dilution to the two-phase aqueous solution was 1:5, and the total volume of the mixed solution was 10 mL. The pH was adjusted to 8 and shaken at 15 ^o C and 125 rpm for 1 h. After settling at 15 ^° C for 3 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Fresh spirulina was filtered and concentrated to make the fixed matter content reach 75 g/L, 3% NaCl was added, and the fresh spirulina was dried at the inlet and outlet temperatures of 130 and 75 ^o C respectively to obtain dry spirulina. Spirulina dry biomass (2.5 g) and 100 mL of NaCl solution with a salt concentration of 80 g/L were added to a 250 mL flask (liquid-solid ratio of 40). The pH was adjusted to 9 and extracted at 100 rpm and 5 ^o C for 30 h. The extract carrying Spirulina cells was homogenized at 40000 rpm for 5 s, diluted 6 times, and added to 35 wt% polyethylene glycol 600 and 30 wt% C ₆ H ₅ Na ₃ O ₇ solution. The total volume ratio of the algae liquid dilution to the two-phase aqueous solution was 1:3, and the total volume of the mixed solution was 9 mL. The pH was adjusted to 7 and shaken at 15 ^o C and 125 rpm for 1 h. After settling at 10 ^° C for 24 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Compared with Example 7, in Comparative Experimental Example 1, fresh spirulina was filtered and concentrated to make the fixed matter content reach 75 g/L, and no NaCl was added. The fresh spirulina was dried under the conditions of inlet and outlet temperatures of 130 and 75 ^° C, respectively, to obtain dry spirulina. Spirulina dry biomass (2.5 g) and 100 mL of deionized water were added to a 250 mL flask (liquid-solid ratio of 40). The pH was adjusted to 9 and extracted at 100 rpm and 5 ^° C for 30 h. The extract containing Spirulina cells was centrifuged at 8000 rpm for 15 min (if not centrifuged, the cells could not be separated in the subsequent two-phase aqueous process), the supernatant was diluted 6 times, and then added to 35 wt% polyethylene glycol 600 and 30 wt% phosphate solution (refer to Liu et al. Aqueous two-phase countercurrent distribution for the separation of c-phycocyanin and allophycocyanin from Spirulina platensis , 2012, 9 (20) 111-117), the total volume ratio of the supernatant dilution to the two-phase aqueous solution was 1:3, the total volume of the mixed solution was 9 mL, the pH was adjusted to 7, and the mixture was shaken at 15 ^° C and 125 rpm for 1 h. After sedimentation at 10 ^° C for 24 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Compared with Example 7, in Comparative Experiment 2, fresh spirulina was filtered and concentrated to make the fixed matter content reach 75 g/L, and no NaCl was added. The fresh spirulina was dried under the conditions of inlet and outlet temperatures of 130 and 75 ^° C, respectively, to obtain dry spirulina. Spirulina dry biomass (2.5 g) and 100 mL of NaCl solution with a salt concentration of 80 g/L were added to a 250 mL flask (liquid-solid ratio of 40). The pH was adjusted to 9 and extracted at 100 rpm and 5 ^° C for 30 h. The extract carrying spirulina cells was homogenized at 40,000 rpm for 5 s, diluted 6 times, and added to 35 wt% polyethylene glycol 600 and 30 wt% C ₆ H ₅ Na ₃ O ₇ solution. The total volume ratio of the algae liquid dilution to the two-phase aqueous solution was 1:3, and the total volume of the mixed solution was 9 mL. The pH was adjusted to 7 and shaken at 15 ^° C and 125 rpm for 1 h. After settling at 10 ^° C for 24 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Fresh spirulina was filtered and concentrated to make the fixed matter content reach 75 g/L, 3% NaCl was added, and the fresh spirulina was dried at the inlet and outlet temperatures of 130 and 75 ^o C respectively to obtain dry spirulina. Spirulina dry biomass (2.5 g) and 100 mL of pure water without NaCl were added to a 250 mL flask (liquid-solid ratio of 40). The pH was adjusted to 9 and extracted at 100 rpm and 5 ^o C for 30 h. The extract carrying Spirulina cells was homogenized at 40000 rpm for 5 s, diluted 6 times, and added to 35 wt% polyethylene glycol 600 and 30 wt% C ₆ H ₅ Na ₃ O ₇ solution. The total volume ratio of the algae liquid dilution to the two-phase aqueous phase was 1:3, and the total volume of the mixed solution was 9 mL. The pH was adjusted to 7 and shaken at 15 ^o C and 125 rpm for 1 h. After settling at 10 ^o C for 24 h, the extraction rate and purity of phycocyanin in the upper phase were determined.

Compared with Example 7, in Comparative Experiment 4, fresh spirulina was filtered and concentrated to make the fixed matter content reach 75 g/L, 3% NaCl was added, and the fresh spirulina was dried under the conditions of inlet and outlet temperatures of 130 and 75 ^° C, respectively, to obtain dry spirulina. Take spirulina dry biomass (2.5 g) and 100 mL of NaCl solution with a salt concentration of 80 g/L and add them to a 250 mL flask (liquid-solid ratio of 40). Adjust the pH to 9 and extract at 100 rpm and 5 ^° C for 30 h. The extract carrying spirulina cells was not homogenized, but directly diluted 6 times and added to 35 wt% polyethylene glycol 600 and 30 wt% C ₆ H ₅ Na ₃ O ₇ solution. The total volume ratio of the algae liquid dilution to the two-phase aqueous solution was 1:3, and the total volume of the mixed solution was 9 mL. Adjust the pH to 7 and shake at 15 ^° C and 125 rpm for 1 h. After settling at 10 ^° C for 24 h, a large number of algal cells were present in the upper phase. The algal cells were removed by centrifugation at 8000 rpm for 15 min, and the extraction rate and purity of phycocyanin in the upper phase were determined.

Compared with Example 7, in Comparative Experiment 5, fresh spirulina was filtered and concentrated to make the fixed matter content reach 75 g/L, 3% NaCl was added, and the fresh spirulina was dried under the conditions of inlet and outlet temperatures of 130 and 75 ^° C, respectively, to obtain dry spirulina. Spirulina dry biomass (2.5 g) and 100 mL of NaCl solution with a salt concentration of 80 g/L were added to a 250 mL flask (liquid-to-solid ratio of 40). The pH was adjusted to 9 and extracted at 100 rpm and 5 ^° C for 30 h. The extract containing Spirulina cells was homogenized at 40,000 rpm for 5 s, diluted 6 times, and added to 35 wt% polyethylene glycol 600 and 30 wt% phosphate solution (refer to Liu et al. Aqueous two-phase countercurrent distribution for the separation of c-phycocyanin and allophycocyanin from Spirulina platensis , 2012, 9 (20) 111-117). The total volume ratio of the algae liquid dilution to the two aqueous phase was 1:3, and the total volume of the mixed solution was 9 mL. The pH was adjusted to 7 and the mixture was shaken at 15 ^° C and 125 rpm for 1 h. After sedimentation at 10 ^° C for 24 h, there were a large number of algae cells in the upper phase. The upper phase was centrifuged at 8000 rpm for 15 min to remove the algae, and the extraction rate and purity of phycocyanin in the upper phase were determined.

The absorbance of the extract was measured using a spectrophotometer. The phycocyanin content and purity were calculated using the following formula:

Where [PC] is the content of phycocyanin in the extract (mg/mL). A is the absorbance measured at the corresponding wavelengths (620 nm, 652 nm, and 280 nm). V is the total volume of the protein solution (mL), m is the mass of the algae powder sample (mg), PC is the phycocyanin content (g/100g), PC _E is the phycocyanin content obtained under different operations (g/100g), PC _T is the total phycocyanin content (g/100g), ER is the phycocyanin extraction rate (%), and EP is the phycocyanin purity (%).

Table 1 Determination of phycocyanin extraction rate and purity

	S1 Salt added to the algae solution before spray drying	S2 Salt Solvent Treatment	S3 Homogenization	Selection of S4 Phase Forming Salt	Extraction rate (%)	purity(%)
Example 1	0.5% NaCl	60 g/L NaCl	20000 rpm homogenization 3 s	15 wt% polyethylene glycol 2000 and 8 wt% C 6 H 5 Na 3 O 7 solution	91.3	524.1
Example 2	2% NaCl	60 g/L KCl	40000 rpm homogenization 15 s	30 wt% polyethylene glycol 800 and 40 wt% C 6 H 5 K 3 O 7 solution	92.2	527.4
Example 3	1% NaCl	40 g/L MgCl	30000 rpm homogenization 5 s	25 wt% polyethylene glycol 1000 and 30 wt% C 6 H 5 K 3 O 7 solution	91.4	515.4
Example 4	3% KCl	80 g/L KCl	10000 rpm homogenization 10 s	35 wt% polyethylene glycol 400 and 10 wt% C 6 H 5 Na 3 O 7 solution	94.1	509.4
Example 5	1% KCl	20 g/L MgCl 2	40000 rpm homogenization 15 s	20 wt% polyethylene glycol 6000 and 20 wt% C 12 H 10 Mg 3 O 14 solution	93.7	498.8
Example 6	5% MgCl 2	20 g/L MgCl 2	20000 rpm homogenization 3 s	40 wt% polyethylene glycol 4000 and 20 wt% C 12 H 10 Mg 3 O 14 solution	93.9	519.6
Example 7	3% NaCl	80 g/L NaCl	40000 rpm homogenization 5 s	35 wt% polyethylene glycol 600 and 30 wt% C 6 H 5 Na 3 O 7 solution	92.9	513.6
Comparative Example 1	none	none	None, centrifuge at 8000 rpm for 15 min to remove algae	35 wt% polyethylene glycol 600 and 30 wt% phosphate solution	69.1	65.6
Comparative Example 2	none	80 g/L NaCl	40000 rpm homogenization 5 s	35 wt% polyethylene glycol 600 and 30 wt% C 6 H 5 Na 3 O 7 solution	79.6	320.1
Comparative Example 3	3% NaCl	none	40000 rpm homogenization 5 s	35 wt% polyethylene glycol 600 and 30 wt% C 6 H 5 Na 3 O 7 solution	72.0	101.2
Comparative Example 4	3% NaCl	80 g/L NaCl	none	35 wt% polyethylene glycol 600 and 30 wt% C 6 H 5 Na 3 O 7 solution, the upper phase was centrifuged at 8000 rpm for 15 min to remove the algae	91.6	167.3
Comparative Example 5	3% NaCl	80 g/L NaCl	40000 rpm homogenization 5 s	35 wt% polyethylene glycol 600 and 30 wt% phosphate solution, the upper phase was centrifuged at 8000 rpm for 15 min to remove the algae	83.8	103.5

Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (9)

1. A method for extracting and purifying phycocyanin from Spirulina platensis, characterized in that it comprises the following steps:

   S1 pre-processing:

   Adding metal chloride salt to the algae liquid before spray drying, and then spray drying;

   S2, adding the spray-dried spirulina into a salt solvent to extract phycocyanin to obtain an extract containing algal cells;

   S3, homogenizing the extract carrying algae cells obtained in S2; the homogenization condition is to homogenize at a speed of 10000-40000 rpm for a time of 3-15 s;

   S4, Aqueous Two-Phase Extraction

   The homogenized extract is diluted and then added into a two-phase aqueous solution composed of a phase-forming salt and polyethylene glycol for extraction. The phase-forming salt is any one of C ₆ H ₅ K ₃ O ₇ , C ₆ H ₅ Na ₃ O ₇ or C ₁₂ H ₁₀ Mg ₃ O ₁₄ . The upper phase is collected to obtain phycocyanin.
2. The method for extracting and purifying phycocyanin from Spirulina platensis as claimed in claim 1, characterized in that the solid content of the algae liquid fed during spray drying in S1 is 50-150 g/L.
3. The method for extracting and purifying phycocyanin from Spirulina platensis as claimed in claim 2, characterized in that the fresh Spirulina is spray-dried under the conditions of an inlet temperature of 120-180°C and an outlet temperature of 60-100°C in S1.
4. The method for extracting and purifying phycocyanin from Spirulina platensis according to any one of claims 1 to 3, characterized in that in S1, 0.5-5wt% of a metal chloride salt is added to the algae liquid before spray drying, and the metal chloride salt is any one of NaCl, KCl or _MgCl2 .
5. The method for extracting and purifying phycocyanin from Spirulina platensis as claimed in claim 4, characterized in that the liquid-to-solid ratio during extraction in S2 is 10-40 v/w, the salt concentration is 20-100 g/L, the pH is 5-10, the extraction temperature is 5-25 ^° C, and the extraction time is 6-30 h.
6. The method for extracting and purifying phycocyanin from Spirulina platensis according to claim 4, characterized in that stirring is required during extraction in S2, and the stirring speed is 50-200 rpm

   7. The method for extracting and purifying phycocyanin from Spirulina platensis according to claim 4, characterized in that the salt solvent in S2 is any one of NaCl, KCl or _MgCl2 .
7. The method for extracting and purifying phycocyanin from Spirulina platensis as claimed in claim 1, characterized in that the rotation speed in S3 is 30000 rpm and the homogenization time is 5-10 s.
8. The method for extracting and purifying phycocyanin from Spirulina platensis as claimed in claim 1, characterized in that the aqueous two-phase extraction is carried out at a temperature of 5-25 ^° C and a pH of 5-9 in S4.
9. The method for extracting and purifying phycocyanin from Spirulina platensis as claimed in claim 1, characterized in that the molecular weight of the polyethylene glycol in S4 is 2000-6000, the concentration is 15-40 wt%; and the salt concentration is 8-40 wt%.