A method for preparing phycocyanobilin and related applications thereof

Abstract

This invention discloses a method for preparing phycocyanin and its related applications, relating to the field of active substance extraction technology. Through a specific sequence of membrane filtration, cell disruption, and chromatography, this invention efficiently and selectively separates and purifies high-purity phycocyanin from the complex composition of *E. coli* fermentation broth. Compared to the cumbersome traditional process relying on algal extraction followed by chemical lysis, this method avoids the seasonality and geographical limitations of raw materials, achieving high recovery rates and highly selective enrichment of phycocyanin. The process flow is clear, the conditions are mild, and it is easy to scale up, demonstrating good industrial scalability potential. This invention not only provides a stable, economical, and efficient technical route for the large-scale preparation of phycocyanin but also lays a solid foundation for its in-depth application in high-value-added fields such as food, pharmaceuticals, and biological detection.

Description

Technical Field

[0001] This invention relates to the field of active substance extraction technology, and more specifically, to a method for preparing phycocyanin and its related applications. Background Technology

[0002] Phycocyanin is a linear tetrapyrrole-structured blue fluorescent pigment with a molecular weight of 586 Da. As the chromophore of phycocyanin, it possesses excellent antioxidant, anti-inflammatory, and fluorescent labeling bioactivities, showing broad application prospects in food, pharmaceuticals, cosmetics, and bioassay. Currently, the production of phycocyanin mainly relies on extracting phycocyanin from algae such as Spirulina and then obtaining it through chemical cleavage. This method is not only cumbersome and costly, but also limited by the seasonality and regionality of algae cultivation, making it difficult to meet the needs of large-scale, stable industrial production.

[0003] In recent years, the technology of producing phycocyanin through fermentation using genetically engineered Escherichia coli has been considered a highly promising alternative due to its advantages such as short cycle time, low cost, ease of process control, and large-scale scaling. However, the E. coli fermentation system is complex, containing a large amount of bacterial cells, host proteins, nucleic acids, and metabolic byproducts, which poses significant technical challenges to the efficient isolation and purification of high-purity phycocyanin molecules from this system. Existing conventional extraction methods often suffer from low recovery rates and insufficient product purity, severely restricting the industrialization of this technology.

[0004] Therefore, developing a process specifically for extracting and purifying phycocyanin from Escherichia coli fermentation broth is crucial for promoting its industrial application.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing phycocyanin and its related applications.

[0007] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a method for preparing phycocyanin, comprising the following steps: obtaining a fermentation broth of Escherichia coli expressing phycocyanin; performing nanofiltration on the fermentation broth; filtering the retentate obtained by nanofiltration; centrifuging the permeate obtained by filtration; collecting the bacterial cell precipitate and performing cell disruption treatment; centrifuging or / or microfiltration on the product after cell disruption treatment; collecting the clarified liquid; and performing chromatographic purification on the clarified liquid and collecting the elution fraction containing phycocyanin.

[0008] Secondly, embodiments of the present invention provide products prepared by the preparation method described in the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide the application of the preparation method as described in the foregoing embodiments or the product as described in the foregoing embodiments in the preparation of fluorescent probes, antioxidants, antitumor adjuvants or colorants.

[0010] The present invention has the following beneficial effects: The preparation process provided by this invention can efficiently and specifically separate and purify high-purity phycocyanin from the complex composition of Escherichia coli fermentation broth. Compared with the cumbersome traditional process that relies on algal extraction followed by chemical lysis, this method avoids the seasonality and geographical limitations of raw materials, achieving high recovery rate and highly selective enrichment of phycocyanin. The process flow is clear, the conditions are mild, and it is easy to scale up, demonstrating good potential for industrial scalability. This invention not only provides a stable, economical, and efficient technical route for the large-scale preparation of phycocyanin, but also lays a solid foundation for its in-depth application in high-value-added fields such as food, medicine, and biological detection. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0012] On one hand, embodiments of the present invention provide a method for preparing phycocyanin, which includes the following steps: Fermentation broth of Escherichia coli expressing phycocyanin was obtained, the fermentation broth was subjected to nanofiltration, and the retentate obtained by nanofiltration was filtered. The permeate obtained from filtration is centrifuged, and the bacterial cell precipitate is collected for cell disruption. The product after the cell wall disruption treatment is centrifuged or / or microfiltered, and the clarified liquid is collected. The clarified solution was purified by chromatography, and the elution fraction containing phycocyanin was collected.

[0013] The technical solutions provided in the embodiments of this application do not have special requirements for the fermentation broth of Escherichia coli expressing phycocyanin; any fermentation broth of Escherichia coli capable of expressing phycocyanin can be used.

[0014] In an optional embodiment, the nanofiltration membrane has a molecular weight cutoff of 300-500 Da, specifically any one or any two of 300, 350, 400, 450 and 500 Da.

[0015] In an optional embodiment, the nanofiltration unit operates at a pressure of 1.0–2.5 MPa and a temperature of 5–15°C. Specifically, the operating pressure can be any one or a range between any two of the following: 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.1, 2.2, 2.3, 2.4, and 2.5 MPa. The temperature can be any one or a range between any two of the following: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15°C.

[0016] In an optional embodiment, the filter membrane has a molecular weight cutoff of 1 to 3 kDa, specifically any one or any two of 1, 1.5, 2, 2.5 and 3 kDa.

[0017] In an optional embodiment, the operating pressure of the filter is 0.5~3.0 MPa, and the temperature is 5~16°C. Specifically, the operating pressure can be any one or a range between any two of 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, and 3 MPa. Specifically, the temperature can be any one or a range between any two of 5, 6, 8, 10, 12, 13, 14, 15, and 16°C.

[0018] In an optional embodiment, the centrifugation speed of the permeate is 8000~13000 rpm, specifically any one or a range between 8000, 9000, 10000, 11000, 12000 and 13000 rpm. The centrifugal force is 10000~16000g, specifically any one or a range between 10000, 11000, 12000, 13000, 14000, 15000 and 16000g.

[0019] In an optional embodiment, the cell wall disruption process is performed by repeated freeze-thaw cycles and / or high-pressure homogenization; In an optional embodiment, the repeated freeze-thaw step includes freezing at -80°C to -75°C and then thawing at 4 to 25°C, repeating the cycle 2 to 4 times.

[0020] In an optional embodiment, the centrifugation speed of the product after cell wall disruption is 8000~13000 rpm, specifically any one or a range between 8000, 9000, 10000, 11000, 12000 and 13000 rpm. The centrifugal force is 10000~16000g, specifically any one or a range between 10000, 11000, 12000, 13000, 14000, 15000 and 16000g.

[0021] In an optional embodiment, the pressure of the high-pressure homogenization is 600~1000 Bar, specifically any one or any two of 600, 700, 800, 900 and 1000 Bar, and the cycle is repeated 1 to 3 times.

[0022] In an optional embodiment, the pore size of the microfiltration membrane is 0.01~1μm, specifically any one or any two of 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1μm.

[0023] In an optional embodiment, the chromatographic purification uses a reverse-phase chromatography column.

[0024] In an optional embodiment, the reversed-phase chromatography column is packed with a reversed-phase C18 column with a particle size of 1–10 μm, a pore size of 100–150 Å, and a column size of (3–5) mm × (40–60) mm. Specifically, the particle size can be any one or any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μm. The pore size can be any one or any combination of 100, 110, 120, 130, 140, and 150 Å. The column size can be (3–4) mm × (45–55) mm.

[0025] In an optional embodiment, the elution mode of the reversed-phase chromatography column is gradient elution, with mobile phase A being an aqueous phase and mobile phase B being an organic phase, wherein the organic phase includes any one of methanol, acetonitrile, and ethanol.

[0026] In an optional embodiment, the elution procedure of the reversed-phase chromatography column is as follows: From 0 to 10 minutes, the volume percentage of mobile phase B increased from 15% to 25% to 45% to 55%. Over 10–20 minutes, the volume percentage of mobile phase B increased from 45%–55% to 90%–99%. For 20-25 minutes, the volume percentage of mobile phase B is maintained at 90%~99%.

[0027] In an optional embodiment, the elution procedure of the reversed-phase chromatography column is as follows: From 0 to 10 minutes, the volume percentage of mobile phase B increased from 18% to 22% to 48% to 52%. In 10–20 minutes, the volume percentage of mobile phase B increased from 48%–52% to 93%–97%. For 20-25 minutes, the volume percentage of mobile phase B was maintained at 93%~97%.

[0028] In an optional embodiment, the elution procedure of the reversed-phase chromatography column is as follows: From 0 to 15 minutes, the volume percentage of mobile phase B increased from 20% to 30% to 55% to 65%. Within 15–25 minutes, the volume percentage of mobile phase B increased from 55%–65% to 90%–100%.

[0029] In an optional embodiment, the elution procedure of the reversed-phase chromatography column is as follows: From 0 to 15 minutes, the volume percentage of mobile phase B increased from 24% to 26% to 58% to 62%. Within 15–25 minutes, the volume percentage of mobile phase B increased from 58%–62% to 98%–100%.

[0030] In an optional embodiment, the chromatographic purification is performed using an anion exchange chromatography column.

[0031] In an optional embodiment, the packing material of the anion exchange chromatography column is ethylene-divinylbenzene copolymer, and the column size is (3-5) mm × (40-60) mm; the column size can be (3-5) mm × (45-55) mm; the pore size is 50-200 nanometers, specifically any one or any two of 50, 60, 80, 120, 140, 160, 180, and 200 nanometers.

[0032] In an optional embodiment, the chromatographic purification step includes: adjusting the pH of the clarified solution to 7.5-8.5, loading the solution onto the ion exchange chromatography column, eluting with elution buffer, and collecting the elution fraction containing phycocyanin. In an optional embodiment, the eluent is a Tris-HCl buffer containing 0-1M NaCl, with a pH of 7.5-8.5. The concentration of NaCl can be any one or a range between 0, 0.2, 0.4, 0.6, 0.8, and 1M. Specifically, the pH can be any one or a range between 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, and 8.5.

[0033] In an optional embodiment, the preparation method further includes: concentrating the elution fraction containing phycocyanin to crystallize the phycocyanin, and then filtering and / or drying it.

[0034] In optional embodiments, the concentration method includes vacuum concentration and / or freeze-drying concentration.

[0035] On the other hand, embodiments of the present invention provide products prepared by the preparation method described in any of the foregoing embodiments.

[0036] Furthermore, embodiments of the present invention provide the application of the preparation method as described in any of the foregoing embodiments or the product as described in any of the foregoing embodiments in the preparation of fluorescent probes, antioxidants, antitumor adjuvants or colorants.

[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0038] The *E. coli* fermentation broth used in this example is derived from a genetically engineered *E. coli* strain suitable for industrial production. This *E. coli* strain can synthesize PCBs. It is based on BL21(DE3) as the starting strain and uses pRSFDuet-1 as the expression vector to express the heme oxygenase gene ho1 and the ferroreductase gene pcyA derived from Synechocystis sp. PCC6803 via the fusion of short peptide tags RIDD and RIAD. It also integrates and expresses the hemBCD and hemEFGH genes. The nucleotide sequences of hemB in the hemBCD gene are shown in GeneID: 945017, hemC in GeneID: 947759, and hemD in GeneID: 948587. Similarly, the nucleotide sequences of hemE in the hemEFGH gene are shown in GeneID: 948497, hemF in GeneID: 946908, hemG in GeneID: 948331, and hemH in GeneID: 947532. The fermentation broth of this *E. coli* was prepared in strictly aseptic, controlled stirred fermenters (typically 5 L and 50 L). Fermentation began with the inoculation of the seed culture into the fermenter containing a carbon source, nitrogen source, inorganic salts, selective antibiotics, and an inducer.

[0039] Example 1 A method for preparing phycocyanin includes the following steps.

[0040] (1) Membrane concentration and primary filtration: Nanofiltration: Take 10L of E. coli fermentation broth (biosynthesized phycocyanin), and use an organic separation membrane experimental machine (the machine is available on the market). First, concentrate it to 2L (5 times concentration) using a 300Da membrane column at a pressure of 1.1MPa and a membrane column temperature of 11.0℃, and collect the retentate (concentrated liquid).

[0041] Filtration: The filter is then used with a 1kDa membrane column at a pressure of 1.1 MPa and a membrane column temperature of 12℃ to remove macromolecular impurities and collect the filtrate (permeate).

[0042] (2) Collection and cell disruption of bacteria Centrifugation: Centrifuge the above filtrate at 8000 rpm (13100 g) for 10 minutes using a Xiangli high-speed refrigerated centrifuge and collect the bacterial precipitate.

[0043] Cell wall disruption treatment: Place the bacterial precipitate in a -80℃ low temperature freezer, freeze it overnight at -78℃, and then thaw it at room temperature (25℃). This freeze-thaw process is repeated 3 times.

[0044] (3) Fine filtration Add an appropriate amount of pure water to the mixture after cell wall disruption, centrifuge again (12000 rpm, 29,500 × g, 10 min), collect the supernatant, and filter it through a 0.22 μm microporous membrane to obtain a clear phycocyanin solution (clarified liquid).

[0045] (4) Chromatographic purification The clarified solution was loaded onto a preparative C18 reversed-phase chromatography column (Waters XBridge BEH C18; column type: reversed-phase C18 column; particle size: 3.5 μm; pore size: 130 Å; column dimensions: 4.6 mm × 50 mm; column temperature: 20–40 °C). Gradient elution was used: mobile phase A was ultrapure water, and mobile phase B was methanol. The gradient elution procedure is as follows: 0-10 min, 20% mobile phase B → 50% mobile phase B; 10-20 min, 50% mobile phase B → 95% mobile phase B; 20-25 min, 95% mobile phase B.

[0046] The flow rate was 5 mL / min, and the detection wavelengths were 280 nm and 650 nm (characteristic absorption peak of phycocyanin). The blue elution peak with absorption at 650 nm was collected.

[0047] (5) Concentration and crystallization: The collected eluent was concentrated to a smaller volume under reduced pressure at 40°C, and then placed in a refrigerator at 4°C overnight to precipitate blue crystals. The crystals were filtered, washed with a small amount of pre-cooled methanol, and dried under vacuum to obtain pure phycocyanin in the form of a blue powder.

[0048] HPLC analysis showed a purity of 98.5%.

[0049] Example 2 A method for preparing phycocyanin is largely the same as in Example 1, except that in step (1), the operating pressure of the 300 Da membrane concentration is adjusted to 1.5 MPa and the temperature is adjusted to 12°C; the operating pressure of the 1 kDa ultrafiltration is adjusted to 2.5 MPa and the temperature is adjusted to 14°C. The final product purity is approximately 98.2%.

[0050] Example 3 A method for preparing phycocyanin is largely the same as in Example 1, except that in step (2), the centrifugal force is changed to 15000 g; and the freeze-thaw cycle is changed to 2 times (freezing at -80℃ and thawing at 4℃). The final product purity is approximately 97.8%.

[0051] Example 4 A method for preparing phycocyanin is largely the same as in Example 1, except that step (4) uses an anion exchange column (manufacturer and brand: Hamilton; model: PRP-X100; column size: 4.6 mm × 50 mm; packing material: styrene-divinylbenzene copolymer; packing material pore size: porous, pore size of 100 nm; column temperature: 20~35℃). The clarified solution from step (3) is first diluted with Tris-HCl buffer (pH 7.2) to make the phycocyanin negatively charged. After loading the sample, gradient elution is performed using Tris-HCl buffer containing 0.5 M NaCl, and the target peak is collected.

[0052] The final product has a purity of approximately 97.0%.

[0053] Example 5 A method for preparing phycocyanin is largely the same as in Example 1, except that the gradient elution procedure in step (4) is changed to: 0-15 min, 25% mobile phase B → 60% mobile phase B; 15-25 min, 60% mobile phase B → 100% mobile phase B.

[0054] The final product has a purity of approximately 90.0%.

[0055] Example 6 A method for preparing phycocyanin includes the following steps.

[0056] Take 500L of E. coli fermentation broth and scale up the equipment accordingly.

[0057] (1) Industrial-grade nanofiltration system (500 Da) and ultrafiltration system (3 kDa) are used for continuous concentration and primary filtration. Ultrafiltration system: operating temperature: 6℃; feed pressure: 0.5 MPa; cross-flow velocity: 1.4 m / s; Nanofiltration system: operating temperature: 8℃; operating pressure: 2.2 MPa; cross-flow velocity: 1.3 m / s.

[0058] (2) A tubular centrifuge (speed (RPM), 11,000 rpm; relative centrifugal force (RCF) ~16,000 ×g; feed flow rate: adjusted according to the processing volume, in this example it is 60 L / h; single run time: 50 minutes) was used to continuously collect bacterial cells.

[0059] (3) The cell walls were broken by high pressure homogenizer (pressure 800 bar, 2 cycles) instead of freeze-thaw method to improve efficiency.

[0060] (4) The cell wall-breaking liquid is clarified by a ceramic membrane microfiltration system (membrane pore size: 50 nm; membrane material / modification: α-alumina (α-Al2O3) with a hydrophilic coating (such as TiO2); transmembrane pressure (TMP): 0.1 - 0.3 Mpa; cross-flow velocity: 0.5 m / s).

[0061] (5) The clarified liquid was purified by entering an industrial-grade preparative chromatography system (the same column as in Example 1) and the elution conditions were the same as in Example 1.

[0062] (6) The eluent is concentrated by a vacuum thin-film evaporator and then spray-dried to obtain phycocyanin product.

[0063] HPLC analysis showed a purity of 96.5%, making it suitable for use as a food additive and cosmetic ingredient.

[0064] Comparative Example 1 A method for preparing phycocyanin includes the following steps.

[0065] Fermentation broth from the same source as in Example 1 was directly extracted using an organic solvent (such as methanol), and then separated by simple silica gel column chromatography, as detailed below.

[0066] 1. Mix with methanol at a volume ratio of 1:2 and stir for 1 hour under light-protected conditions. Maintain the temperature below 20℃. After completion, separate the supernatant by centrifugation to obtain a crude extract containing phycocyanin and other lipid-soluble components, then freeze-dry the crude extract powder. 2. Dissolve the concentrated crude extract powder in a small amount of methanol, then add approximately 1-2 times its weight of coarse silica gel (80 mesh), mix thoroughly, and evaporate the solvent completely to obtain a loose powder. Add this powder evenly to the top of the column bed. 3. Start with the low-polarity solvent petroleum ether and gradually increase the proportion of methanol. Specifically, start with a petroleum ether:methanol ratio of 9:1 (volume ratio) and gradually transition to 7:3.

[0067] Results and drawbacks: Although the method is simple, impurities are not completely removed. HPLC analysis shows that the product purity is only 65%, and it is accompanied by obvious discoloration and odor.

[0068] Comparative Example 2 Preparation method: Fermentation broth from the same source as in Example 1 was directly extracted with methanol. The extract was concentrated to dryness by rotary evaporation to obtain a crude extract. No column chromatography purification steps were performed.

[0069] Effects and limitations: This method is the simplest and fastest. However, the resulting product is a dark paste with complex components. HPLC shows that the purity of phycocyanin is less than 10%, containing a large amount of impurities such as chlorophyll, lipids, and cell debris, which cannot meet the requirements for subsequent high-purity applications.

[0070] Comparative Example 3 Preparation method: Fermentation broth from the same source as in Example 1 was used. First, the cell walls were disrupted using a repeated freeze-thaw cycle, followed by extraction with methanol. After concentration, the extract was separated using simple silica gel column chromatography, as in Comparative Example 1.

[0071] Results and limitations: Freeze-thaw cell disruption improved the leaching rate of phycocyanin, resulting in a slight increase in the overall product yield compared to Comparative Example 1 (approximately 5%). However, due to the lack of enhancement in the purification steps, the co-eluting of pigments and lipid-soluble impurities remained severe, and the HPLC purity only slightly improved to 70%. Discoloration and off-odor issues persisted.

[0072] Comparative Example 4 Preparation method: Fermentation broth from the same source as in Example 1 was extracted with methanol. After concentration, the extract was eluted using a fine silica gel column gradient (methanol-water fractional elution at volume ratios of 100:0, 80:20, 60:40, and 50:50), and the target fractions were collected in real time using thin-layer chromatography (TLC) under monitoring.

[0073] Results and limitations: This method achieves significant purification results, with HPLC showing a product purity of up to 75%, and marked improvements in appearance and odor. However, the process is extremely lengthy, consumes a large amount of elution solvent, and has a narrow target fraction collection window, resulting in complex operation, low overall yield (<50%), and high production costs, making it unsuitable for large-scale preparation.

[0074] Comparative Example 5 Preparation method: Fermentation broth from the same source as in Example 1 was extracted with methanol. Instead of silica gel column chromatography, purification was attempted using a reversed-phase C18 solid-phase extraction column (packing type: high-purity silica-based C18; particle size: 40-60 μm; pore size: 100 Å; column size: diameter 50 mm, length 150 mm). Impurities were removed by washing with water and then with a low-concentration methanol aqueous solution. Finally, the target product was eluted with high-concentration methanol.

[0075] Advantages and disadvantages: This method is relatively simple and fast. Product purity can reach 88%, and the elution solvent may cause denaturation of some products. The recovery rate is stable. However, the reversed-phase C18 solid-phase extraction column is somewhat expensive.

[0076] In summary, the technical solution provided by this invention can efficiently and specifically purify high-purity phycocyanin from Escherichia coli fermentation broth, significantly outperforming traditional methods. The parameter adjustments in each embodiment are all within the protection range and all achieve ideal results, demonstrating the robustness and scalability of this invention.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing phycocyanin, characterized in that, It includes the following steps: Fermentation broth of Escherichia coli expressing phycocyanin was obtained, the fermentation broth was subjected to nanofiltration, and the retentate obtained by nanofiltration was filtered. The permeate obtained from filtration is centrifuged, and the bacterial cell precipitate is collected for cell disruption. The product after the cell wall disruption treatment is centrifuged or / or microfiltered, and the clarified liquid is collected. The clarified solution was purified by chromatography, and the elution fraction containing phycocyanin was collected.

2. The preparation method according to claim 1, characterized in that, The chromatographic purification was performed using a reverse-phase chromatography column; And / or, the reversed-phase chromatography column is a reversed-phase C18 chromatographic column with a particle size of 1-10 μm, a pore size of 100-150 Å, and a column size of (3-5) mm × (40-60) mm; And / or, the elution mode of the reversed-phase chromatography column is gradient elution, with mobile phase A being an aqueous phase and mobile phase B being an organic phase, wherein the organic phase includes any one of methanol, acetonitrile, and ethanol.

3. The preparation method according to claim 2, characterized in that, The elution program of the reversed-phase chromatography column is (1) or (2): (1) From 0 to 10 min, the volume percentage of mobile phase B increased from 15% to 25% to 45% to 55%; Over 10–20 minutes, the volume percentage of mobile phase B increased from 45%–55% to 90%–99%. For 20-25 minutes, the volume percentage of mobile phase B is maintained at 90%~99%; (2) From 0 to 15 min, the volume percentage of mobile phase B increased from 20% to 30% to 55% to 65%; Within 15–25 minutes, the volume percentage of mobile phase B increased from 55%–65% to 90%–100%.

4. The preparation method according to claim 1, characterized in that, The chromatographic purification was performed using an anion exchange chromatography column; And / or, the packing material of the anion exchange chromatography column is styrene-divinylbenzene copolymer with a pore size of 50~200 nanometers and a column size of (3~5) mm × (40~60) mm; And / or, the chromatographic purification step includes: adjusting the pH of the clarified solution to 7.5-8.5, loading the solution onto the ion exchange chromatography column, eluting with elution buffer, and collecting the elution fraction containing phycocyanin; And / or, the elution buffer is: Tris-HCl buffer containing 0~1M NaCl, with a pH of 7.5~8.

5.

5. The preparation method according to claim 1, characterized in that, The nanofiltration membrane has a molecular weight cutoff of 300-500 Da; and / or the nanofiltration operates at a pressure of 1.0-2.5 MPa and a temperature of 5-15°C.

6. The preparation method according to claim 1, characterized in that, The filter membrane used for filtration has a molecular weight cutoff of 1~3kDa; And / or, the operating pressure of the filter is 0.5~3.0 MPa, and the temperature is 5~16℃.

7. The preparation method according to claim 1, characterized in that, The centrifugation speed of the permeate is 8000~13000 rpm, and the centrifugal force is 10000~16000 g; And / or, the cell wall breaking process is performed by repeated freeze-thaw cycles and / or high-pressure homogenization; And / or, the repeated freeze-thaw steps include: freezing at -80°C to -75°C and then thawing at 4 to 25°C, repeating 2 to 4 times.

8. The preparation method according to claim 1, characterized in that, The pore size of the microfiltration membrane is 0.01~1μm; And / or, the preparation method further includes: concentrating the elution fraction containing phycocyanin to crystallize the phycocyanin, and filtering and / or drying it; And / or, the concentration methods include vacuum concentration and / or freeze-drying concentration.

9. The product prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the preparation method according to any one of claims 1 to 8 or the product according to claim 9 in the preparation of fluorescent probes, antioxidants, antitumor adjuvants or colorants.