阅读说明：本技术 一种高效分离纯化海洋微藻藻胆蛋白的方法及藻胆蛋白 (Method for efficiently separating and purifying marine microalgae phycobiliprotein and phycobiliprotein ) 是由 郑伊奕 范建华 季亮 钱宇慧 凌睿婧 马娆 王启要 于 2021-04-16 设计创作，主要内容包括：本发明提供了一种高效分离纯化海洋微藻藻胆蛋白的方法,其特征在于,包括：取新鲜海洋微藻藻液于离心管,将藻液稀释至5×10～6-5×10～7cells/mL；二次离心,去上清,获得藻体；将藻体重悬于PEG/盐双水相体系中,获得双水相藻体重悬液；然后经反复冻融,获得微藻细胞破碎液；接着,待双水相体系分配平衡后取上相,即为初步纯化的藻胆蛋白提取液；接着,依次经羟基磷灰石柱纯化和银离子交换柱纯化获得藻胆蛋白纯化液；接着,进行透析除盐,冷冻干燥获得微藻藻胆蛋白成品。本发明采用冻融法破碎双水相藻体重悬液,可一步实现藻体的破碎与藻胆蛋白的初步纯化,可简化分离纯化步骤,有效提高回收率。(The invention provides a method for efficiently separating and purifying phycobiliprotein of marine microalgae, which is characterized by comprising the following steps: taking fresh marine microalgae solution in a centrifuge tube, and diluting the solution to 5 × 10 6 ‑5×10 7 cells/mL; centrifuging for the second time, and removing the supernatant to obtain algae; resuspending the algae in a PEG/salt aqueous two-phase system to obtain aqueous two-phase algae suspension; then repeatedly freezing and thawing to obtain microalgae cell crushing liquid; then, after the two aqueous phase system is distributed and balanced, taking the upper phase, namely the primarily purified phycobiliprotein extracting solution; then purifying by hydroxyapatite columnPurifying with silver ion exchange column to obtain purified phycobiliprotein solution; and then dialyzing to remove salt, and freeze-drying to obtain a microalgae phycobiliprotein finished product. The invention adopts a freeze-thawing method to crush the aqueous two-phase algae heavy suspension, can realize the crushing of algae and the primary purification of phycobiliprotein by one step, can simplify the separation and purification steps and effectively improve the recovery rate.)

1. A method for efficiently separating and purifying marine microalgae phycobiliprotein is characterized by comprising the following steps:

step one, taking fresh marine microalgae solution into a centrifuge tube, and diluting the algae solution to 5 multiplied by 10⁶-5×10⁷cells/mL; centrifuging for the second time, and removing the supernatant to obtain algae; resuspending the algae in a PEG/salt aqueous two-phase system to obtain aqueous two-phase algae suspension;

step two, repeatedly freezing and thawing the aqueous two-phase algae heavy suspension in the step one to obtain microalgae cell crushing liquid, and centrifuging for later use;

step three, taking the upper phase after the distribution of the aqueous two-phase system in the step two is balanced, namely obtaining a primarily purified phycobiliprotein extracting solution;

and step four, purifying, dialyzing and desalting the phycobiliprotein extracting solution obtained in the step three to obtain microalgae phycobiliprotein.

2. The method for efficiently separating and purifying phycobiliproteins from marine microalgae according to claim 1, wherein the fourth step further comprises the following steps:

A. purifying the phycobiliprotein extracting solution in the third step by adopting a hydroxyapatite column to obtain a phycobiliprotein purifying solution 1;

B. further purifying the phycobiliprotein purification solution 1 in the step A by adopting an anion exchange column to obtain a phycobiliprotein purification solution 2;

C. and D, dialyzing the phycobiliprotein solution 2 in the step B for desalting, and freeze-drying to obtain a microalgae phycobiliprotein finished product.

3. The method for separating and purifying phycobiliproteins of marine microalgae according to claim 1 or 2, wherein the marine microalgae includes but is not limited to spirulina, rhodophyta, cryptophyta.

4. The method for separating and purifying phycobiliprotein from marine microalgae according to claim 1 or 2, wherein the PEG/salt aqueous two-phase system in the first step is a PEG/potassium sodium tartrate system with the same volume, the length of the system line is 15-35%, the volume ratio of the algae to the PEG/salt aqueous two-phase system is 0.2-0.4, and the pH value is 6.8-8.0.

5. The method for efficiently separating and purifying the phycobiliprotein from marine microalgae according to claim 1 or 2, wherein the number of times of freeze thawing in the second step is preferably 5-10, the freezing and thawing operation steps are preferably freezing and icing at-80 to-20 ℃ and complete thawing at 0-4 ℃.

6. The method for efficiently separating and purifying phycobiliproteins from marine microalgae according to claim 1 or 2, wherein the aqueous two-phase system distribution balancing step in the third step is: and standing the microalgae cell disruption solution in the step two for 1-5 hours at 3-5 ℃ until obvious layering occurs.

7. The method for efficiently separating and purifying phycobiliprotein from marine microalgae according to claim 2, wherein the step of purifying with hydroxyapatite column in step A comprises: balancing a hydroxyapatite chromatography column by using 0.01M, pH phosphate buffer solution with the volume of 7.0 to 3 to 5 times of the column volume, after balancing, loading the primarily purified phycobiliprotein extracting solution to the well balanced hydroxyapatite column, wherein the loading amount is 1/3 to 2/3 of the column bed volume, after loading is finished, performing gradient elution by using the phosphate buffer solution which contains 0.1 to 0.2M of sodium chloride and has the pH of 6.5 to 7.2, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH is 7.0, and the elution linear velocity is 200-300 cm/h.

8. The method for efficiently separating and purifying phycobiliprotein from marine microalgae according to claim 2, wherein the step of further purifying by anion exchange column in the step B comprises: preparing Tris-HCl Buffer solution Buffer A and Tris-HCl-NaCl Buffer solution Buffer B, and filtering; balancing the ion exchange column by using Buffer A Buffer solution; directly loading the phycobiliprotein purification solution 1, performing gradient elution by using Buffer B, and collecting eluent; the ion exchange column was washed with saline solution and then back-flushed with alkaline solution.

9. The method for efficiently separating and purifying phycobiliproteins from marine microalgae according to claim 2, wherein the specific dialysis step in step C is: placing in a dialysis bag with cut-off of 100-300KDa, and dialyzing with 0.01-0.02M, pH phosphate buffer solution of 6.0-7.5 as dialysate for 12-15 h.

10. A phycobiliprotein, which is prepared by the method for efficiently separating and purifying the phycobiliprotein of marine microalgae according to any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of biochemical separation, and particularly relates to a method for efficiently separating and purifying phycobiliprotein of marine microalgae and phycobiliprotein.

Background

Microalgae generally refers to a general term for microorganisms that contain chlorophyll a and are capable of photosynthesis, and belongs to a group of protists. The phycobilisome is a main light-capturing antenna compound in marine microalgae such as spirulina, red algae, cryptophyceae and the like, and is composed of phycobiliprotein and connexin, wherein the phycobiliprotein is formed by connecting phycobilin chromogen and apoprotein through covalent bonds. The different phycobiliproteins have basically similar structures, and each of the different phycobiliproteins comprises two polypeptide chains with similar structures, namely alpha subunit and beta subunit, and the molecular weight of the polypeptide chains is about 3 ten thousand daltons, and each subunit is combined with one molecule of phycocyanin. Phycobiliproteins can be classified into Phycocyanin (PC), Allophycocyanin (APC), Phycoerythrin (PE), and Phycoerythrin (PEC) according to absorption spectrum properties. Wherein the purity of phycocyanin, phycoerythrin and allophycocyanin is higher, the selling price is higher, and the purity is calculated by A₆₂₀/A₂₈₀、A₅₆₅/A₂₈₀And A₆₅₀/A₂₈₀Characterized by being graded as follows according to different purities: food grade>0.7, pharmaceutical grade>3.0 and reagent grade>4.0. Phycobiliprotein has the functions of oxidation resistance, inflammation resistance, aging resistance, cancer resistance, immunofluorescence and the like, is widely used as natural pigments (food, cosmetics, dyes and the like), medical and health products and fluorescent reagents in molecular biology research, and has huge potential market demands.

For the extraction and separation of intracellular soluble proteins such as phycobiliprotein, it is necessary to break down algal cells, release the proteins in a dissolved state while maintaining the activity of the proteins, and then separate and purify the proteins. In the crude extract of phycobiliprotein, the content of hetero-protein is very high, and for separating to obtain the commercial application standard phycobiliprotein, the hetero-protein is usually firstly removed by such as rivanol precipitation, salting out method, isoelectric point method or crystallization method, and then purified by column chromatography, including light-based limestone adsorption chromatography, cellulose series ion exchange chromatography, affinity chromatography, molecular anion exclusion chromatography and the like. The phycobiliprotein purification method has the advantages of less disposable capacity and easy pollution, low purity obtained by single-column purification, complex column recovery operation and low efficiency, and thus, the production cost is too high to realize large-scale industrial preparation.

In the aspect of phycobiliprotein separation and purification, the research of related technologies in China is late, the research foundation is weak, the problems of complex separation and purification process, high cost and low yield exist, the development and utilization force and depth are small at present, and most of the phycobiliprotein is only simply eaten. The complicated separation and purification process limits the application of phycobiliprotein. Therefore, it is necessary to develop a simple and efficient method suitable for large-scale preparation, separation and purification of phycobiliproteins, and provide a technical basis for industrial production of phycobiliproteins from marine microalgae.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides a method for efficiently separating and purifying marine microalgae phycobiliprotein, which is simple to operate, high in yield and suitable for large-scale production. The second purpose of the invention is to provide a phycobiliprotein.

In order to achieve the purpose, the invention adopts the following technical scheme:

as a first aspect of the invention, a method for efficiently separating and purifying phycobiliprotein of marine microalgae comprises the following steps:

step one, taking fresh marine microalgae solution into a centrifuge tube, and diluting the algae solution to 5 multiplied by 10⁶-5×10⁷cells/mL; centrifuging twice, removing supernatant to obtainAlgae bodies; resuspending the algae in a PEG/salt aqueous two-phase system to obtain aqueous two-phase algae suspension;

step two, repeatedly freezing and thawing the aqueous two-phase algae heavy suspension in the step one to obtain microalgae cell crushing liquid, and centrifuging for later use;

step three, taking the upper phase after the distribution of the aqueous two-phase system in the step two is balanced, namely obtaining a primarily purified phycobiliprotein extracting solution;

and step four, purifying, dialyzing and desalting the phycobiliprotein extracting solution obtained in the step three to obtain microalgae phycobiliprotein.

According to the invention, the method for efficiently separating and purifying the phycobiliprotein of the marine microalgae further comprises the following steps:

step A, purifying the phycobiliprotein extracting solution in the step III by adopting a hydroxyapatite column to obtain a phycobiliprotein purified solution 1;

step B, further purifying the phycobiliprotein purified liquid 1 in the step A by adopting an anion exchange column to obtain a phycobiliprotein purified liquid 2;

and C, dialyzing the phycobiliprotein solution 2 in the step B for desalting, and freeze-drying to obtain a microalgae phycobiliprotein finished product.

According to the present invention, marine microalgae include, but are not limited to, spirulina, rhodophyta, cryptophyta.

According to the invention, the centrifugation speed in the first step is 4000-10000rpm, and the centrifugation time is 5-10 min.

According to the invention, the PEG/salt aqueous two-phase system in the first step is a PEG/potassium sodium tartrate system with the same volume, the length of the system line is 15-35%, the volume ratio of the algae to the PEG/salt aqueous two-phase system is 0.2-0.4, and the pH value is 6.8-8.0.

Preferably, the length of the system line of the PEG/potassium sodium tartrate system is 20%, the volume ratio of the algae to the PEG/salt aqueous two-phase system is 0.3, and the pH value is 7.2.

According to the invention, the number of times of freezing and thawing in the second step is preferably 5-10, the operation steps of freezing and thawing are preferably-80 to-20 ℃, and the thawing is complete at 0-4 ℃.

Preferably, the number of times of freezing and thawing in the second step is 6-8, the operation steps of freezing and thawing are preferably-80 to-20 ℃, and the thawing is complete at 4 ℃.

According to the invention, the distribution balance step of the double water phase system in the third step is as follows: standing the microalgae cell disruption solution at 3-5 deg.C for 1-5h until obvious layering occurs.

Further, the microalgae cell disruption solution is kept stand for 3 hours at 4 ℃.

According to the invention, step A, the phycobiliprotein extracting solution in step III is purified by adopting a hydroxyapatite column, and gradient elution is carried out to obtain phycobiliprotein purified solution 1.

Further, the step A of purifying by using a hydroxyapatite column comprises the following steps: balancing a hydroxyapatite chromatography column by using 0.01M, pH phosphate buffer solution with the volume of 7.0 to 3 to 5 times of the column volume, after balancing, loading the primarily purified phycobiliprotein extracting solution to the well balanced hydroxyapatite column, wherein the loading amount is 1/3 to 2/3 of the column bed volume, after loading is finished, performing gradient elution by using the phosphate buffer solution which contains 0.1 to 0.2M of sodium chloride and has the pH of 6.5 to 7.2, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH is 7.0, and the elution linear velocity is 200-300 cm/h.

According to the invention, the anion exchange column is an Agarosix FF-DEAE anion exchange column.

According to the invention, the step of further purifying by using an anion exchange column in the step B is as follows: preparing Tris-HCl Buffer solution Buffer A and Tris-HCl-NaCl Buffer solution Buffer B, and filtering; balancing the ion exchange column by using Buffer A Buffer solution; directly loading the phycobiliprotein purification solution 1, performing gradient elution by using Buffer B, and collecting eluent; washing the ion exchange column with a saline solution, and then back-flushing with an alkaline solution; the pH value of the Tris-HCl buffer solution is 6-6.8, and the conductivity is 4-8 mu s/cm; the pH value of the Tris-HCl-NaCl buffer solution is 5-6.8, and the conductivity is 4-8.5 mu s/cm.

According to the invention, the specific dialysis steps of step C are: placing in a dialysis bag with cut-off of 100-300KDa, and dialyzing with 0.01-0.02M, pH phosphate buffer solution of 6.0-7.5 as dialysate for 12-15 h.

As a second aspect of the invention, the phycobiliprotein is prepared by the method for efficiently separating and purifying the phycobiliprotein of the marine microalgae.

The method for efficiently separating and purifying the phycobiliprotein of the marine microalgae has the beneficial effects that:

(1) the traditional extraction process usually adopts microalgae dry powder, but the invention directly takes fresh microalgae mud as raw material, thereby greatly reducing high energy consumption and high cost caused by the drying process, and further reducing the production cost on the basis of green environmental protection.

(2) Different from the traditional method for separating and purifying phycobiliprotein by crushing firstly and then purifying the phycobiliprotein firstly, the invention adopts a freeze-thaw method to crush the aqueous two-phase phycobiliprotein heavy suspension, can realize the crushing of phycosomes and the primary purification of phycobiliprotein in one step, can simplify the separation and purification steps, and effectively improves the recovery rate.

(3) The freeze thawing-aqueous two-phase one-step primary purification method adopted by the invention has the advantages of simple extraction process and low cost, and is suitable for large-scale industrial production of phycobiliprotein.

(4) The method for separating and purifying the phycobiliprotein of the marine microalgae adopted by the invention has low operation temperature and can effectively ensure that the biological activity of the phycobiliprotein is not damaged.

Detailed Description

The present invention will be further illustrated by reference to the following specific examples. The specific techniques or conditions not mentioned in the examples are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The reagent or the apparatus is not indicated to the manufacturer, and is a conventional product available from a normal distributor.

The absorbances of phycocyanin, allophycocyanin and phycoerythrin in the following examples were measured by UV-visible spectrophotometer, and the phycocyanin purity was calculated according to the formula P₁＝A₆₂₀/A₂₈₀The purity of allophycocyanin is calculated according to the formula P2 ═ A₆₅₀/A₂₈₀The purity of phycoerythrin is calculated according to the formula P3 ═ A₅₆₅/A₂₈₀The calculation of phycocyanin concentration is based on the formula [ PC]＝(A₆₂₀-0.7×A₆₅₀) /7.38, allophycocyanin concentration calculationAccording to the formula [ APC ]]＝(A₆₅₀-0.19×A₆₂₀) 5.65 calculation of phycoerythrin concentration according to the formula [ PE]＝(A₅₆₅-2.8×[PC]-1.34×[APC]) /12.7, wherein A₂₈₀、A₅₆₅、A₆₂₀、A₆₅₀Absorbance at wavelengths 280, 565, 620, 650nm, respectively.

The anion exchange column used in the following examples was an Agarosix FF-DEAE anion exchange column. The medium of the anion exchange column is composed of agarose gel with the particle size of 50-150 mu m and the crosslinking degree of 6%; preferably, the particle size is 90 μm; the agarose gel relies on secondary chains such as hydrogen bonds between sugar chains to maintain a network structure, and the density of the network structure depends on the concentration of agarose. In general, agarose gels are structurally stable and can be used under a variety of conditions (e.g., water, saline solutions with a pH in the range of 4-9); the agarose gel begins to melt above 40 deg.C, cannot be autoclaved, and can be treated by chemical sterilization.

Example 1

(1) Taking fresh spirulina liquid into a centrifuge tube, diluting the spirulina liquid with water to 5 × 10⁶cells/mL; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10min to obtain algae; suspending the algae in an isometric PEG/potassium sodium tartrate system with a line length of 20%, a volume ratio of 0.3 and a pH value of 7.2 to obtain a double-aqueous-phase algae suspension;

(2) repeatedly freezing and thawing the aqueous two-phase algae suspension obtained in the step (1) for 6 times at-20 ℃, completely thawing at 4 ℃ to obtain microalgae cell disruption solution, and centrifuging at 10000rpm for 10min for later use;

(3) standing the microalgae cell disruption solution at 4 ℃ for 3h, and taking an upper phase after the double aqueous phase system is obviously layered, namely obtaining a primarily purified phycobiliprotein extracting solution;

(4) balancing a hydroxyapatite chromatography column by using phosphate buffer solution with the volume of 0.01M, pH being 7.0 times and 4 times of the column volume, after balancing, loading the primarily purified phycobiliprotein extracting solution to the well-balanced hydroxyapatite column, wherein the loading amount is 2/3 times of the volume of a column bed, after loading, performing gradient elution by using phosphate buffer solution with the pH value of 7.2 and containing 0.2M sodium chloride, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH value is 7.0, and the elution linear velocity is 300cm/h, thus obtaining phycobiliprotein purified solution 1;

(5) preparing a Tris-HCl Buffer solution Buffer A with the pH value of 6.8 and the conductivity of 6 mu s/cm and a Tris-HCl-NaCl Buffer solution Buffer B with the pH value of 6.8 and the conductivity of 8.5 mu s/cm, and filtering; washing the pump with 1 column volume of deionized water, and balancing the ion exchange column with 5 column volumes of Buffer A Buffer solution; directly loading phycobiliprotein purified liquid 1 with 2.5 column volumes, performing Buffer B gradient elution, and collecting 45-100% Tris-HCl-NaCl eluent with pH of 6.0 and conductivity of 4 mus/cm to obtain phycobiliprotein purified liquid 2;

(6) placing in dialysis bag with cut-off of 100KDa, dialyzing with 0.01M, pH phosphate buffer solution of 6.5 as dialysate for 12h, and centrifuging the desalted solution at 8000rpm for 30min to obtain phycobiliprotein solution.

The experimental data for example 1 are shown in table 1.

Table 1 experimental data for example 1

Example 2

(1) Taking fresh spirulina liquid into a centrifuge tube, diluting the spirulina liquid with water to 1 × 10⁷cells/mL; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10 min; suspending the algae in an isometric PEG/potassium sodium tartrate system with a line length of 20%, a volume ratio of 0.3 and a pH value of 7.2 to obtain a double-aqueous-phase algae suspension;

(2) repeatedly freezing and thawing the aqueous two-phase algae suspension obtained in the step (1) for 8 times at-80 ℃, completely thawing at 4 ℃ to obtain microalgae cell disruption solution, and centrifuging at 10000rpm for 10min for later use;

(3) standing the microalgae cell disruption solution at 4 ℃ for 3h, and taking an upper phase after the double aqueous phase system is obviously layered, namely obtaining a primarily purified phycobiliprotein extracting solution;

(4) balancing a hydroxyapatite chromatography column by using phosphate buffer solution with the volume of 0.01M, pH being 7.0 times and 4 times of the column volume, after balancing, loading the primarily purified phycobiliprotein extracting solution to the well-balanced hydroxyapatite column, wherein the loading amount is 2/3 times of the volume of a column bed, after loading, performing gradient elution by using phosphate buffer solution with the pH value of 7.2 and containing 0.2M sodium chloride, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH value is 7.0, and the elution linear velocity is 300cm/h, thus obtaining phycobiliprotein purified solution 1;

(5) preparing a Tris-HCl Buffer solution Buffer A with the pH value of 6.8 and the conductivity of 6 mu s/cm and a Tris-HCl-NaCl Buffer solution Buffer B with the pH value of 6.8 and the conductivity of 8.5 mu s/cm, and filtering; washing the pump with 1 column volume of deionized water, and balancing the ion exchange column with 5 column volumes of Buffer A Buffer solution; directly loading phycobiliprotein purified liquid 1 with 2.5 column volumes, performing Buffer B gradient elution, and collecting 45-100% Tris-HCl-NaCl eluent with pH of 6.0 and conductivity of 4 mus/cm to obtain phycobiliprotein purified liquid 2;

(6) placing in dialysis bag with cut-off of 100KDa, dialyzing with 0.01M, pH phosphate buffer solution of 6.5 as dialysate for 12h, and centrifuging the desalted solution at 8000rpm for 30min to obtain phycobiliprotein solution.

The experimental data for example 2 are shown in table 2.

Table 2 experimental data for example 2

Example 3

(1) Taking fresh porphyridium algae liquid into a centrifuge tube, diluting the algae liquid with water according to a proportion to 5 multiplied by 10⁶cells/mL; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10 min; suspending the algae in an isometric PEG/potassium sodium tartrate system with a line length of 20%, a volume ratio of 0.3 and a pH value of 7.2 to obtain a double-aqueous-phase algae suspension;

(2) repeatedly freezing and thawing the aqueous two-phase algae suspension obtained in the step (1) for 6 times at-20 ℃, completely thawing at 4 ℃ to obtain microalgae cell disruption solution, and centrifuging at 10000rpm for 10min for later use;

(3) standing the microalgae cell disruption solution at 4 ℃ for 3h, and taking an upper phase after the double aqueous phase system is obviously layered, namely obtaining a primarily purified phycobiliprotein extracting solution;

(4) balancing a hydroxyapatite chromatography column by using phosphate buffer solution with the volume of 0.01M, pH being 7.0 times and 4 times of the column volume, after balancing, loading the primarily purified phycobiliprotein extracting solution to the well-balanced hydroxyapatite column, wherein the loading amount is 2/3 times of the volume of a column bed, after loading, performing gradient elution by using phosphate buffer solution with the pH value of 7.2 and containing 0.2M sodium chloride, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH value is 7.0, and the elution linear velocity is 300cm/h, thus obtaining phycobiliprotein purified solution 1;

(5) preparing a Tris-HCl Buffer solution Buffer A with the pH value of 6.8 and the conductivity of 6 mu s/cm and a Tris-HCl-NaCl Buffer solution Buffer B with the pH value of 6.8 and the conductivity of 8.5 mu s/cm, and filtering; washing the pump with 1 column volume of deionized water, and balancing the ion exchange column with 5 column volumes of Buffer A Buffer solution; directly loading phycobiliprotein purified liquid 1 with 2.5 column volumes, performing Buffer B gradient elution, and collecting 45-100% Tris-HCl-NaCl eluent with pH of 6.0 and conductivity of 4 mus/cm to obtain phycobiliprotein purified liquid 2;

(6) placing in dialysis bag with cut-off of 100KDa, dialyzing with 0.01M, pH phosphate buffer solution of 6.5 as dialysate for 12h, and centrifuging the desalted solution at 8000rpm for 30min to obtain phycobiliprotein solution.

The experimental data for example 3 are shown in table 3.

Table 3 experimental data for example 3

Name of solution	B-PE concentration (mg/L)	Purity of spectroscopy
			Phycobiliprotein extract	124	2.43
Phycobiliprotein purification solution 1	102	3.24
			Phycobiliprotein purification solution 2	82	3.75
Phycobiliprotein solution	77	4.02

Example 4

(1) Taking fresh porphyridium algae liquid into a centrifuge tube, diluting the algae liquid with water according to a proportion to 1 × 10⁷cells/mL; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10 min; suspending the algae in an isometric PEG/potassium sodium tartrate system with a line length of 20%, a volume ratio of 0.3 and a pH value of 7.2 to obtain a double-aqueous-phase algae suspension;

(2) repeatedly freezing and thawing the aqueous two-phase algae suspension obtained in the step (1) for 8 times at-80 ℃, completely thawing at 4 ℃ to obtain microalgae cell disruption solution, and centrifuging at 10000rpm for 10min for later use;

(3) standing the microalgae cell disruption solution at 4 ℃ for 3h, and taking an upper phase after the double aqueous phase system is obviously layered, namely obtaining a primarily purified phycobiliprotein extracting solution;

(4) balancing a hydroxyapatite chromatography column by using phosphate buffer solution with the volume of 0.01M, pH being 7.0 times and 4 times of the column volume, after balancing, loading a primarily purified phycobiliprotein extracting solution to the well-balanced hydroxyapatite column, wherein the loading amount is 2/3 times of the volume of a column bed, after loading is finished, performing gradient elution by using phosphate buffer solution with the pH value of 7.2 and containing 0.2M sodium chloride, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH value is 7.0, and the elution linear velocity is 300cm/h, so as to obtain phycobiliprotein purified solution 1;

(5) preparing a Tris-HCl Buffer solution Buffer A with the pH value of 6.8 and the conductivity of 6 mu s/cm and a Tris-HCl-NaCl Buffer solution Buffer B with the pH value of 6.8 and the conductivity of 8.5 mu s/cm, and filtering; washing the pump with 1 column volume of deionized water, and balancing the ion exchange column with 5 column volumes of Buffer A Buffer solution; directly loading phycobiliprotein purified liquid 1 with 2.5 column volumes, performing Buffer B gradient elution, and collecting 45-100% Tris-HCl-NaCl eluent with pH of 6.0 and conductivity of 4 mus/cm to obtain phycobiliprotein purified liquid 2;

(6) placing in dialysis bag with cut-off of 100KDa, dialyzing with 0.01M, pH phosphate buffer solution of 6.5 as dialysate for 12h, and centrifuging the desalted solution at 8000rpm for 30min to obtain phycobiliprotein solution.

The experimental data for example 4 are shown in table 4.

Table 4 experimental data for example 4

Name of solution	B-PE concentration (mg/L)	Purity of spectroscopy
			Phycobiliprotein extract	137	2.16
Phycobiliprotein purification solution 1	128	2.96
			Phycobiliprotein purification solution 2	94	4.12
Phycobiliprotein solution	82	4.22

According to the results of the above examples 1-4, the purity of phycocyanin > 4, allophycocyanin > 4 and phycoerythrin > 4 can be obtained by the above method, and can meet the requirements of reagent grade.

Example 5

(1) Taking fresh porphyridium algae liquid into a centrifuge tube, diluting the algae liquid with water according to a proportion to 5 multiplied by 10⁶cells/mL; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10 min; suspending the algae in an isometric PEG/potassium sodium tartrate system with a line length of 20%, a volume ratio of 0.3 and a pH value of 7.2 to obtain a double-aqueous-phase algae suspension;

(2) repeatedly freezing and thawing the aqueous two-phase algae suspension obtained in the step (1) for 4 times at-20 ℃, completely thawing at 4 ℃ to obtain microalgae cell disruption solution, and centrifuging at 10000rpm for 10min for later use;

(3) standing the microalgae cell disruption solution at 4 ℃ for 3h, and taking an upper phase after the double aqueous phase system is obviously layered, namely obtaining a primarily purified phycobiliprotein extracting solution;

(4) balancing a hydroxyapatite chromatography column by using phosphate buffer solution with the volume of 0.01M, pH being 7.0 times and 4 times of the column volume, after balancing, loading a primarily purified phycobiliprotein extracting solution to the well-balanced hydroxyapatite column, wherein the loading amount is 2/3 times of the volume of a column bed, after loading is finished, performing gradient elution by using phosphate buffer solution with the pH value of 7.2 and containing 0.2M sodium chloride, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH value is 7.0, and the elution linear velocity is 300cm/h, so as to obtain phycobiliprotein purified solution 1;

(5) preparing a Tris-HCl Buffer solution Buffer A with the pH value of 6.8 and the conductivity of 6 mu s/cm and a Tris-HCl-NaCl Buffer solution Buffer B with the pH value of 6.8 and the conductivity of 8.5 mu s/cm, and filtering; washing the pump with 1 column volume of deionized water, and balancing the ion exchange column with 5 column volumes of Buffer A Buffer solution; directly loading phycobiliprotein purified liquid 1 with 2.5 column volumes, performing Buffer B gradient elution, and collecting 45-100% Tris-HCl-NaCl eluent with pH of 6.0 and conductivity of 4 mus/cm to obtain phycobiliprotein purified liquid 2;

(6) placing in dialysis bag with cut-off of 100KDa, dialyzing with 0.01M, pH phosphate buffer solution of 6.5 as dialysate for 12h, and centrifuging the desalted solution at 8000rpm for 30min to obtain phycobiliprotein solution.

The experimental data for example 5 are shown in table 5.

Table 5 experimental data for example 5

Name of solution	B-PE concentration (mg/L)	Purity of spectroscopy
			Phycobiliprotein extract	76	1.99
Phycobiliprotein purification solution 1	61	2.52
			Phycobiliprotein purification solution 2	35	3.12
Phycobiliprotein solution	27	3.86

Example 6

(1) Taking fresh porphyridium algae liquid into a centrifuge tube, diluting the algae liquid with water according to a proportion to 1 × 10⁶cells/mL; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10 min; suspending the algae in an isometric PEG/potassium sodium tartrate system with a line length of 20%, a volume ratio of 0.3 and a pH value of 7.2 to obtain a double-aqueous-phase algae suspension;

(2) repeatedly freezing and thawing the aqueous two-phase algae suspension obtained in the step (1) for 6 times at-20 ℃, completely thawing at 4 ℃ to obtain microalgae cell disruption solution, and centrifuging at 10000rpm for 10min for later use;

(3) standing the microalgae cell disruption solution at 4 ℃ for 3h, and taking an upper phase after the double aqueous phase system is obviously layered, namely obtaining a primarily purified phycobiliprotein extracting solution;

(4) balancing a hydroxyapatite chromatography column by using phosphate buffer solution with the volume of 0.01M, pH being 7.0 times and 4 times of the column volume, after balancing, loading a primarily purified phycobiliprotein extracting solution to the well-balanced hydroxyapatite column, wherein the loading amount is 2/3 times of the volume of a column bed, after loading is finished, performing gradient elution by using phosphate buffer solution with the pH value of 7.2 and containing 0.2M sodium chloride, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH value is 7.0, and the elution linear velocity is 300cm/h, so as to obtain phycobiliprotein purified solution 1;

(5) preparing a Tris-HCl Buffer solution Buffer A with the pH value of 6.8 and the conductivity of 6 mu s/cm and a Tris-HCl-NaCl Buffer solution Buffer B with the pH value of 6.8 and the conductivity of 8.5 mu s/cm, and filtering; washing the pump with 1 column volume of deionized water, and balancing the ion exchange column with 5 column volumes of Buffer A Buffer solution; directly loading phycobiliprotein purified liquid 1 with 2.5 column volumes, performing Buffer B gradient elution, and collecting 45-100% Tris-HCl-NaCl eluent with pH of 6.0 and conductivity of 4 mus/cm to obtain phycobiliprotein purified liquid 2;

(6) placing in dialysis bag with cut-off of 100KDa, dialyzing with 0.01M, pH phosphate buffer solution of 6.5 as dialysate for 12h, and centrifuging the desalted solution at 8000rpm for 30min to obtain phycobiliprotein solution.

The experimental data for example 6 are shown in table 6.

Table 6 experimental data of example 6

Name of solution	B-PE concentration (mg/L)	Purity of spectroscopy
			Phycobiliprotein extract	18	1.87
Phycobiliprotein purification solution 1	15	2.34
			Phycobiliprotein purification solution 2	/	/
Phycobiliprotein solution	/	/

Example 7

(1) Taking fresh porphyridium algae liquid into a centrifuge tube, diluting the algae liquid with water according to a proportion to 5 multiplied by 10⁶cells/mL；4Centrifuging at 000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10 min; suspending the algae in an isometric PEG/potassium sodium tartrate system with a line length of 20%, a volume ratio of 0.3 and a pH value of 7.2 to obtain a double-aqueous-phase algae suspension;

(2) repeatedly freezing and thawing the aqueous two-phase algae suspension obtained in the step (1) for 4 times at-20 ℃, completely thawing at 4 ℃ to obtain microalgae cell disruption solution, and centrifuging at 10000rpm for 10min for later use;

(3) standing the microalgae cell disruption solution at 4 ℃ for 3h, and taking an upper phase after the double aqueous phase system is obviously layered, namely obtaining a primarily purified phycobiliprotein extracting solution;

(4) balancing a hydroxyapatite chromatography column by using phosphate buffer solution with the volume of 0.01M, pH being 7.0 times and 4 times of the column volume, after balancing, loading a primarily purified phycobiliprotein extracting solution to the well-balanced hydroxyapatite column, wherein the loading amount is 2/3 times of the volume of a column bed, after loading is finished, performing gradient elution by using phosphate buffer solution with the pH value of 7.2 and containing 0.2M sodium chloride, the concentration gradients of the phosphate buffer solution are respectively 0.02, 0.05 and 0.1M, the pH value is 7.0, and the elution linear velocity is 300cm/h, so as to obtain phycobiliprotein purified solution 1;

(5) preparing a Tris-HCl Buffer solution Buffer A with the pH value of 6.8 and the conductivity of 6 mu s/cm and a Tris-HCl-NaCl Buffer solution Buffer B with the pH value of 6.8 and the conductivity of 8.5 mu s/cm, and filtering; washing the pump with 1 column volume of deionized water, and balancing the ion exchange column with 5 column volumes of Buffer A Buffer solution; directly loading phycobiliprotein purified liquid 1 with 2.5 column volumes, performing Buffer B gradient elution, and collecting 45-100% Tris-HCl-NaCl eluent with pH of 6.0 and conductivity of 4 mus/cm to obtain phycobiliprotein purified liquid 2;

(6) placing in dialysis bag with cut-off of 100KDa, dialyzing with 0.01M, pH phosphate buffer solution of 6.5 as dialysate for 12h, and centrifuging the desalted solution at 8000rpm for 30min to obtain phycobiliprotein solution.

The experimental data for example 7 are shown in table 7.

Table 7 experimental data for example 7

Name of solution	B-PE concentration (mg/L)	Purity of spectroscopy
			Phycobiliprotein extract	76	1.99
Phycobiliprotein purification solution 1	61	2.52
			Phycobiliprotein purification solution 2	35	3.12
Phycobiliprotein solution	27	3.86

According to the test results of the above examples 5-7, the purity of phycocyanin, allophycocyanin and phycoerythrin obtained by the above method is more than 3, and can meet the requirements of pharmaceutical grade.

In conclusion, the invention takes fresh marine microalgae as a raw material, can further realize the crushing of the algae and the preliminary purification of the phycobiliprotein by a freeze thawing-aqueous two-phase extraction primary purification method, can prepare the high-purity phycobiliprotein by further combining column chromatography, has simple extraction process and low cost compared with the traditional chromatography process, and is suitable for the large-scale industrial production of the phycobiliprotein.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.