CN112645999B - Selenium-enriched spirulina protein separation and purification method - Google Patents

Abstract

The invention discloses a method for separating and purifying selenium-enriched spirulina protein, which comprises the following operation steps: (1) Adding PBS buffer solution into the algae powder, repeatedly freezing, thawing, performing ultrasound to obtain algae cell disruption solution, centrifuging, collecting supernatant, salting out to saturation, centrifuging to obtain crude protein, adding PBS buffer solution to dissolve crude protein, dialyzing and desalting, and vacuum freeze-drying to obtain selenium-enriched spirulina total protein powder; (2) Dissolving in PBS buffer solution, ultrasonic treating, centrifuging, filtering, and collecting filtrate to obtain selenium-rich spirulina protein solution; (3) The weak anion DEAE was selected as the ion exchanger and the sample was separated by gradient elution. The method can separate 5 protein groups, has good separation effect and high purity, and the selenium content of the total protein reaches 66.15 percent; the 5 protein components separated by the method have obvious difference in antioxidant activity, and the method can separate target proteins with high activity, and has relatively simple operation and relatively low cost.

Description

Selenium-enriched spirulina protein separation and purification method

Technical Field

The invention relates to the technical field of separation and purification of selenium-enriched spirulina proteins, in particular to a separation and purification method of selenium-enriched spirulina proteins.

Background

In 1973, selenium was formally listed as one of 14 microelements required by human physiology, and both insufficient and excessive selenium intake can have serious effects on the body. Currently, selenium has biological activities mainly including: (1) The selenium cysteine is an active center of glutathione peroxidase, and the glutathione peroxidase participates in an antioxidant process of an organism, can remove free radicals and peroxidized lipid generated by oxidative stress, and prevents the damage of oxygen free radicals to the organism; (2) Selenium participates in the regulation of the immune system of the organism, and can protect the organism by activating the activity of lymphocyte enzymes, improving the proliferation of B cells and T cells of the human body, promoting the phagocytosis and the activity of phagocytes, strengthening a lymph specific system and a non-specific immune system, and can realize the effects of preventing and resisting cancers and tumors and inhibit the occurrence of liver cancer and breast cancer by regulating oxidative stress and immunity; (3) Selenium is a good natural antidote, and can be used as a non-metal ion with negative charges to combine with harmful heavy metal ions with positive charges in a body to form a metal-selenium-protein complex to be discharged out of the body, so that the toxic effects of heavy metals such as arsenic, mercury, chromium and the like in the body are antagonized and weakened. Therefore, if the human body lacks selenium, various diseases such as keshan disease, bone joint disease, various cancers and the like can be caused.

Selenium is an essential trace element for maintaining normal operation of the body, and the lack of selenium in the human body can cause various diseases. Because the human body cannot synthesize the needed organic selenium, diet is a main way for people to obtain selenium. However, the distribution of selenium is uneven worldwide, and the natural plants have poor selenium enrichment ability, and the normal needs of the human body cannot be generally satisfied only by the selenium content in the natural foods, so how to obtain safe and effective selenium supplements is attracting attention. Wherein, the spirulina has the advantages of rich nutrition, large scale, simple culture, high selenium tolerance and the like, the selenium-rich spirulina is widely applied and accepted as a selenium-supplementing functional food, and 45 to 70 percent of selenium exists in the protein component in an organic form. Researches show that the selenium is added and the spirulina contains rich bioactive components, so that the selenium-enriched spirulina has particularly diverse and obvious active functions, and mainly comprises a plurality of aspects of antioxidation, anticancer, liver tissue regeneration promotion, immunoregulation and the like. Selenium protein is the main existence form of organic selenium, the bioavailability and biological function of selenium depend on the existence form to a great extent, and meanwhile, the protein is used as the biological function active ingredient with the largest algae content, so that various selenium-containing proteins and research biological functions thereof are separated, and a new idea is provided for improving the functional research and economic benefit of selenium-enriched spirulina.

At present, no patent for separating and purifying selenium-enriched spirulina protein exists in the method for separating and purifying selenium-enriched spirulina protein, and the reported papers mainly relate to separating and purifying selenium-enriched spirulina protein and allophycocyanin. The yellow peak is subjected to cell disruption by using a repeated freeze thawing wall breaking method, then 30-80% ammonium sulfate is used for salting out and precipitating to obtain total protein of the algae, and then HA column and DEAE-5 column are used for combining, separating and purifying phycocyanin. Huang Shi separation of selenocyano from phycocyanin by HA column and DEAE-52 column chromatography, li Nongle separation of phycocyanin by DEAE-52 column chromatography and SePhadxeG-10 column chromatography.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a method for separating and purifying selenium-enriched spirulina protein, which aims to obtain the method for separating and purifying the selenium-enriched spirulina with high purity and simple operation.

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

a method for separating and purifying selenium-enriched spirulina protein comprises the following operation steps:

(1) Adding PBS buffer solution into the algae powder, freezing, melting in water bath, repeatedly freezing and melting for 4 times, performing ultrasonic pyrolysis on algae bodies to obtain algae cell disruption solution, centrifuging the obtained algae cell disruption solution, taking supernatant, salting out the supernatant with ammonium sulfate at 4 ℃ overnight until the saturation is 80%, centrifuging, collecting precipitate to obtain crude protein, adding a small amount of PBS buffer solution into the crude protein to dissolve the crude protein, dialyzing with distilled water to desalt, and performing vacuum freeze-drying to obtain selenium-enriched spirulina total protein powder;

(2) Weighing the selenium-enriched spirulina total protein powder obtained in the step (1) to PBS buffer solution (pH 7.0,50mM, without containing 2% polyvinylpyrrolidone and 0.5% dithiothreitol), dissolving, centrifuging after ultrasonic dissolution promotion, filtering supernatant by using a 0.45 μm and 0.22 μm water-based filter membrane in sequence, and collecting filtrate to obtain selenium-enriched spirulina protein solution;

(3) Selecting weak anion DEAE as ion exchanger (i.e. DEAE anion exchange chromatography column), balancing chromatography column with PBS buffer solution [ slender column (2.5 cm. Times.60 cm) ], loading 40mL of selenium-enriched spirulina protein solution (3.0 mg/mL of selenium-enriched spirulina protein solution is obtained after dilution of selenium-enriched spirulina protein solution obtained in step (2)), eluting the combined column with PBS buffer solution, sequentially eluting and separating sample with 0.12M, 0.2M, 0.4M, 0.6M and 0.8M NaCl-PBS buffer solution (pH 7.0) respectively, taking elution volume as abscissa and absorbance at 280nm wavelength as ordinate, plotting protein elution curve, collecting sample of same NaCl-PBS buffer solution (pH 7.0) elution gradient as one component, i.e. 0.12M, 0.2M, 0.4M, 0.6M, 0.8M-PBS respectively corresponding to Se-SP1, se-SP2, SP3 Se-SP4, se-SP4, se-SP, se-5 Da, concentrating, and concentrating, separating to obtain purified Se-enriched protein, concentrating, and vacuum dialyzing, concentrating, and concentrating to obtain Se-enriched protein, and concentrating to obtain Se-enriched protein.

Preferably, the molar concentration of the PBS buffer in the step (1) is 0.1M, 2% polyvinylpyrrolidone and 0.5% dithiothreitol are contained, and the pH value is 6.8.

Preferably, the freezing temperature in the step (1) is-20 ℃ and the time is 3 hours; the ultrasonic cracking time is 30min; the centrifugation is carried out at 10000rpm and 4 ℃ for 30min.

Preferably, 5g of algae powder in step (1) is added to 100ml of LPBS buffer.

Preferably, the freeze drying time in step (1) is 36h at-25 ℃.

Preferably, 0.2g of the selenium-enriched spirulina total protein powder in the step (2) is added into 50ml of buffer solution.

Preferably, the centrifugal speed in the step (2) is equal to or higher than 8000rpm, and the centrifugal speed is 4 ℃ for 20min.

Preferably, in the step (2), the solution is fully dissolved for 30min at the temperature of 4 ℃ and is ultrasonically dissolved for 5min.

Preferably, the PBS buffer in step (2) and the PBS buffer in step (3) are of the same specification, and the PBS buffer has a concentration of 50mM and a pH of 7.0, and does not contain 2% polyvinylpyrrolidone and 0.5% dithiothreitol.

Preferably, the gradient elution separation sample in step (3) has a flow rate of 1mL/min.

Compared with the prior art, the invention has the following beneficial effects:

the method can separate 5 protein groups, has good separation effect and high purity, and the selenium content of the total protein reaches 66.15 percent; the 5 protein components separated by the method have obvious difference in antioxidant activity, and the method can separate target proteins with high activity, and has relatively simple operation and relatively low cost.

Drawings

Fig. 1 is a full-wave scan of A1, A2, A3, A4 selenium enriched spirulina protein solution.

FIG. 2 is an elution profile for different salt concentration gradients; wherein (a) is a gradient of 0.1M, 0.12M, 0.2M, 0.3M, 0.4M, 0.6M, 0.8M, 1.0M; (b) 0.12M, 0.2M, 0.4M, 0.6M, 0.8M gradient.

FIG. 3 is an elution profile for different pH buffers; wherein, the (a), (b) and (c) diagrams correspond to PBS buffers with pH values of 6.8, 7.0 and 7.2 respectively.

FIG. 4 shows elution curves of different loading concentrations, wherein (a), (b) and (c) correspond to loading solutions with protein concentrations of 4.5mg/mL, 3.0mg/mL and 1.5mg/mL, respectively.

FIG. 5 is an elution profile for different column sizes, wherein (a) and (b) correspond to the long and short columns (2.5 cm. Times.60 cm) and the short and thick columns (6 cm. Times.30 cm), respectively.

FIG. 6 shows the SDA-PEGA electrophoretic distribution of different protein fractions.

FIG. 7 shows the ability of different protein fractions to scavenge ABTS and DPPH radicals.

Detailed Description

The following detailed description, in conjunction with the accompanying drawings, describes in detail, but it is to be understood that the scope of the invention is not limited to the specific embodiments.

The selenium-enriched spirulina powder used in the examples is self-cultured in a laboratory in the following manner: spirulina platensis species were supplied by North sea Bada Biotechnology Co., ltd, cultured in Zarouk medium formulation of its company, with the addition of sodium selenite (Na) at a concentration of 30mg/kg ₂ SeO ₃ ) Culturing spirulina in a pipeline type photobioreactor with capacity of 5 tons, inoculating the spirulina seeds in logarithmic growth phase, culturing in outdoor sunlight for 7 days, measuring the OD value of the density of the spirulina every day, and ending the culture of the selenium-enriched spirulina when the OD value reaches 1.2. Filtering the culture solution with 300 mesh bolting silk, washing with water twice to collect algae mud, passing throughAnd performing vacuum freeze drying to obtain selenium-enriched spirulina powder, and preserving the spirulina powder at room temperature.

NaCl-PBS buffer (pH 7.0): the final concentration of PBS buffer from 0.05 to M to pH7.0, and contains 0.1M, 0.12M, 0.2M, 0.3M, 0.4M, 0.6M, 0.8M, 1.0M NaCL.

Example 1

A method for separating and purifying selenium-enriched spirulina protein comprises the following operation steps:

(1) Weighing 5.00g of selenium-enriched spirulina powder, putting into a beaker, adding 100mL of PBS buffer solution (0.1M, containing 2% polyvinylpyrrolidone and 0.5% dithiothreitol, pH 6.8), freezing at 20 ℃ for 3h, thawing in water bath at 37 ℃ for 4 times, repeatedly freezing, and thawing for 4 times, using an ultrasonic breaker to crack the spirulina body for 30min (power 50%, work for 3s and intermittent 2 s), obtaining spirulina cell disruption solution, centrifuging the spirulina cell disruption solution at 4 ℃ for 30min at 10000rpm, taking supernatant, salting out the supernatant with ammonium sulfate at 4 ℃ overnight until the saturation is 80%, centrifuging the supernatant at 10000rpm and 4 ℃ for 30min, collecting precipitate to obtain crude protein, adding a small amount of PBS buffer solution into the crude protein to dissolve the crude protein, desalting the crude protein with distilled water, and then carrying out vacuum freeze-drying at-25 ℃ for 36h to obtain selenium-enriched spirulina total protein powder, and storing at-20 ℃ for use; measuring protein content of the extract by Kjeldahl nitrogen determination method, and measuring selenium content by ICP-MS;

(2) Weighing 0.2g of the selenium-enriched spirulina total protein powder obtained in the step (1) to 50mL of PBS buffer solution (pH 7.0,50mM, without 2% polyvinylpyrrolidone and 0.5% dithiothreitol), fully dissolving for 30min at 4 ℃, centrifuging for 20min at 8000rpm after ultrasonic dissolution for 5min, filtering supernatant by using a 0.45 mu m and 0.22 mu m water-based filter membrane in sequence, and collecting filtrate to obtain selenium-enriched spirulina protein solution, wherein the obtained selenium-enriched spirulina protein solution is Se-SP and is marked as A1;

(3) Selecting weak anion DEAE as an ion exchanger (namely, adopting a DEAE anion exchange chromatographic column), balancing the chromatographic column [ a slender column (2.5 cm multiplied by 60 cm) ] by using PBS buffer solution which does not contain 2% polyvinylpyrrolidone and 0.5% dithiothreitol at pH7.0 and 50mM, loading 40mL of a selenium-enriched spirulina protein solution (3.0 mg/mL of the selenium-enriched spirulina protein solution obtained after dilution of the selenium-enriched spirulina protein solution obtained in the step (2), eluting the combined column by using PBS buffer solution which does not contain 2% polyvinylpyrrolidone and 0.5% dithiothreitol at pH7.0 and 50mM, and sequentially eluting and separating a sample by using NaCl-PBS buffer solution (pH 7.0) at a gradient of 0.12M, 0.2M, 0.4M, 0.6M and 0.8M, wherein the flow rate is 1mL/min, the volume of the selenium-enriched spirulina protein solution is 10 mL/tube, and the absorbance of the protein is plotted at a wavelength of 280nm in an abscissa; collecting samples of the same NaCl-PBS buffer solution (pH 7.0) elution gradient as one component, namely 0.12M, 0.2M, 0.4M, 0.6M and 0.8M NaCl-PBS respectively corresponding to Se-SP1, se-SP1,2, se-SP3, se-SP4 and Se-SP5, separating 5 protein groups, concentrating by using a 3000Da ultrafiltration tube, dialyzing for desalination, and storing at minus 20 ℃; the protein content of the extract was determined by Kjeldahl method and the selenium content by ICP-MS.

Comparative example 1

Influence of centrifugal force on solid-liquid separation effect

Fully dissolving for 30min at 4 ℃ in the step (2), and centrifuging for 20min at different rotation speeds (6000, 10000, 12000 rpm) after ultrasonic dissolution promotion for 5 min; the rest of the operations of the step (1) and the step (2) are the same as those of the embodiment 1, and the step (3) is not adopted; the supernatant was filtered sequentially with 0.45 μm and 0.22 μm aqueous filters, and the filtrates were collected to obtain selenium-enriched spirulina protein solutions, labeled A2 (6000 rpm), A3 (10000 rpm) and A4 (12000 rpm), respectively, and the concentrations were determined using BCA protein quantification kit, and scanned at full wavelength (270-720 nm) using an ultraviolet-visible light analyzer.

Comparative example 2

Influence of elution gradient on separation of selenium-enriched spirulina protein

In step (3), se-SP obtained in step (2), namely A1, was subjected to gradient elution, namely, samples were separated by gradient elution with 0.1M, 0.12M, 0.2M, 0.3M, 0.4M, 0.6M, 0.8M, 1.0M NaCl-PBS buffer (pH 7.0), respectively, at a flow rate of 1mL/min,10 mL/tube, and the rest of the procedure was the same as in example 1. And (3) drawing a protein elution curve by taking the elution volume as an abscissa and the absorbance at the wavelength of 280nm as an ordinate.

Comparative example 3

Influence of the pH of the eluent on the elution of selenium-enriched spirulina proteins

In the step (3), the same procedure as in example 1 was repeated except that PBS buffer (not containing 2% polyvinylpyrrolidone and 0.5% dithiothreitol) was used at pH6.8 mM, pH7.0 mM and pH7.2 mM, respectively, to equilibrate the column [ slim column (2.5 cm. Times.60 cm) ]. And (3) drawing a protein elution curve by taking the elution volume as an abscissa and the absorbance at the wavelength of 280nm as an ordinate.

Comparative example 4

Influence of sample concentration on elution of selenium-enriched spirulina protein

In the step (3), 40mL of a selenium-enriched spirulina protein solution of 1.5mg/mL, 3.0mg/mL and 4.5mg/mL were put on a column, and the other steps were the same as in example 1. And (3) drawing a protein elution curve by taking the elution volume as an abscissa and the absorbance at the wavelength of 280nm as an ordinate.

Comparative example 5

Influence of column size on separation of selenium-enriched spirulina protein

In step (3), weakly anionic DEAE was selected as an ion exchanger (i.e., DEAE anion exchange column of 2.5 cm. Times.60 cm and 6 cm. Times.30 cm, respectively), and the column [ slim column (2.5 cm. Times.60 cm), (6 cm. Times.30 cm) ] was equilibrated with PBS buffer (pH 7.0,50 mM) (containing no 2% polyvinylpyrrolidone and 0.5% dithiothreitol) and the other operations were the same as in example 1. And (3) drawing a protein elution curve by taking the elution volume as an abscissa and the absorbance at the wavelength of 280nm as an ordinate.

Result verification

1. Verification of the separation purification

Under the optimal elution condition, the selenium-enriched spirulina protein is separated and purified. According to the protein elution curve, the proteins of the same component are combined, concentrated and desalted by an ultrafiltration tube of 3000Da, the protein content is measured by using a BCA kit, and the recovery rate of each component is calculated, wherein the formula is as follows:

2. determination of antioxidant Activity of selenium-enriched spirulina protein

The ability of eliminating DPPH free radical and ABTS free radical is used as index to compare the antioxidant activity of selenium-enriched spirulina protein before and after separation.

2.1 DPPH clearance determination of selenium-enriched Spirulina proteins

Referring to Babini literature report method for measuring DPPH clearance, 2mL of protein solution with different concentration gradients is respectively taken, 2mL of 0.1mM DPPH solution is added, after severe shaking, the solution is kept away from light for 30min at 25 ℃, and the absorbance at 517nm is measured. 2mL of PBS buffer was used as a control group, 2mL of absolute ethanol was used as a sample blank group, and 3 parallel experiments were set up and the average value thereof was taken. The DPPH clearance is calculated as follows:

(As, ab, A0 are the absorbance of the sample, blank and control groups, respectively)

2.2 determination of ABTS clearance of selenium-enriched Spirulina proteins

2.45mmol/L ABTS solution is prepared according to the method reported by Qiao Shen and stored in a dark place for later use. The ABTS solution was diluted with PBS buffer to absorbance at 734nm of 0.700.+ -. 0.020 before use. 1mL of protein solution with different concentration gradients is respectively taken and put into a test tube, 4mL of ABTS diluent is added, the reaction is accurately carried out for 6min at 30 ℃ in dark, and the absorbance at 734nm is measured. 1mL of phosphate buffer was used as a control group instead of the sample, distilled water was used as a blank group instead of the ABTS, and 3 parallel experiments were set up and the average value thereof was taken. ABTS radical scavenging was calculated as follows.

(As, ab, A0 are the absorbance of the sample, blank and control groups, respectively)

Experimental results

Influence of centrifugal force on solid-liquid separation of selenium-enriched spirulina protein crude extract

Pigment adsorption seriously affects the separation efficiency of a chromatographic column, and the invention combines centrifugal separation and membrane filtration to remove green impurities in selenium-enriched spirulina crude protein solution. As can be seen from FIG. 1, the absorbance of the supernatant liquid treated at four rotational speeds (6000 rpm, 8000rpm, 10000rpm and 12000 rpm) was successively lowered in the wavelength ranges of 300 to 550nm and 650 to 700 nm. Wherein, compared with the 6000rpm experimental group, the A436.2 and A677 of the supernatant of the 8000rpm experimental group are 0.5918 and 0.0719 respectively, which are reduced by 88.5 percent and 82.92 percent, and the phycocyanin purity A620/A280 is improved from 0.95 to 1.2, which is not much different from the other two groups of experiments (10000 rpm and 12000 rpm). The chlorophyll has characteristic absorption peaks at the wavelength 677nm, and the absorption regions of chlorophyll-red light and chlorophyll-blue-violet light are respectively in the wavelength ranges of 430-450 nm and 640-670 nm, so that the green impurities in the selenium-enriched spirulina protein crude extract can be better removed by adopting centrifugal force above 8000rpm, and the purity of the phycocyanin can be improved.

The principle of ion exchange chromatography is that the charge carried by the separated material is combined with the opposite charge carried by the ion exchanger, whereas the binding between the charged molecule and the stationary phase is reversible. Therefore, the ion intensity and the pH value of the eluent are changed, so that the sample and the eluent are subjected to ion exchange to achieve the purpose of separating substances. Phycocyanin is used as main component of spirulina, its isoelectric point is 4.6, pH is kept stable at 6-8, and its negative charge is carried out. Therefore, the present invention selects weakly anionic DEAE as the ion exchanger, and optimizes the elution conditions in terms of elution gradient, loading concentration, pH of the eluent (PBS buffer, PBS buffer containing no 2% polyvinylpyrrolidone and 0.5% dithiothreitol), and column size.

First, the elution gradient of NaCl-PBS buffer was optimized: elution gradients of 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 and 1.0M were used, and the elution curves are shown in FIG. 2 (a), and the other 6 gradients except 1.0M all showed elution peaks. The eluting peak at 0.1M is relatively wide and short, and the solution is colorless; the elution volume at 0.2M was maximum, the solution was blue-violet, presumably phycocyanin; the elution peaks at 0.3M and 0.4M are sharp and short, the protein concentration is low, the solution is light blue, and the allophycocyanin is presumed to be allophycocyanin; the elution peak was high and sharp at 0.6M and 0.8M, and the solution was colorless. On the basis, the gradient of 0.1M is increased to 0.12M, the gradient of 0.3M and the gradient of 0.4M are combined to 0.4M, the gradient of 1.0M is deleted for separation, and the elution curves are shown in the graph in FIG. 2 (b), and the elution peaks of 0.12M, 0.6M and 0.8M have no obvious change; the elution peak at 0.2M is widened and short, and a more obvious tailing is generated; whereas the 0.4M elution peak became sharp and the protein content was significantly increased.

Next, under the optimal elution gradient, three PBS buffers (pH 6.8, 7.0, 7.2, 50 mM) with different pH were used for elution according to isoelectric point and pH distribution characteristics of phycocyanin, and the separation effect is shown in fig. 3: at PBS buffer ph=6.8, significant tailing and tailing peaks appear (fig. 3 (a)); at PBS buffer ph=7.0, 5 good elution peaks can be obtained (fig. 3 (b)); however, when the pH of the PBS buffer was raised to 7.2, only one elution peak occurred and the protein was not separated (FIG. 3 (c)).

The concentration of the sample to be added influences the purity and recovery rate of protein separation, the concentration of the sample is too low, the affinity effect is poor, and the peak is difficult to appear; the sample concentration is too high and the retention time is long. Thus, protein loading concentrations were screened and optimized for optimal elution gradient and eluent pH conditions, with the results shown in fig. 4; the sample adding volume of 40mL is kept, and the sample adding concentration is reduced to 4.5mg/mL, 3.0mg/mL and 1.5mg/mL in sequence, namely, the sample adding amount is 180mg, 120mg and 60mg respectively; when the sample concentration is adjusted from 4.5mg/mL to 3.0mg/mL, 5 elution peaks are more sharp, the protein content is increased, the tailing of the 0.2M elution peak is reduced, and when the sample concentration is adjusted from 3.0mg/mL to 1.5mg/mL, double peaks appear in both the 0.4M elution peak and the 0.6M elution peak; in addition, a significant reduction in the five elution peak-to-peak values was noted.

The exchange capacity is affected by the effective surface area of the exchanger that interacts with the separation sample, the larger the effective surface area the higher its exchange capacity. For this, a short crude column of 6cm×30cm was selected for purification to improve the efficiency of separation and purification, and the elution profile thereof is shown in fig. 5; however, during the whole elution, only one elution peak appears under a 0.2M gradient, the peak is very high and sharp, and the selenium-rich protein is not separated. Therefore, the short thick column is not suitable for separation and purification of selenium-enriched spirulina proteins.

From the above experimental results, it was found that under the optimal separation conditions: PBS buffer pH7.0, elution gradients 0.12M, 0.2M, 0.4M, 0.6M and 0.8M, sample concentration 3.0mg/L and selection of the column (2.5 cm. Times.60 cm) allowed separation of 5 protein fractions designated Se-SP1 (0.12M), se-SP2 (0.2M), se-SP3 (0.4M), se-SP4 (0.6M) and Se-SP5 (0.8M), respectively. The 5 protein fractions were analyzed by SDS-PAGE and the results are shown in FIG. 6. The 5 component bands were significantly different compared to the Se-SP electrophoresis band, indicating good separation. Based on this, the selenium content and recovery rate were measured, and the results are shown in Table 1. The re-dissolved supernatant (Se-SP) of the selenium-enriched spirulina crude protein has the selenium content of 11.66+/-0.15 mug/g and accounts for 32.09 percent of the total protein selenium content. The selenium content and recovery rate of Se-SP2 in the 5 purified protein components are highest and are respectively 1.99+/-0.03 mug/g and 66.15 percent.

TABLE 1 selenium content and recovery of different protein fractions

	Selenium-enriched crude protein	Se-SP	Se-SP1	Se-SP2	Se-SP3	Se-SP4	Se-SP5
								Recovery (%)	74.73	-	7.28	66.15	6.02	2.55	0.017
Selenium content (μg/g)	36.32	11.66±0.15	0.67±0.02	1.99±0.03	1.03±0.08	1.43±0.05	0.99±0.03
								Selenium ratio (%)	100	32.09	5.75	17.04	8.87	12.28	8.46

Antioxidant activity of selenium-enriched spirulina protein separation component

In combination with the recovery rate of separation and purification, the ability of selecting protein components Se-SP1, se-SP2, se-SP3 and Se-SP4 to remove ABTS and DPPH free radicals is measured, and the result is shown in figure 7, and each purified protein has better antioxidant ability than selenium-rich crude protein; wherein, the ability of Se-SP2 to clear ABTS can be improved from 54.84 percent of 0.05mg/mL to 97.95 percent of 0.15mg/mL, which is far higher than that of Se-SP (17.48 percent to 48.7 percent). The IC50 of the different protein fractions for scavenging free radicals was calculated using SPSS software, and the results are shown in Table 2, wherein the IC50 of Se-SP2, se-SP3 and Se-SP4 for scavenging ABTS was 0.051, 0.113 and 0.112mg/mL, respectively, and the IC50 for scavenging DPPH was 0.582, 0.273 and 0.265mg/mL, respectively. Thus, the comprehensive analysis of Se-SP3 and Se-SP4 shows better antioxidant activity.

TABTS and DPPH radical scavenging IC50 for different protein fractions

Note that: a. b, c, d represent the degree of difference between the respective protein components

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims (6)

1. The separation and purification method of the selenium-enriched spirulina protein is characterized by comprising the following operation steps:

(1) Adding PBS buffer solution into the algae powder, freezing, thawing, repeatedly freezing and thawing, performing ultrasonic lysing to obtain algae cell disruption solution, centrifuging the obtained algae cell disruption solution, collecting supernatant, salting out the supernatant with ammonium sulfate overnight to saturation, centrifuging, collecting precipitate to obtain crude protein, adding PBS buffer solution into the crude protein to dissolve the crude protein, dialyzing and desalting, and vacuum freeze-drying to obtain selenium-enriched spirulina total protein powder;

(2) Weighing the selenium-enriched spirulina total protein powder obtained in the step (1) to PBS buffer solution, dissolving, centrifuging after ultrasonic dissolution promotion, filtering the supernatant, and collecting the filtrate to obtain a selenium-enriched spirulina protein solution;

(3) Selecting weak anion DEAE as an ion exchanger, balancing a chromatographic column by using PBS buffer solution, loading 40mL of 3.0mg/mL selenium-enriched spirulina protein solution onto the column, eluting the binding column by using PBS buffer solution, sequentially and respectively eluting and separating samples by using 0.12M, 0.2M, 0.4M, 0.6M and 0.8M NaCl-PBS buffer solution in a gradient way, taking the elution volume as an abscissa, drawing a protein elution curve by taking absorbance at a wavelength of 280nm as an ordinate, collecting samples with the same NaCl-PBS buffer solution elution gradient as a component, namely 0.12M, 0.2M, 0.4M, 0.6M and 0.8M NaCl-PBS respectively corresponding to Se-SP1, se-SP1,2, se-SP3, se-SP4 and Se-SP5, separating 5 protein groups, concentrating, desalting, and performing vacuum freeze drying to obtain purified selenium-containing protein components;

wherein the freezing temperature in the step (1) is-20 ℃ and the time is 3 hours; the ultrasonic cracking time is 30min; the centrifugation is carried out at 10000rpm and 4 ℃ for 30min; the molar concentration of the PBS buffer solution is 0.1M, 2% polyvinylpyrrolidone and 0.5% dithiothreitol are contained, and the pH value is 6.8;

wherein, the centrifugal speed in the step (2) is more than or equal to 8000rpm, and the centrifugal speed is more than or equal to 4 ℃ for 20min;

wherein the PBS buffer solutions in the step (2) and the step (3) are of the same specification, the concentration of the PBS buffer solution is 50mM, the pH value is 7.0, and the PBS buffer solution does not contain 2% polyvinylpyrrolidone and 0.5% dithiothreitol.

2. The method according to claim 1, characterized in that: in the step (1), 5g of algae powder is added into 100mLPBS buffer.

3. The method according to claim 1, characterized in that: the freeze-drying time in the step (1) is 36h, and the temperature is minus 25 ℃.

4. The method according to claim 1, characterized in that: in the step (2), 0.2g of selenium-enriched spirulina total protein powder is added into 50ml of buffer solution.

5. The method according to claim 1, characterized in that: and (3) fully dissolving for 30min at the temperature of 4 ℃ in the step (2), and ultrasonically dissolving for 5min.

6. The method according to claim 1, characterized in that: the flow rate of the gradient elution separation sample in the step (3) is 1mL/min.