Method for extracting high-purity phycocyanin from spirulina

Abstract

The invention relates to the technical field of bioactive substance extraction, in particular to a method for extracting high-purity phycocyanin from spirulina. The method comprises the following steps: (1) mixing spirulina powder with a phosphate buffer solution to obtain an algae suspension; (2) adding a compound enzyme into the algae suspension, and oscillating for enzymolysis to obtain enzymatic hydrolysate; the enzymatic hydrolysate is homogenized, and a homogenized solution is obtained; (3) centrifuging the homogenate, taking supernatant, filtering, and taking filtrate to obtain crude extract; (4) adding ammonium sulfate crystals into the crude extract, standing for 4-6 hours, centrifuging, taking precipitate, mixing the precipitate with a phosphate buffer solution, and performing ultrafiltration to obtain ultrafiltrate; and (5) enabling the ultrafiltrate to pass through a column, eluting, collecting a fraction of an A620 main peak, and freeze-drying to obtain phycocyanin. According to the method disclosed by the invention, high-purity phycocyanin can be extracted, and a basis is provided for efficient extraction of phycocyanin.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction technology, and in particular to a method for extracting high-purity phycocyanin from Spirulina. Background Technology

[0002] Phycocyanin is a water-soluble natural pigment protein unique to spirulina. It has biological activities such as anti-oxidation, enhancing immunity, and scavenging free radicals. At the same time, due to its natural blue color and high safety, it is in great demand in the fields of food coloring and pharmaceutical research and development.

[0003] Existing methods for extracting phycocyanin from Spirulina have the following drawbacks: 1) Traditional alkali dissolution methods easily lead to protein denaturation and low protein activity retention. 2) Ultrasonic disruption method results in insufficient cell wall disruption rate and low extraction rate; 3) High-purity cyanobacterial protein cannot be obtained by salting out alone; 4) Organic solvent extraction methods pose a risk of residue and do not meet the requirements for food-grade raw materials.

[0004] Therefore, it is of great significance to develop an efficient, high-purity, and environmentally friendly method for extracting phycocyanin. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting phycocyanin with high extraction rate, high purity, and safety. Specifically, it relates to a method for extracting high-purity phycocyanin from Spirulina.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for extracting high-purity phycocyanin from Spirulina, comprising the following steps: (1) Mix spirulina powder with phosphate buffer to obtain an algal suspension; (2) Add the compound enzyme to the algal suspension and shake to hydrolyze for 30-50 min to obtain the enzymatic hydrolysate; homogenize the enzymatic hydrolysate 2-4 times to obtain the homogenized solution; (3) Centrifuge the homogenized liquid, take the supernatant, filter, take the filtrate, and obtain the crude extract; (4) Add ammonium sulfate crystals to the crude extract to make the saturation 25-30%, let it stand for 4-6 hours, centrifuge, take the precipitate, mix the precipitate with phosphate buffer, and ultrafilter to obtain the ultrafiltrate. (5) Pass the ultrafiltrate through a column, elute, and collect A. 620 The fraction from the main peak was freeze-dried to obtain phycocyanin.

[0007] Preferably, the mass-to-volume ratio of the spirulina powder to the phosphate buffer in step (1) is 1g:15~20mL; The initial concentration of the phosphate buffer solution is 0.05~0.1 mol / L; The pH of the phosphate buffer solution is 6.8 to 7.2.

[0008] Preferably, the mass ratio of spirulina powder to compound enzyme is 100:0.5~1.2; The complex enzyme includes cellulase and pectinase, and the mass ratio of cellulase to pectinase in the complex enzyme is 2~4:1.

[0009] Preferably, the rotation speed of the oscillating enzymatic hydrolysis is 150~200 rpm; The temperature for the oscillating enzymatic hydrolysis is 35~39℃.

[0010] Preferably, the pressure of the homogenization is 30~40MPa.

[0011] Preferably, the centrifugation speed in step (3) is 8000~12000 rpm and the centrifugation time is 15~25 min; The filtration in step (3) is membrane filtration, and the pore size of the membrane is 0.2~0.3μm.

[0012] Preferably, the centrifugation speed in step (4) is 7000~9000 rpm and the centrifugation time is 8~12 min; The initial concentration of the phosphate buffer solution in step (4) is 0.05~0.1mol / L, and the pH is 6.8~7.2.

[0013] Preferably, the ultrafiltration membrane in step (4) has a molecular weight cutoff of 25~35kDa.

[0014] Preferably, the column used in step (5) is a DEAE-52 chromatography column; The elution in step (5) is a gradient elution, and the eluent is a phosphate buffer containing 0~0.3 mol / L sodium chloride; The freeze-drying temperature in step (5) is -50~-40℃, and the vacuum degree of freeze-drying is ≤10Pa.

[0015] The present invention also provides a method for extracting phycocyanin from Spirulina using the method, wherein the phycocyanin has a purity ≥3.9.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The cell wall breaking method of the present invention is enzymatic hydrolysis + high pressure homogenization, which can make the cell wall breaking rate of Spirulina ≥95%, and can make Spirulina release a large amount of phycocyanin, which is significantly improved compared with the 65% cell wall breaking rate of ultrasonic waves.

[0017] (2) The extraction solvent of the present invention is phosphate buffer, without the addition of any organic solvent, which avoids the destruction of protein activity by acids, bases and organic solvents, and can extract phycocyanin with high protein activity.

[0018] (3) The crude extract can be purified by salting out, ultrafiltration and column chromatography to obtain a purity of / Phycocyanin with a concentration of ≥3.9.

[0019] (4) The method of the present invention is simple to operate, has mild conditions, low energy consumption, and is suitable for industrial-scale production. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] The spirulina powder used in this embodiment of the invention was purchased from Shanxi Ruilin Panier Biotechnology Co., Ltd. The cellulase and pectinase used in the embodiments of this invention are both food grade and were purchased from Jiangsu Swiss Bioengineering Co., Ltd.

[0022] Example 1

[0023] Take 100g of spirulina powder and add it to 0.08mol / L phosphate buffer (pH 7.0) at a ratio of 1g:18mL. Stir to obtain an algal suspension. Add 0.8g of a complex enzyme (cellulase and pectinase in a mass ratio of 3:1) to the algal suspension and hydrolyze at 37℃ and 180rpm for 40min to obtain an enzymatic hydrolysate. Homogenize the enzymatic hydrolysate three times in a homogenizer at 35MPa for 2min each time to obtain a homogenized solution.

[0024] Centrifuge the homogenized liquid at 10,000 rpm for 20 min, collect the supernatant, filter it through a 0.22 μm filter membrane, collect the filtrate, and obtain the crude extract.

[0025] Add ammonium sulfate crystals to the crude extract to achieve a saturation of 25%, let stand at 4°C for 5 hours, centrifuge at 8000 rpm for 10 minutes, and collect the precipitate. Reconstitute the precipitate with 100 mL of 0.08 mol / L phosphate buffer (pH 7.0), and then filter the reconstituted solution through a 30 kDa ultrafiltration membrane to obtain the ultrafiltrate.

[0026] The ultrafiltrate was loaded onto a DEAE-52 chromatography column and eluted with a gradient of phosphate buffer (pH 7.0) containing 0–0.3 mol / L sodium chloride (NaCl) at a flow rate of 1 mL / min. The peak fraction was absorbed to obtain a purified solution.

[0027] The purified solution was placed at a temperature of -40℃ and a vacuum degree of 10Pa and freeze-dried for 30 hours to obtain a dark blue phycocyanin powder, which was then sealed and stored away from light.

[0028] Example 2

[0029] Take 100g of spirulina powder and add it to 0.1mol / L phosphate buffer (pH 7.2) at a ratio of 1g:15mL. Stir to obtain an algal suspension. Add 0.5g of a complex enzyme (cellulase and pectinase in a mass ratio of 4:1) to the algal suspension and hydrolyze at 38℃ and 150rpm for 50min to obtain an enzymatic hydrolysate. Homogenize the enzymatic hydrolysate twice in a homogenizer at a pressure of 40MPa for 2min each time to obtain a homogenized solution.

[0030] Centrifuge the homogenized liquid at 12000 rpm for 15 min, collect the supernatant, filter it through a 0.22 μm filter membrane, collect the filtrate, and obtain the crude extract.

[0031] Add ammonium sulfate crystals to the crude extract to achieve a saturation of 30%, let stand at 4°C for 6 hours, centrifuge at 7000 rpm for 12 minutes, and collect the precipitate. Reconstitute the precipitate with 100 mL of 0.1 mol / L phosphate buffer (pH 7.2), and then filter the reconstituted solution through a 30 kDa ultrafiltration membrane to obtain the ultrafiltrate.

[0032] The ultrafiltrate was loaded onto a DEAE-52 chromatography column and eluted with a gradient of phosphate buffer (pH 7.0) containing 0–0.3 mol / L sodium chloride (NaCl) at a flow rate of 1 mL / min. The peak fraction was absorbed to obtain a purified solution.

[0033] The purified solution was placed at a temperature of -50℃ and a vacuum degree of 10Pa and freeze-dried for 24 hours to obtain a dark blue phycocyanin powder, which was then sealed and stored away from light.

[0034] Example 3

[0035] Take 100g of spirulina powder and add it to 0.05mol / L phosphate buffer (pH 6.8) at a ratio of 1g:20mL. Stir to obtain an algal suspension. Add 1.2g of a complex enzyme (cellulase and pectinase in a mass ratio of 2:1) to the algal suspension and hydrolyze at 35℃ and 200rpm for 30min to obtain an enzymatic hydrolysate. Homogenize the enzymatic hydrolysate three times in a homogenizer at 30MPa for 2min each time to obtain a homogenized solution.

[0036] Centrifuge the homogenized liquid at 8000 rpm for 25 min, collect the supernatant, filter it through a 0.22 μm filter membrane, collect the filtrate, and obtain the crude extract.

[0037] Add ammonium sulfate crystals to the crude extract to achieve a saturation of 30%, let stand at 6°C for 4 hours, centrifuge at 9000 rpm for 8 minutes, and collect the precipitate. Reconstitute the precipitate with 100 mL of 0.05 mol / L phosphate buffer (pH 6.8), and then filter the reconstituted solution through a 30 kDa ultrafiltration membrane to obtain the ultrafiltrate.

[0038] The ultrafiltrate was loaded onto a DEAE-52 chromatography column and eluted with a gradient of phosphate buffer (pH 7.0) containing 0–0.3 mol / L sodium chloride (NaCl) at a flow rate of 1 mL / min. The peak fraction was absorbed to obtain a purified solution.

[0039] The purified solution was placed at a temperature of -45℃ and a vacuum degree of 10Pa and freeze-dried for 30 hours to obtain a dark blue phycocyanin powder, which was then sealed and stored away from light.

[0040] Comparative Example 1

[0041] Comparative Example 1 was set up according to the method of Example 1. The difference from Example 1 is that no complex enzyme was added to the algal suspension, and no homogenization was performed. Instead, the algal suspension was directly treated with 100W ultrasound for 20 min, centrifuged at 10000 rpm, the supernatant was collected, filtered through a 0.22 μm filter membrane, and the filtrate was collected to obtain the crude extract. The remaining steps were the same as in Example 1.

[0042] Comparative Example 2

[0043] Following the steps of Example 1 in preparing the crude extract, the crude extract of Comparative Example 2 was prepared. After obtaining the crude extract, dipotassium hydrogen phosphate and deionized water were added to it, making the mass fraction of dipotassium hydrogen phosphate 40% and the mass fraction of the crude extract 20%. The mixture was stirred to obtain an aqueous two-phase system. The pH of the aqueous phase system was adjusted to 8.0 and stirred again. The mixture was then allowed to stand at 25°C for 10 min to separate the phases, resulting in an upper phase and a lower phase. Equal amounts of dipotassium hydrogen phosphate and deionized water were added to the upper phase, and the mixture was stirred again. The mixture was then allowed to stand at 25°C for 8 min to separate the phases, resulting in an upper phase and a lower phase. The lower phase was added to 0.08 mol / L phosphate buffer (pH 7.0) and dialyzed at 4°C for 24 h. After that, it was placed under vacuum freeze-drying conditions at -40°C and a vacuum degree of 10 Pa to obtain phycocyanin.

[0044] Comparative Example 3

[0045] Take 100g of Spirulina powder and add it to 0.08mol / L phosphate buffer (pH 7.0) at a ratio of 1g:18mL. Stir to obtain an algal suspension. Treat the algal suspension with 100W ultrasound for 20min, centrifuge at 10000rpm, collect the supernatant, filter through a 0.22μm filter membrane, and collect the filtrate to obtain the crude extract. Add dipotassium hydrogen phosphate and deionized water to the crude extract to make the mass fraction of dipotassium hydrogen phosphate 40% and the mass fraction of the crude extract 20%. Mix well to obtain an aqueous two-phase system. Adjust the pH of the aqueous phase system to 8.0, mix well, and let it stand at 25℃ for 10min to separate the phases into upper and lower phases. Add equal amounts of dipotassium hydrogen phosphate and deionized water to the upper phase, mix well, and let it stand at 25℃ for 8min to separate the phases into upper and lower phases. After adding 0.08 mol / L phosphate buffer (pH 7.0) to the lower phase and dialyzing at 4°C for 24 h, the mixture was then freeze-dried under vacuum at -40°C and 10 Pa to obtain phycocyanin.

[0046] Experimental Example 1

[0047] The homogenized solutions obtained in Examples 1-3 were compared with the ultrasonically treated algal suspension obtained in Example 1, and the cell wall disruption rate of Spirulina was observed under a microscope. Cell wall disruption rate = [number of broken cells / (number of broken cells + number of intact cells)] × 100%. The results are shown in Table 1.

[0048] Table 1. Fragmentation of Spirulina cell walls by different methods

[0049] It can be seen that the method of the present invention can achieve a cell wall breakage rate of over 95% for Spirulina, which is significantly higher than the 65% of Comparative Example 1.

[0050] Experiment Example 2

[0051] Weigh the phycocyanin obtained in Examples 1-3 and Comparative Examples 1-3, and calculate the yield of phycocyanin prepared by different methods. Phycocyanin yield (mg / g) = mass of phycocyanin obtained from extraction and purification (mg) / mass of Spirulina sample (g).

[0052] Phycocyanin extracted was detected by ultraviolet-visible spectrophotometer. and The absorbance, and calculated according to the formula phycocyanin purity = / The results are shown in Table 2.

[0053] Table 2. Effects of different methods on the purity of phycocyanin obtained from extraction and purification.

[0054] As can be seen, using Examples 1-3 of the present invention, phycocyanin with a yield of 210.5-234.5 mg / g and a purity as high as 3.95-4.16 can be obtained. The yield and purity of phycocyanin are higher than those of Comparative Example 1 treated with ultrasonic cell disruption, higher than those of Comparative Example 2 treated with aqueous two-phase extraction, and also higher than those of Comparative Example 3 treated with ultrasonic + aqueous two-phase extraction.

[0055] As can be seen from the above embodiments, the present invention provides a method for extracting high-purity phycocyanin from Spirulina. The method of the present invention includes the following steps: (1) mixing Spirulina powder with phosphate buffer to obtain an algal suspension; (2) adding a complex enzyme to the algal suspension, shaking and hydrolyzing for 30-50 min to obtain an enzymatic hydrolysate; homogenizing the enzymatic hydrolysate 2-4 times to obtain a homogenized solution; (3) centrifuging the homogenized solution, taking the supernatant, filtering, taking the filtrate to obtain a crude extract; (4) adding ammonium sulfate crystals to the crude extract to make its saturation 25-30%, letting it stand for 4-6 h, centrifuging, taking the precipitate, mixing the precipitate with phosphate buffer, ultrafiltration to obtain an ultrafiltrate; (5) passing the ultrafiltrate through a column, eluting, and collecting A. 620 The fraction from the main peak is freeze-dried to obtain phycocyanin. The method of this invention can effectively disrupt the cell wall of Spirulina, improving the yield and purity of phycocyanin. The yield of phycocyanin can reach 210.5~234.5 mg / g, and the purity can reach 3.95~4.16, which is higher than that of existing extraction methods. This provides a basis for the efficient extraction of phycocyanin.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting high purity phycocyanin from Spirulina, characterized by, The method comprises the following steps: (1) mixing spirulina powder with phosphate buffer to obtain an algae suspension; (2) adding a complex enzyme to the algae suspension, oscillating for 30-50 min to obtain an enzyme hydrolysate; homogenizing the enzyme hydrolysate 2-4 times to obtain a homogenate; (3) centrifuging the homogenate, taking the supernatant, filtering, and taking the filtrate to obtain a crude extract; (4) adding ammonium sulfate crystals to the crude extract to a saturation degree of 25-30%, standing for 4-6 h, centrifuging, taking the precipitate, mixing the precipitate with phosphate buffer, and ultrafiltering to obtain an ultrafiltrate; (5) The ultrafiltrate is passed through a column, eluted, and the A 620 The main peak fraction is freeze-dried to obtain phycocyanin.

2. The method of claim 1, wherein, The mass-volume ratio of the spirulina powder to the phosphate buffer in step (1) is 1 g: 15-20 mL; The initial concentration of the phosphate buffer is 0.05-0.1 mol / L; The pH of the phosphate buffer is 6.8-7.

2.

3. The method of claim 1, wherein, The mass ratio of the spirulina powder to the complex enzyme is 100: 0.5-1.2; The complex enzyme comprises cellulase and pectinase, and the mass ratio of the cellulase to the pectinase in the complex enzyme is 2-4:

1.

4. The method of claim 1, wherein, The oscillation speed of the oscillation enzyme hydrolysis is 150-200 rpm; The oscillation temperature of the oscillation enzyme hydrolysis is 35-39 ℃.

5. The method of claim 1, wherein, The pressure of the homogenization is 30-40 MPa.

6. The method of claim 1, wherein, The centrifugation speed in step (3) is 8000-12000 rpm, and the centrifugation time is 15-25 min; The filtration in step (3) is membrane filtration, and the pore size of the membrane is 0.2-0.3 μm.

7. The method of claim 1, wherein, The centrifugation speed in step (4) is 7000-9000 rpm, and the centrifugation time is 8-12 min; The initial concentration of the phosphate buffer in step (4) is 0.05-0.1 mol / L, and the pH is 6.8-7.

2.

8. The method of claim 1, wherein, The molecular weight cut-off of the ultrafiltration membrane in step (4) is 25-35 kDa.

9. The method of claim 1, wherein, The column in step (5) is a DEAE-52 chromatography column; The elution in step (5) is gradient elution, and the eluent is a phosphate buffer containing 0-0.3 mol / L of sodium chloride; The temperature of the freeze-drying in step (5) is -50--40 ℃, and the vacuum degree of the freeze-drying is ≤10 Pa.

10. The method of any one of claims 1 to 9, wherein the phycocyanin is extracted from Spirulina, characterized in that, The purity of the phycocyanin is ≥3.9.

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