Method for extracting phycocyanin from spirulina

Abstract

The invention discloses a method for extracting phycocyanin from spirulina, and belongs to the field of phycocyanin preparation. The method comprises the following steps: taking spirulina, adding water to obtain a spirulina suspension, performing freeze thawing, intermittent ultrasonic treatment and centrifugation, and collecting supernate to obtain a crude extract of phycocyanin; adding a (NH4) 2SO4 solution into the protein crude extract, standing, and centrifuging to obtain a salting-out precipitate; adding the salting-out precipitate into a mixture of ethylene glycol and inorganic salt, magnetically stirring and layering to obtain a protein solution; and carrying out ultrafiltration, centrifugation and freeze drying on the protein solution to obtain the spirulina phycocyanin. The purity of the phycocyanin can reach 1.0, the extraction rate can reach 69.87%, and the purity and the extraction rate of the phycocyanin extracted through the method are both improved. The extraction method provided by the invention has the advantages of simple process flow, low cost, high efficiency and strong practicability, and is suitable for large-scale industrial production of high-purity phycocyanin.

Description

Technical Field

[0001] The invention relates to the field of phycocyanin preparation, in particular to a method for extracting phycocyanin from spirulina. Background Art

[0002] Spirulina is a low-level aquatic plant belonging to the Cyanophyta, Cyanobacteria, Oscillatoriaceae, Spirulina or Arthrospira genus. It is native to Africa and North America and is a low-level aquatic algae plant that has existed on Earth for more than 3 billion years. It is blue-green in appearance and spiral-shaped under microscope observation, hence the name Spirulina. Spirulina is rich in phycocyanin, vitamins, carotene and polysaccharides, and is an ideal food for humans. Its protein content is as high as 60% to 70%. It is a single-cell spiral filamentous blue algae that has been cultivated on a large scale in recent years. Because Spirulina is rich in nutrients and rich in various medicinal properties, it has attracted widespread attention.

[0003] Phycocyanin is a blue powder, non-toxic, soluble in water, insoluble in alcohol and oil. It is a pigment-protein complex present in cyanobacteria and spirulina cells. Phycocyanin is a natural colorant that is used in chewing gum, ice cream, beverages, candy and cosmetics. Phycocyanin is also used as a biochemical tracer due to its fluorescent properties. In addition to being able to capture light energy to provide energy for algae cells, phycocyanin also has many physiological activities such as anti-oxidation, enhancing the body's immunity, anti-cancer, anti-inflammatory and liver protection. It is widely used in food, cosmetics and medicine. Comprehensive understanding and mastering of the physiological regulation function and mechanism of phycocyanin has important guiding significance for the development and utilization of phycocyanin.

[0004] Phycocyanin extraction and purification methods are mainly divided into three steps: cell wall breaking, crude extraction and purification. Among them, the methods of cell disruption include swelling method, ultrasonic cell wall breaking method, freeze-thaw method, mechanical cell wall breaking method, chemical reagent method, lysozyme method, etc. In the experiment, one or two methods are often used in combination to break the cell wall of phycocyanin. Phycocyanin extraction generally adopts salting-out method, isoelectric precipitation method, ultrafiltration method and adsorption method. Among them, adsorption method is to use adsorbent to adsorb impurities or proteins to extract proteins. Commonly used adsorbents include activated carbon and chitosan. Compared with the salting-out method, this method has the advantages of cheap reagents, no environmental pollution and less dosage. A small amount of adsorbent can adsorb impurities without a large amount of ammonium sulfate, but its disadvantage is that it is necessary to accurately control the dosage and adsorption time of the adsorbent, otherwise the target protein is easily adsorbed away, reducing the extraction rate. In general, there are many research reports on the extraction of phycocyanin from Spirulina in the prior art, but its extraction rate is not high, and the extraction cost of high-purity phycocyanin is high and expensive, and large-scale production in industry is still the key and difficult point. Summary of the invention

[0005] The object of the present invention is to provide a method for extracting phycocyanin from Spirulina to solve the problems existing in the above-mentioned prior art. The purity and extraction rate of the phycocyanin extracted by the method of the present invention are both increased. The extraction method of the present invention has a simple technological process, low cost, high efficiency and strong practicability, and is applicable to large-scale industrial production of high-purity phycocyanin.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for extracting phycocyanin from Spirulina, comprising the following steps: taking Spirulina and adding water to obtain a Spirulina suspension, subjecting it to freeze-thaw, intermittent ultrasonic treatment, and centrifugation, and collecting the supernatant to obtain a crude extract of phycocyanin;

[0008] Adding an (NH4)2SO4 solution to the crude protein extract, standing and centrifuging to obtain a salting-out precipitate;

[0009] Adding the salting-out precipitate to a mixture of ethylene glycol and inorganic salt, magnetically stirring, and layering to obtain a protein solution;

[0010] Subjecting the protein solution to ultrafiltration, centrifugation, and freeze-drying to obtain the Spirulina phycocyanin.

[0011] Optionally, the conditions of the intermittent ultrasonic treatment are: the ultrasonic power is 200W, the ultrasonic treatment is for 10s, the rest is for 10s, and the cycle is until the total time is 50 - 60min.

[0012] Optionally, the material-liquid ratio of the Spirulina to water is (1 - 10) g: 100 mL.

[0013] Optionally, the time of the freeze-thaw is 1.5 - 2.5h.

[0014] Optionally, in the mixture of ethylene glycol and inorganic salt, the concentration of ethylene glycol is 13% - 17%, and the concentration of inorganic salt is 10% - 11%.

[0015] Optionally, the inorganic salt includes potassium phosphate and sodium phosphate, and the mass ratio of the potassium phosphate to the sodium phosphate is 1: (1 - 1.5).

[0016] Optionally, the cut-off molecular weight of the ultrafiltration is 12KDa - 25KDa.

[0017] The present invention also provides a phycocyanin, which is obtained by the method described above.

[0018] The present invention discloses the following technical effects:

[0019] The present invention combines the freeze-thaw and intermittent ultrasonic treatment technologies to provide a method for extracting phycocyanin from Spirulina. The purity of the extracted phycocyanin can reach 1.0 or above, and the extraction rate can reach 69.87% or above. The purity and extraction rate of the phycocyanin extracted by the method of the present invention are both increased.

[0020] The extraction method of the present invention has a simple process flow, low cost, high efficiency, and strong practicability, and is suitable for large-scale industrial production of high-purity phycocyanin. Detailed implementation manners

[0021] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0025] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0026] The Spirulina used in the present invention was purchased from Hebei Zhongzhisheng Biotechnology Co., Ltd., CAS: 724424-92-4.

[0027] Phycocyanin has a maximum absorption peak at 620 nm, protein has a maximum absorption peak at 280 nm, and allophycocyanin has a maximum absorption peak at 650 nm. According to this property, a UV-visible spectrophotometer can be used to measure the absorbance of the protein solution at 280 nm, 620 nm, and 650 nm, so as to calculate the purity and extraction rate of phycocyanin.

[0028] The purity of phycocyanin is represented by P, and P = A 620 / A 280 ; the concentration of phycocyanin is represented by C, and C (mg / mL) = (A 620 - 0.474×A 650 ) / 5.34; the extraction rate of phycocyanin is represented by Y, and Y (%) = (C×V0) / (m0×1000)×100%, where C is the concentration of phycocyanin (mg / mL), V0 is the volume of the crude extract (mL), and m0 is the mass of the Spirulina powder (g).

[0029] Example 1

[0030] A method for extracting phycocyanin from Spirulina includes the following steps:

[0031] Take Spirulina powder, add water according to the solid-liquid ratio of 5 g: 100 mL, fully mix to obtain a Spirulina powder suspension, freeze-thaw the Spirulina powder suspension at -20 °C and 37 °C for 1 time, the freeze-thaw time is 2 h, and then perform intermittent ultrasonic treatment under the conditions of a power of 200 W, working for 10 s, pausing for 10 s, cycling until the total time reaches 60 min; then centrifuge at 5000 rpm for 30 min, and collect the supernatant to obtain the crude extract of phycocyanin;

[0032] Add (NH4)2SO4 solution to the crude protein extract to 80% saturation, stand at 3 °C for 10 h, and centrifuge to obtain the salting-out precipitate;

[0033] Aqueous two-phase extraction: Prepare a mixture of ethylene glycol and inorganic salts (a mixture of potassium phosphate and sodium phosphate in a mass ratio of 1:1) with a pH of 6.0, an ethylene glycol concentration of 15%, and an inorganic salt concentration of 11%, add the salting-out precipitate, stir magnetically, and then transfer to a separating funnel for layering. The upper layer is the protein solution;

[0034] Ultrafiltration: Place the protein solution in an ultrafiltration centrifuge tube with a molecular weight cut-off of 20 KDa, centrifuge, and freeze-dry to obtain Spirulina phycocyanin. This step can effectively remove ethylene glycol from the protein solution without destroying the active protein, increase the protein concentration and ensure that the protein purity does not decrease or even increases.

[0035] The purity of the prepared phycocyanin is 1.3, and the extraction rate is 72.15%.

[0036] Example 2

[0037] Take Spirulina powder, add water according to the ratio of solid to liquid of 1 g: 100 mL, and mix well to obtain a Spirulina powder suspension. Place the Spirulina powder suspension at -20 °C and 37 °C for 1 freeze-thaw cycle with a freeze-thaw time of 2.5 h. Then, perform intermittent ultrasonic treatment under the conditions of a power of 200 W, working for 10 s, pausing for 10 s, and cycling until the total time reaches 50 min. Then, centrifuge at 5000 rpm for 30 min, and collect the supernatant to obtain a crude extract of phycocyanin.

[0038] Add (NH4)2SO4 solution to the crude protein extract to 80% saturation, let it stand at 3 °C for 12 h, and then centrifuge to obtain a salting-out precipitate.

[0039] Aqueous two-phase extraction: Prepare a mixture of ethylene glycol and inorganic salts (a 1:1.5 mixture of potassium phosphate and sodium phosphate) with a pH of 6.0, an ethylene glycol concentration of 13%, and an inorganic salt concentration of 10%. Add the salting-out precipitate, stir magnetically, and then transfer it to a separatory funnel for layering. The upper layer is the protein solution.

[0040] Ultrafiltration: Place the protein solution in an ultrafiltration centrifugal tube with a molecular weight cut-off of 12 KDa, centrifuge, and freeze-dry to obtain Spirulina phycocyanin.

[0041] The purity of the prepared phycocyanin is 1.0, and the extraction rate is 69.87%.

[0042] Example 3

[0043] Take Spirulina powder, add water according to the ratio of solid to liquid of 10 g: 100 mL, and mix well to obtain a Spirulina powder suspension. Place the Spirulina powder suspension at -20 °C and 37 °C for 1 freeze-thaw cycle with a freeze-thaw time of 1.5 h. Then, perform intermittent ultrasonic treatment under the conditions of a power of 200 W, working for 10 s, pausing for 10 s, and cycling until the total time reaches 60 min. Then, centrifuge at 5000 rpm for 30 min, and collect the supernatant to obtain a crude extract of phycocyanin.

[0044] Add (NH4)2SO4 solution to the crude protein extract to 80% saturation, let it stand at 3 °C for 14 h, and then centrifuge to obtain a salting-out precipitate.

[0045] Aqueous two-phase extraction: Prepare a mixture of ethylene glycol and inorganic salts (a 1:1 mixture of potassium phosphate and sodium phosphate) with a pH of 6.0, an ethylene glycol concentration of 17%, and an inorganic salt concentration of 11%. Add the salting-out precipitate, stir magnetically, and then transfer it to a separatory funnel for layering. The upper layer is the protein solution.

[0046] Ultrafiltration: Place the protein solution in an ultrafiltration centrifugal tube with a molecular weight cut-off of 25 KDa, centrifuge, and freeze-dry to obtain phycocyanin from Spirulina platensis.

[0047] The purity of the prepared phycocyanin is 1.1, and the extraction rate is 71.39%.

[0048] Comparative Example 1

[0049] A method for extracting phycocyanin from Spirulina platensis is the same as that in Example 1, except that the conditions of intermittent ultrasonic treatment are modified to a power of 300 W, working for 30 s, pausing for 30 s, and cycling until the total time reaches 60 min. The specific steps are as follows.

[0050] Take Spirulina platensis dry powder, add water according to a solid-liquid ratio of 5 g:100 mL, and mix well to obtain a Spirulina platensis powder suspension. Freeze-thaw the suspension at -20 °C and 37 °C once for 2 h, and then perform intermittent ultrasonic treatment under the conditions of a power of 300 W, working for 30 s, pausing for 30 s, and cycling until the total time reaches 60 min; then centrifuge at 5000 rpm for 30 min, and collect the supernatant to obtain the crude extract of phycocyanin;

[0051] Add (NH4)2SO4 solution to the crude protein extract to 80% saturation, let it stand at 3 °C for 10 h, and centrifuge to obtain the salting-out precipitate;

[0052] Aqueous two-phase extraction: Prepare a mixture of ethylene glycol and inorganic salts (a 1:1 mixture of potassium phosphate and sodium phosphate) with a pH of 6.0, an ethylene glycol concentration of 15%, and an inorganic salt concentration of 11%. Add the salting-out precipitate, stir magnetically, and then transfer it to a separating funnel for layering. The upper layer is the protein solution;

[0053] Ultrafiltration: Place the protein solution in an ultrafiltration centrifugal tube with a molecular weight cut-off of 20 KDa, centrifuge, and freeze-dry to obtain phycocyanin from Spirulina platensis.

[0054] The purity of the prepared phycocyanin is 0.6, and the extraction rate is 54.27%.

[0055] Comparative Example 2

[0056] A method for extracting phycocyanin from Spirulina platensis is the same as that in Example 1, except that the conditions of intermittent ultrasonic treatment are modified to a power of 200 W, working for 5 s, pausing for 5 s, and cycling until the total time reaches 60 min. The specific steps are as follows.

[0057] Take the dry powder of Spirulina, add water according to the ratio of solid to liquid of 5 g: 100 mL, and mix well to obtain a Spirulina powder suspension. Place the Spirulina powder suspension at -20 °C and 37 °C for one freeze-thaw cycle with a freeze-thaw time of 2 h. Then, perform intermittent ultrasonic treatment under the conditions of a power of 200 W, working for 5 s and pausing for 5 s in a cycle until the total time reaches 60 min. Then, centrifuge at 5000 rpm for 30 min and collect the supernatant to obtain the crude extract of phycocyanin;

[0058] Add (NH4)2SO4 solution to the crude protein extract to 80% saturation, let it stand at 3 °C for 10 h, and then centrifuge to obtain the salting-out precipitate;

[0059] Aqueous two-phase extraction: Prepare a mixture of ethylene glycol and inorganic salts (a 1:1 mixture of potassium phosphate and sodium phosphate) with a pH of 6.0, an ethylene glycol concentration of 15%, and an inorganic salt concentration of 11%. Add the salting-out precipitate, stir magnetically, and then transfer it to a separating funnel for stratification. The upper layer is the protein solution;

[0060] Ultrafiltration: Place the protein solution in an ultrafiltration centrifuge tube with a molecular weight cut-off of 20 KDa, centrifuge, and freeze-dry to obtain Spirulina phycocyanin.

[0061] The purity of the prepared phycocyanin is 0.7, and the extraction rate is 52.34%.

[0062] Comparative Example 3

[0063] A method for extracting phycocyanin from Spirulina is the same as that in Example 1, except that instead of using intermittent ultrasonic treatment, continuous ultrasonic treatment is used. The specific steps are as follows.

[0064] Take the dry powder of Spirulina, add water according to the ratio of solid to liquid of 5 g: 100 mL, and mix well to obtain a Spirulina powder suspension. Place the Spirulina powder suspension at -20 °C and 37 °C for one freeze-thaw cycle with a freeze-thaw time of 2 h. Then, perform ultrasonic treatment at a power of 200 W for 60 min. Then, centrifuge at 5000 rpm for 30 min and collect the supernatant to obtain the crude extract of phycocyanin;

[0065] Add (NH4)2SO4 solution to the crude protein extract to 80% saturation, let it stand at 3 °C for 10 h, and then centrifuge to obtain the salting-out precipitate;

[0066] Aqueous two-phase extraction: Prepare a mixture of ethylene glycol and inorganic salts (a 1:1 mixture of potassium phosphate and sodium phosphate) with a pH of 6.0, an ethylene glycol concentration of 15%, and an inorganic salt concentration of 11%. Add the salting-out precipitate, stir magnetically, and then transfer it to a separating funnel for stratification. The upper layer is the protein solution;

[0067] Ultrafiltration: Place the protein solution in an ultrafiltration centrifuge tube with a molecular weight cut-off of 20 KDa, centrifuge, and freeze-dry to obtain phycocyanin from Spirulina platensis.

[0068] The purity of the prepared phycocyanin is 0.6, and the extraction rate is 55.34%.

[0069] Comparative Example 4

[0070] A method for extracting phycocyanin from Spirulina platensis is the same as that in Example 1, except that the Spirulina powder suspension is not subjected to freeze-thaw, and the specific steps are as follows.

[0071] Take Spirulina dry powder, add water according to a solid-liquid ratio of 5 g: 100 mL, and mix well to obtain a Spirulina powder suspension. Then, perform intermittent ultrasonic treatment under the conditions of a power of 200 W, working for 10 s, pausing for 10 s, and cycling until the total time reaches 60 min; then centrifuge at 5000 rpm for 30 min, and collect the supernatant to obtain a crude extract of phycocyanin;

[0072] Add (NH4)2SO4 solution to the crude protein extract to 80% saturation, let it stand at 3 °C for 10 h, and centrifuge to obtain a salting-out precipitate;

[0073] Aqueous two-phase extraction: Prepare a mixture of ethylene glycol and inorganic salts (a 1:1 mixture of potassium phosphate and sodium phosphate) with a pH of 6.0, an ethylene glycol concentration of 15%, and an inorganic salt concentration of 11%. Add the salting-out precipitate, stir magnetically, and then transfer it to a separatory funnel for layering. The upper phase is the protein solution;

[0074] Ultrafiltration: Place the protein solution in an ultrafiltration centrifuge tube with a molecular weight cut-off of 20 KDa, centrifuge, and freeze-dry to obtain phycocyanin from Spirulina platensis.

[0075] The purity of the prepared phycocyanin is 0.7, and the extraction rate is 50.58%.

[0076] From the data comparison between Example 1 and Comparative Examples 1-3, it can be seen that in Comparative Examples 1-3, due to the change in the intermittent and working time, the purity and extraction rate of phycocyanin are significantly lower than those in Example 1. From the data comparison between Example 1 and Comparative Example 4, it can be seen that in Comparative Example 4, due to the lack of freeze-thaw treatment of the Spirulina powder suspension, the purity and extraction rate of phycocyanin are significantly lower than those in Example 1. Only by using the intermittent ultrasonic treatment with a power of 200 W, working for 10 s, pausing for 10 s, and cycling until the total time reaches 60 min combined with freeze-thaw conditions provided by the present invention can the effect of improving the purity and extraction rate of phycocyanin be achieved.

[0077] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting phycocyanin from Spirulina, characterized in that, The following steps are involved: Adding spirulina to water to obtain a spirulina suspension, subjecting the suspension to freeze-thaw, intermittent ultrasonic treatment, and centrifugation, and collecting the supernatant to obtain a crude extract of phycocyanin; Add (NH4)2SO4 solution to the crude protein extract, let stand, and centrifuge to obtain a salting-out precipitate; adding the salting-out precipitate into a mixture of ethylene glycol and an inorganic salt, stirring with a magnetic force, and separating the layers to obtain a protein solution; The protein solution is subjected to ultrafiltration, centrifugation and freeze-drying to obtain the Spirulina phycocyanin.

2. The method according to claim 1, wherein The conditions of the intermittent ultrasonic treatment are as follows: ultrasonic power is 200 W, ultrasonic treatment is performed for 10 seconds, and rest is performed for 10 seconds, and the cycle is repeated until the total time is 50-60 minutes.

3. The method according to claim 1, wherein The solid-liquid ratio of the spirulina to water is (1-10) g:100 mL.

4. The method according to claim 1, wherein The freeze-thaw time is 1.5-2.5h.

5. The method according to claim 1, wherein In the mixture of ethylene glycol and inorganic salt, the concentration of ethylene glycol is 13%-17%, and the concentration of inorganic salt is 10%-11%.

6. The method according to claim 5, wherein The inorganic salt includes potassium phosphate and sodium phosphate, and the mass ratio of the potassium phosphate to the sodium phosphate is 1:(1-1.5).

7. The method according to claim 1, wherein The molecular weight cut-off of the ultrafiltration is 12KDa-25KDa.

8. A phycocyanin, characterized in that, The phycocyanin is extracted by the method according to any one of claims 1 to 7.