WO2023015792A1

WO2023015792A1 - Method for rapidly extracting phycocyanin

Info

Publication number: WO2023015792A1
Application number: PCT/CN2021/137050
Authority: WO
Inventors: 黄成谭; 潘军; 叶蕾; 黄敏
Original assignee: 海南绿藻世界生物科技有限公司
Priority date: 2021-08-12
Filing date: 2021-12-10
Publication date: 2023-02-16
Also published as: CN113480638A; CN113480638B

Abstract

The present invention relates to the technical field of protein extraction, in particular to a method for rapidly extracting phycocyanin. The method comprises the following steps: mixing aquaculture water containing microalgae with a protein protectant, and performing a first centrifugation to obtain concentrated algae liquid; mixing the concentrated algae liquid with a cell cryoprotectant, and performing repeated freezing and thawing to obtain thawed concentrated algae liquid; mixing the thawed concentrated algae liquid with the protein protectant and a surfactant, and crushing cells to obtain crushed algae liquid; subjecting the crushed algae liquid to a second centrifugation to obtain supernatant; mixing the supernatant with ammonium sulfate and polypropylene glycol, and performing stirring; subjecting the stirred solution to a third centrifugation to obtain a precipitate; and washing the precipitate with an ethanol aqueous solution, performing a fourth centrifugation, and freeze-drying the precipitate. The extraction method of the present invention has simple and rapid operation and a low cost and the obtained phycocyanin has a high purity.

Description

A kind of rapid extraction method of phycocyanin

This application claims the priority of the Chinese patent application submitted to the China Patent Office on August 12, 2021, with the application number 202110925095.8 and the title of the invention "A Method for Rapid Extraction of Phycocyanin", the entire contents of which are incorporated herein by reference Applying.

technical field

The invention relates to the technical field of protein extraction, in particular to a rapid extraction method for phycocyanin.

Background technique

Microalgae is one of the oldest species on the earth. It is rich in nutrients and has a wide range of applications in animal husbandry, environmental protection, medicine, cosmetics, food and other fields. It is also praised by the World Food and Agriculture Organization as the most ideal for human beings in the 21st century. The most perfect food. At the same time, microalgae are the bait (or bait of bait) of aquaculture animals for life or specific developmental stages, and as one of the important foundations, they support the aquaculture industry to a large extent. Nannochloropsis grows rapidly, has small cell particles, is rich in EPA and other unsaturated fatty acids, has comprehensive nutrition, and has the characteristics of thicker cell walls. Microalgae is the primary productivity of the water ecosystem, rich in protein, polyunsaturated fatty acids, carotenoids, vitamins and other nutrients; while providing food for aquatic animals, microalgae can also absorb excess water in the water through photosynthesis. Ammonium nitrogen, phosphorus and other substances can stabilize and improve water quality, so it is widely used in aquaculture.

Phycocyanin is an important nutrient component in microalgae, a kind of photosynthetic accessory pigment commonly found in microalgae cells, and a kind of compound composed of open-chain tetrachloride and apoprotein. It is an important photosynthetic natural pigment in microalgae cells, and it can preferentially transmit light energy to the photosystem with almost high efficiency in photosynthesis. Many studies have shown that phycocyanin has biological properties such as improving the body's radiation resistance, anti-oxidation, anti-inflammation, anti-cancer, anti-aging, antibacterial, protecting nerve tissue from damage, enhancing body immunity, protecting the liver, and immunofluorescence. Activity, has been widely used in food, cosmetics, medicine, molecular probes and other fields. Phycocyanin is ubiquitous in microalgae, and its content is as high as 20% in the algae flour of Amputum algae. It is rich in eight essential amino acids for the human body, has great development value in nature, and is a protein resource for food and bait. Phycocyanin has two important uses: ①The crude product can be used as natural food coloring (sky blue); ②The pure product can be used as a fluorescent molecular probe, which is mainly used for early detection of cancer, routine detection or prepared as a rapid detection kit. Rely on imports.

There are still several major technical problems in the processing of phycocyanin to varying degrees: such as low extraction rate, high cost, and unstable properties. These problems often affect the color and efficacy of phycocyanin, and affect the commodity value of the product. At present, more than two methods of breaking the algae cell wall are used, such as: repeated freezing and thawing combined with ultrasonic treatment. Although the phycocyanin can be released from the microalgae to a large extent, the method is cumbersome and time-consuming. , high energy consumption, high equipment cost and the variability of proteins in ultrasonic treatment. The large-scale purification technology of phycocyanin is not yet mature, and there are salting-out method, chromatography, two-phase extraction and so on. The salting-out method is mild and easy to concentrate, but the purity of phycocyanin obtained by this method is not high. The large-scale purification technology of phycocyanin mostly adopts chromatographic purification technology, which is easy to scale up and large-scale production. However, this technology faces problems such as expensive chromatographic column packing, harsh purification conditions, and difficult product recovery and preparation. At present, the vast majority of phycocyanin extraction research is still in the laboratory stage, and the methods are relatively cumbersome and complicated, with low yield, low purity, and high cost, making it difficult to realize industrialization. The extraction and purification of phycocyanin mainly use gel adsorption and ion chromatography, but the operation is complex and needs to be combined with each other, which is not conducive to the purification of phycocyanin.

Contents of the invention

In view of this, the present invention provides a rapid extraction method of phycocyanin. The extraction method is simple and fast in operation, low in cost, and the obtained phycocyanin has high purity.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a fast extraction method of phycocyanin, comprising the following steps:

Step (1) mixing cultured water containing microalgae with a protein protectant, and performing a first centrifugation to obtain concentrated algae liquid;

Step (2) mixing the concentrated algae liquid with a cell cryoprotectant, and repeatedly freezing and thawing to obtain the thawed concentrated algae liquid;

Step (3) mixing the thawed concentrated algae liquid with a protein protectant and a surfactant, and crushing the cells to obtain the crushed algae liquid;

Step (4) performing a second centrifugation on the crushed algae liquid to obtain a supernatant;

Step (5) mixing the supernatant with ammonium sulfate and polypropylene glycol, stirring;

In step (6), the stirred feed liquid is subjected to a third centrifugation to obtain a precipitate;

In step (7), the obtained precipitate is washed with an aqueous ethanol solution, and after the fourth centrifugation, the precipitate is freeze-dried.

Preferably, the protein protecting agent is sodium azide.

Preferably, in step (1), in the culture water containing microalgae, the amount of the protein protection agent added is 0.1-0.5 mg/L.

In the specific example provided by the present invention, in the culture water containing microalgae, the protein protection agent is added in an amount of 0.2 mg/L.

Preferably, the rotational speed of the first centrifugation is 5000-10000 rpm, and the water content of the concentrated algae liquid is 50%-70%.

In the specific embodiment provided by the present invention, the rotational speed of the first centrifugation is 8000 rpm, and the water content of the concentrated algae solution is 58%.

As preferably, the cell cryoprotectant is glycerol;

Preferably, in step (2), in the concentrated algae liquid, the added amount of the cell cryoprotectant is 5-10 g/kg.

In the specific example provided by the present invention, the added amount of the cell cryoprotectant is 10 g/kg in the concentrated algae liquid.

Preferably, the procedure of repeated freezing and thawing is: freezing at -20 to -10°C for 12 to 24 hours, and thawing at 10 to 30°C for 6 to 12 hours, repeated 2 to 3 times.

Preferably, the procedure of repeated freezing and thawing is: freezing at -18°C for 12 to 24 hours, and thawing at room temperature for 6 to 12 hours, repeated 2 to 3 times.

In the specific example provided by the present invention, the procedure of repeated freezing and thawing is: freezing at -18°C for 12 hours, thawing at room temperature for 6 hours, and repeating twice.

As preferably, surfactant is Tween 80 and Dow's DF104 polyether defoamer;

As a preference, in step (3), in the concentrated algae liquid after thawing, the addition amount of protein protection agent is 1-2mg/L, the addition amount of Tween 80 is 1-5mL/L, Dow DF104 polyether defoaming The dosage of the agent is 1-2mL/L.

In the specific example provided by the present invention, in the concentrated algae liquid after thawing, the addition amount of protein protection agent is 2mg/L, the addition amount of Tween 80 is 5mL/L, the addition amount of Dow DF104 polyether defoamer The amount added is 1mL/L.

Preferably, the rotational speed of the second centrifugation is 2000-3000 rpm, and the time is 40-60 minutes.

In a specific embodiment provided by the present invention, the rotational speed of the second centrifugation is 2000 rpm, and the time is 40 minutes.

As preferably, in step (5), in the supernatant, the addition of ammonium sulfate is 10-15g/L, and the addition of polypropylene glycol is 5-30mL/L;

In the specific embodiment provided by the invention, in the supernatant, the add-on of ammonium sulfate is 10g/L, and the add-on of polypropylene glycol is 20mL/L

Preferably, in step (5), the stirring speed is 500-1000 rpm, and the stirring time is 1-2 hours.

In a specific embodiment provided by the present invention, the stirring speed is 1000 rpm, and the stirring time is 1 h.

Preferably, the molecular weight of polypropylene glycol is 1500-2000.

In the specific example provided by the present invention, the molecular weight of polypropylene glycol is 1500.

Preferably, the rotational speed of the third centrifugation is 5000-10000 rpm, and the time is 20-40 minutes.

In a specific embodiment provided by the present invention, the rotation speed of the third centrifugation is 5000 rpm, and the time is 30 minutes.

As preferably, the concentration of ethanol aqueous solution is 20%～50%;

Preferably, the concentration of the aqueous ethanol solution is 30%.

Preferably, the rotation speed of the fourth centrifugation is 5000-10000 rpm, and the time is 20-40 minutes.

In a specific embodiment provided by the present invention, the rotation speed of the fourth centrifugation is 5000 rpm, and the time is 30 minutes.

In the present invention, the microalgae are selected from algae of Cyanophyta, Chlorophyta, Chrysophyta or Rhodophyta. Such as Chlorella, Nannochloropsis and so on.

The invention provides a rapid extraction method of phycocyanin. The method comprises the following steps: mixing cultured water containing microalgae with a protein protectant, and performing first centrifugation to obtain a concentrated algae liquid; mixing the concentrated algae liquid with a cell cryoprotectant, and repeatedly freezing and thawing to obtain a concentrated algae liquid after thawing Algae liquid; mix the thawed concentrated algae liquid with protein protectant and surfactant, and then crush the cells to obtain the broken algae liquid; perform second centrifugation on the broken algae liquid to obtain the supernatant; The liquid is mixed with ammonium sulfate and polypropylene glycol, and stirred; the stirred material liquid is subjected to a third centrifugation to obtain a precipitate; the obtained precipitate is washed with an aqueous ethanol solution, and the precipitate is freeze-dried after the fourth centrifugation. The technical effect that the present invention has is:

In the present invention, sodium azide is used as a protein protecting agent to avoid protein denaturation and decomposition during the centrifugation process and form impurities. Glycerol has a cytoprotective effect, avoiding the formation of large ice crystals during the freezing process and destroying the protein structure.

In step (2), the microalgae undergoes repeated freezing and dissolving, which can destroy the hard cell wall of the microalgae, which is more conducive to the homogenization and crushing efficiency.

In step (3), Tween 80 and Dow DF104 polyether defoamer form a mixed surfactant, which can precipitate phycocyanin in the supernatant when used in combination.

The combined use of ammonium sulfate and polypropylene glycol in step (5) can precipitate the phycocyanin dissolved in the supernatant to form a particle precipitate.

In step (7), the residual ammonium sulfate, polypropylene glycol and other impurities can be washed away by using the aqueous ethanol solution, so that the purity of phycocyanin can be improved.

Through the multi-step simple and quick extraction and purification operation of the present invention, the purity of the freeze-dried phycocyanin dry powder can reach 99%, the protein capture rate is 87%, and the protein activity is 98%.

Detailed ways

The invention discloses a rapid extraction method of phycocyanin, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

A method for rapidly extracting microalgae phycocyanin provided by the present invention specifically comprises:

1. Add sodium azide to the cultured microalgae (microalgae, chlorella, Nannochloropsis, etc.) at a rate of 0.1-0.5 mg/L. Then the microalgae liquid is centrifuged through a disc centrifuge to extract the algae cells to form a concentrated algae liquid. The rotating speed of the centrifuge is 5000-10000rpm, and the water content of the separated algae liquid is 50%-70%.

2. Add glycerol (100% purity, 5-10g/kg concentrated algae) to the separated concentrated algae liquid, stir evenly, and place in a -18°C refrigerator for freezing for 12-24 hours.

3. Thaw the frozen concentrated algae liquid at room temperature for 6-12 hours.

4. Repeat the second step and the third step 2-3 times.

5. Add sodium azide (addition amount: 1-2 mg/L), Tween 80 (addition amount: 1-5 ml/L) and Dow DF104 polyether defoamer (1- 2mL/L), mix well, and place in a homogenizer for cell disruption.

6. Centrifuge the crushed algae liquid through a disc centrifuge at a low speed of 2000-3000rpm for 40-60min, collect the supernatant and remove the precipitate.

7. Add solid ammonium sulfate (addition amount 10-15g/L) and polypropylene glycol (addition amount 5-30mL/L) to the collected supernatant, and stir for 1-2h.

8. The stirred feed liquid is centrifuged at a high speed by a disc centrifuge at a speed of 5000-10000rpm, centrifuged for 20-40min, and the precipitate is collected.

9. Wash the precipitate with 30% ethanol solution, and centrifuge again at 5000-10000 rpm for 20-40 minutes to collect the precipitate.

10. Freeze-dry the precipitate to obtain phycocyanin freeze-dried powder.

All reagents and instruments used in the present invention can be purchased from the market.

Below in conjunction with embodiment, further set forth the present invention:

Example 1

1. Add sodium azide to the cultivated seawater chlorella (algae density: 8 million cells/mL) at a rate of 0.2 mg/L. Then the microalgae liquid is centrifuged through a disc centrifuge to extract the algae cells to form a concentrated algae liquid. The rotating speed of the centrifuge is 8000rpm, and the water content of the separated algae liquid is 58%.

2. Add glycerol (100% purity, 10 g/kg concentrated algae) to the separated concentrated algae liquid, stir evenly, and place in a -18°C refrigerator for freezing for 12 hours.

3. Thaw the frozen concentrated algae liquid at room temperature for 6 hours.

4. Repeat the second step and the third step 2 times.

5. Add sodium azide (addition amount: 2mg/L), Tween 80 (addition amount: 5ml/L), Dow DF104 polyether defoamer (1mL/L) to the thawed concentrated algae liquid, and mix Evenly, put it into a homogenizer for cell disruption.

6. Centrifuge the crushed algae liquid through a disc centrifuge at a low speed of 2000 rpm for 40 minutes, collect the supernatant, and remove the precipitate.

7. Add solid ammonium sulfate (addition amount 10g/L) and polypropylene glycol (addition amount 20mL/L) to the collected supernatant, and stir for 1 hour.

8. Centrifuge the stirred feed liquid through a disc centrifuge at a high speed of 5000 rpm for 30 minutes to collect the precipitate.

9. Wash the precipitate with 30% ethanol solution, and centrifuge again at 5000 rpm for 30 min to collect the precipitate.

10. Freeze-dry the precipitate to obtain phycocyanin freeze-dried powder.

11. After testing, the purity of phycocyanin is 99.2%, the protein capture rate is 87%, and the protein activity is 98%.

Comparative example 1

With reference to the extraction method of Example 4 in the publication number CN109160947A patent (a preparation method of spirulina phycocyanin and its application), the phycocyanin in seawater chlorella is extracted. details as follows:

Add sodium azide to the cultured seawater chlorella (algae density: 8 million cells/mL) at a rate of 0.2 mg/L. Then the microalgae liquid is centrifuged through a disc centrifuge to extract the algae cells to form a concentrated algae liquid. The rotating speed of the centrifuge is 8000rpm, and the water content of the separated algae liquid is 55%.

Add the separated concentrated algae liquid into phosphate buffer saline (concentration is 0.1mol/L, containing 0.004mol/L sodium azide) according to the volume ratio of material to liquid 1:20g/ml, mix well; place it in 4 Swell at ℃ for 6 hours, cut for 15 minutes with an ultra-fine shearer, centrifuge the cut material, and take the supernatant; slowly add ammonium sulfate to the supernatant until its saturation is 30%, remove other proteins Impurities, centrifuged, take the supernatant; continue to slowly add ammonium sulfate solution to the supernatant until its saturation is 70%, centrifuge to obtain a blue precipitate; freeze-dry the precipitated precipitate for 18 hours to obtain phycocyanin, The capture rate of phycocyanin is 65%, and the purity is 73%.

Comparative example 2

This comparative example refers to Example 1, but no sodium azide is added for protection throughout.

1. Centrifuge the cultured seawater chlorella (algae density: 8 million cells/mL) through a disc centrifuge to extract the algal cells to form a concentrated algae liquid. The rotating speed of the centrifuge is 8000rpm, and the water content of the separated algae liquid is 58%.

3. Thaw the frozen concentrated algae liquid at room temperature for 6 hours.

4. Repeat the second step and the third step 2 times.

5. Add Tween 80 (5ml/L) and Dow DF104 polyether defoamer (1mL/L) to the thawed concentrated algae liquid, mix well, and put it into a homogenizer for cell disruption .

10. Freeze-dry the precipitate to obtain phycocyanin freeze-dried powder.

11. After testing, the purity of phycocyanin is 65%, the protein capture rate is 80%, and the protein activity is 40%.

Comparative example 3

This comparative example refers to Example 1, but does not carry out repeated freezing and thawing, only one-step freezing and thawing is carried out.

3. Thaw the frozen concentrated algae liquid at room temperature for 6 hours.

4. Add sodium azide (addition amount: 2mg/L), Tween 80 (addition amount: 5ml/L), Dow DF104 polyether defoamer (1mL/L) to the thawed concentrated algae liquid, and mix Evenly, put it into a homogenizer for cell disruption.

5. Centrifuge the crushed algae liquid through a disc centrifuge at a low speed of 2000 rpm for 40 minutes, collect the supernatant, and remove the precipitate.

6. Add solid ammonium sulfate (addition amount 10g/L) and polypropylene glycol (addition amount 20mL/L) to the collected supernatant, and stir for 1 hour.

7. Centrifuge the stirred feed liquid through a disc centrifuge at a high speed at 5000 rpm for 30 minutes to collect the precipitate.

8. Wash the precipitate with 30% ethanol solution, and centrifuge again at 5000 rpm for 30 min to collect the precipitate.

9. Freeze-dry the precipitate to obtain phycocyanin freeze-dried powder.

10. After testing, the purity of phycocyanin is 85%, the protein capture rate is 35%, and the protein activity is 89%.

Comparative example 4

This comparative example refers to Example 1, ammonium sulfate and polypropylene glycol in step 7 are replaced with polyaluminum chloride flocculant (PAC)

3. Thaw the frozen concentrated algae liquid at room temperature for 6 hours.

4. Repeat the second step and the third step 2 times.

7. Add polyaluminum chloride flocculant (PAC) (addition amount 2g/L) to the collected supernatant, and stir for 1 hour.

10. Freeze-dry the precipitate to obtain phycocyanin freeze-dried powder.

11. After testing, the purity of phycocyanin is 25%, the protein capture rate is 35%, and the protein activity is 40%.

A rapid extraction method for phycocyanin provided by the present invention has been described in detail above. This article uses specific examples to illustrate the principle and implementation of the present invention. The description of the above embodiments is only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

A rapid extraction method for phycocyanin, characterized in that it comprises the steps of:

Step (1) mixing cultured water containing microalgae with a protein protectant, and performing a first centrifugation to obtain concentrated algae liquid;

Step (2) mixing the concentrated algae liquid with a cell cryoprotectant, and repeatedly freezing and thawing to obtain the thawed concentrated algae liquid;

Step (3) mixing the thawed concentrated algae liquid with a protein protectant and a surfactant, and crushing the cells to obtain the crushed algae liquid;

Step (4) performing a second centrifugation on the crushed algae liquid to obtain a supernatant;

Step (5) mixing the supernatant with ammonium sulfate and polypropylene glycol, stirring;

In step (6), the stirred feed liquid is subjected to a third centrifugation to obtain a precipitate;

In step (7), the obtained precipitate is washed with an aqueous ethanol solution, and after the fourth centrifugation, the precipitate is freeze-dried.
The extraction method according to claim 1, wherein the protein protecting agent is sodium azide;

In step (1), in the culture water containing microalgae, the protein protection agent is added in an amount of 0.1-0.5 mg/L.
The extraction method according to claim 1, characterized in that, the rotational speed of the first centrifugation is 5000-10000 rpm, and the water content of the concentrated algae liquid is 50%-70%.
The extraction method according to claim 1, wherein the cell cryoprotectant is glycerol;

In step (2), in the concentrated algae liquid, the added amount of the cell cryoprotectant is 5-10 g/kg.
The extraction method according to claim 1, characterized in that the procedure of repeated freezing and thawing is: freezing at -20 to -10°C for 12 to 24 hours, thawing at 10 to 30°C for 6 to 12 hours, repeating 2 to 3 times.
extraction method according to claim 1, is characterized in that, described surfactant is Tween 80 and Dow's DF104 polyether defoamer;

Step (3), in the thawed concentrated algae liquid, the added amount of the protein protectant is 1-2mg/L, the added amount of the Tween 80 is 1-5mL/L, and the Dow DF104 polymer The amount of ether defoamer added is 1-2mL/L.
The extraction method according to claim 1, characterized in that, the rotational speed of the second centrifugation is 2000-3000 rpm, and the time is 40-60 minutes.
The extraction method according to claim 1, characterized in that, step (5), in the supernatant, the addition of ammonium sulfate is 10 to 15g/L, and the addition of polypropylene glycol is 5 to 30mL/L;

The rotational speed of the stirring is 500-1000 rpm, and the stirring time is 1-2 hours.
The extraction method according to claim 1, characterized in that, the rotation speed of the third centrifugation is 5000-10000 rpm, and the time is 20-40 minutes.
The extraction method according to any one of claims 1 to 9, wherein the concentration of the aqueous ethanol solution is 20% to 50%;

The rotation speed of the fourth centrifugation is 5000-10000 rpm, and the time is 20-40 minutes.