CN111116737A - High-purity phycocyanin and preparation method thereof - Google Patents
High-purity phycocyanin and preparation method thereof Download PDFInfo
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- CN111116737A CN111116737A CN202010032593.5A CN202010032593A CN111116737A CN 111116737 A CN111116737 A CN 111116737A CN 202010032593 A CN202010032593 A CN 202010032593A CN 111116737 A CN111116737 A CN 111116737A
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- phycocyanin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
Abstract
The invention relates to the field of separation and purification of proteins, and discloses high-purity phycocyanin and a preparation method thereof. The method adopts purified water to extract the spirulina dry powder, so that cell walls and cell membranes of spirulina cells are broken after the spirulina cells fully absorb water and swell, and phycocyanin is dissolved out. In addition, the separation and purification process of the invention completely adopts a physical process of membrane separation, and does not use any chemical reagent, so that the obtained product has the characteristics of high purity and high activity.
Description
Technical Field
The invention relates to the field of separation and purification of proteins, and particularly relates to high-purity phycocyanin and a preparation method thereof.
Background
Spirulina, also known as Arthrospira or cyanobacteria, belongs to the phylum Cyanophyta and mainly includes Spirulina maxima, Spirulina platensis and Spirulina subsalsa. The spirulina contains abundant nutrient components, various microorganisms, protein, fat, carbohydrate, mineral substances and trace elements, and the proportion of various nutrient components is scientific and reasonable, so that the spirulina is an ideal pure natural bioactive health food.
Phycocyanin is a water-soluble natural blue pigment only existing in blue algae such as spirulina, and the content of phycocyanin in spirulina reaches 10% -20%. Phycocyanin has been widely used as a high-grade natural pigment for foods and cosmetics in developed countries such as europe, the united states, japan, and the like. Recent researches show that phycocyanin also has the effects of resisting cancers, promoting blood cell regeneration, resisting oxidation, diminishing inflammation and the like. In addition, phycocyanin has high fluorescence quantum yield and 30 times stronger intensity than common fluorescein, and can be used as a marker to be combined with biotin, avidin, DNA molecules and various monoclonal antibodies to prepare a fluorescent probe. Therefore, the phycocyanin is a natural protein with important application value in the related fields of food, medicine, scientific research and the like.
At present, there are many methods for extracting phycocyanin from spirulina, for example, the prior art discloses a method for extracting high-purity phycocyanin, which comprises adding potassium phosphate buffer solution into spirulina powder, stirring uniformly, freezing, thawing, performing high-pressure homogenization and wall breaking, performing microfiltration membrane filtration on supernatant obtained by centrifugal separation to obtain crude phycocyanin extract, performing decoloration and purification on the crude phycocyanin extract through combined resin, collecting eluent, performing purification through a hydroxyapatite chromatographic column to obtain refined phycocyanin extract, and finally performing high-speed centrifugal spray drying after desalting and concentration to obtain high-purity phycocyanin. However, the method has the disadvantages of complex operation steps, high power consumption, long operation period and the like in the actual industrial production process. In addition, the yield loss is large due to excessive steps, large-scale production is difficult and expensive to implement, and phycocyanin has practical application value only when large-scale industrial production is realized.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect that the large-scale industrial production is difficult in the extraction method of phycocyanin in the prior art, thereby providing a method for extracting phycocyanin more efficiently and economically and in a large scale.
In order to solve the technical problems, the invention provides a preparation method of phycocyanin, which comprises the following steps:
(1) according to the mass ratio of the spirulina dry powder to the purified water of 1: (15-25), soaking for at least 6h, and stirring and extracting for at least 1h to obtain a leaching solution;
(2) centrifuging the leaching solution, removing cell debris, and filtering to obtain filtrate;
(3) sequentially carrying out membrane separation on the filtrate by using ultrafiltration membranes with the molecular weight cutoff of 12-15 KDa and 3-5 KDa respectively to obtain concentrated solutions;
(4) spray drying the concentrated solution to obtain powder, i.e. phycocyanin.
Further, the step (1) is to soak the mixed solution of the spirulina dry powder and the purified water for 8-12 hours at the temperature of less than or equal to 20 ℃, and then stir the mixed solution for 80-120 min at the rotating speed of 180-220 r/min.
Further, in the step (2), the leaching liquor is subjected to centrifugal separation at a rotating speed of 5500-6500 r/min.
Further, in the step (2), the filtration is to filter the obtained centrifugate by using a ceramic membrane of 200-400 nm.
Further, in the step (2), the ceramic membrane passes through the ceramic membrane with the thickness of 200-400 nm at the pressure of 2-4 MPa and the flow rate of 40-60L/h.
Further, in the step (3), firstly, an ultrafiltration membrane with the molecular weight cutoff of 12-15 KDa is passed through at the flow rate of 18-22L/h under the pressure of 5-8 MPa, and then an ultrafiltration membrane with the molecular weight cutoff of 3-5 KDa is passed through at the flow rate of 18-22L/h under the pressure of 8-12 MPa.
Further, in the step (4), the air inlet temperature of the spray drying is 180-190 ℃, and the air outlet temperature is 60-70 ℃.
The invention also provides phycocyanin prepared by the preparation method.
The technical scheme of the invention has the following advantages:
1. the preparation method of the phycocyanin provided by the invention comprises the steps of firstly leaching spirulina dry powder by using purified water, and then obtaining the high-purity phycocyanin through centrifugal separation, ceramic membrane filtration, ultrafiltration membrane purification, concentration and spray drying. Compared with the prior art which adopts a buffer salt solution or a chemical reagent, the method has mild conditions, not only improves the extraction rate of the phycocyanin, but also has small damage to the phycocyanin, ensures the stability of the phycocyanin, and avoids the problems of solvent pollution on products and the degeneration of the phycocyanin. In addition, the separation and purification process of the invention completely adopts a physical process of membrane separation, and no chemical reagent is used, so that the obtained product has the characteristics of high purity and activity and no pollution.
2. The preparation method of the phycocyanin provided by the invention has the advantages that the operations from wall breaking, separation and purification to drying and other steps are simple and easy, the operability is strong, the activity and the extraction rate of the phycocyanin are fully ensured, no pollutant is generated in the production process, the preparation method is environment-friendly, the defects of the traditional preparation method are well made up, and the industrial production is easy.
3. The phycocyanin provided by the invention has stable property, and the purity A620/A280 is more than 2.0, which exceeds the purity requirement of food-grade phycocyanin.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1
The embodiment provides a preparation method of phycocyanin, which comprises the following steps:
(1) mixing spirulina dry powder and purified water at a mass ratio of 1:20, soaking at 20 deg.C for 8h, stirring at 200r/min for 80min to obtain leaching solution containing phycocyanin product;
(2) placing the leaching solution in an automatic slag discharge butterfly centrifuge, continuously centrifuging at the rotating speed of 6000r/min, removing cell debris, and obtaining a centrifugate containing a phycocyanin product;
(3) filtering the centrifugate with a ceramic membrane of 300nm at a flow rate of 50L/h under a pressure of 3MPa to obtain filtrate containing phycocyanin;
(4) passing the filtrate through an ultrafiltration membrane with molecular weight cutoff of 15KDa at a flow rate of 20L/h under 6MPa, washing the obtained permeate with clear water with the volume of 3 times of that of the permeate, collecting the permeate containing phycocyanin products, and passing through an ultrafiltration membrane with molecular weight cutoff of 5KDa at a flow rate of 20L/h under 10MPa to obtain concentrated solution containing phycocyanin products;
(5) and (3) directly spray-drying the concentrated solution, wherein the air inlet temperature of a spray dryer is 185 ℃, the air outlet temperature of the spray dryer is 65 ℃, and collecting powder by adopting two-stage series-connected cyclone separators to obtain the high-purity phycocyanin, wherein the extraction rate is 68.4%, and the purity is A620/A280-2.9.
Example 2
The embodiment provides a preparation method of phycocyanin, which comprises the following steps:
(1) mixing spirulina dry powder and purified water at a mass ratio of 1:25, soaking at 20 deg.C for 6h, stirring at 400r/min for 60min to obtain leaching solution containing phycocyanin;
(2) placing the leaching solution in an automatic residue discharge butterfly centrifuge, continuously centrifuging at the rotating speed of 6500r/min, removing cell debris, and obtaining a centrifugate containing a phycocyanin product;
(3) filtering the centrifugate at a flow rate of 40L/h through a 200nm ceramic membrane under a pressure of 4MPa to obtain filtrate containing phycocyanin;
(4) passing the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 12KDa at a flow rate of 18L/h under a pressure of 8MPa, washing the obtained permeate with clear water with a volume of 3 times of that of the permeate for 3 times, collecting the permeate containing the phycocyanin product, and passing through the ultrafiltration membrane with the molecular weight cutoff of 3KDa at a flow rate of 18L/h under a pressure of 12MPa to obtain a concentrated solution containing the phycocyanin product;
(5) and (3) directly spray-drying the concentrated solution, wherein the air inlet temperature of a spray dryer is 190 ℃, the air outlet temperature of the spray dryer is 70 ℃, and collecting powder by adopting two-stage series-connected cyclone separators to obtain the high-purity phycocyanin, wherein the extraction rate is 61.7%, and the purity is A620/A280-3.0.
Example 3
The embodiment provides a preparation method of phycocyanin, which comprises the following steps:
(1) mixing spirulina dry powder and purified water at a mass ratio of 1:15, soaking at 18 deg.C for 12h, stirring at 200r/min for 120min to obtain leaching solution containing phycocyanin;
(2) placing the leaching liquor in an automatic deslagging butterfly centrifuge, continuously centrifuging at the rotating speed of 5500r/min, removing cell debris, and obtaining a centrifugate containing a phycocyanin product;
(3) filtering the centrifugate with 400nm ceramic membrane at a flow rate of 60L/h under 2MPa to obtain filtrate containing phycocyanin product;
(4) passing the filtrate through an ultrafiltration membrane with molecular weight cutoff of 15KDa at a flow rate of 22L/h under a pressure of 5MPa, washing the obtained permeate with clear water with a volume of 3 times of that of the permeate, collecting the permeate containing phycocyanin products, and passing through the ultrafiltration membrane with molecular weight cutoff of 5KDa at a flow rate of 22L/h under a pressure of 8MPa to obtain a concentrated solution containing phycocyanin products;
(5) and (3) directly spray-drying the concentrated solution, wherein the air inlet temperature of a spray dryer is 180 ℃, the air outlet temperature of the spray dryer is 60 ℃, and collecting powder by adopting two-stage series-connected cyclone separators to obtain the high-purity phycocyanin, wherein the extraction rate is 63.6%, and the purity is A620/A280-2.7.
Comparative example 1
The comparative example provides a preparation method of phycocyanin, which comprises the following steps:
(1) mixing spirulina dry powder with 0.01mol/L phosphate buffer solution with pH of 6.5 at a mass ratio of 1:20, soaking at 20 deg.C for 8h, stirring at 200r/min for 80min to obtain leaching solution containing phycocyanin product;
(2) placing the leaching solution in an automatic slag discharge butterfly centrifuge, continuously centrifuging at the rotating speed of 6000r/min, removing cell debris, and obtaining a centrifugate containing a phycocyanin product;
(3) filtering the centrifugate with a ceramic membrane of 300nm at a flow rate of 50L/h under a pressure of 3MPa to obtain filtrate containing phycocyanin;
(4) passing the filtrate through an ultrafiltration membrane with molecular weight cutoff of 15KDa at a flow rate of 20L/h under 6MPa, washing the obtained permeate with clear water with the volume of 3 times of that of the permeate, collecting the permeate containing phycocyanin products, and passing through an ultrafiltration membrane with molecular weight cutoff of 5KDa at a flow rate of 20L/h under 10MPa to obtain concentrated solution containing phycocyanin products;
(5) and (3) directly spray-drying the concentrated solution, wherein the air inlet temperature of a spray dryer is 185 ℃, the air outlet temperature of the spray dryer is 65 ℃, and collecting powder by adopting two-stage series-connected cyclone separators to obtain the high-purity phycocyanin, wherein the extraction rate is 55.1%, and the purity is A620/A280-2.3.
Comparative example 2
The comparative example provides a preparation method of phycocyanin, which comprises the following steps:
(1) mixing spirulina dry powder and purified water at a mass ratio of 1:20, soaking at 20 deg.C for 8h, stirring at 200r/min for 80min to obtain leaching solution containing phycocyanin product;
(2) placing the leaching solution in an automatic slag discharge butterfly centrifuge, continuously centrifuging at the rotating speed of 6000r/min, removing cell debris, and obtaining a centrifugate containing a phycocyanin product;
(3) filtering the centrifugate with a ceramic membrane of 300nm at a flow rate of 50L/h under a pressure of 3MPa to obtain filtrate containing phycocyanin;
(4) purifying the filtrate with combined resin, wherein the combined resin is D900 type ion exchange resin and BEHP-3 type large mesh nonpolar adsorption resin at a volume ratio of 2:1, to obtain concentrated solution containing phycocyanin;
(5) and (3) directly spray-drying the concentrated solution, wherein the air inlet temperature of a spray dryer is 185 ℃, the air outlet temperature of the spray dryer is 65 ℃, and collecting powder by adopting two-stage series-connected cyclone separators to obtain the high-purity phycocyanin, wherein the extraction rate is 48.1%, and the purity is A620/A280-1.5.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (8)
1. A preparation method of phycocyanin is characterized by comprising the following steps:
(1) according to the mass ratio of the spirulina dry powder to the purified water of 1: (15-25), soaking for at least 6h, and stirring and extracting for at least 1h to obtain a leaching solution;
(2) centrifuging the leaching solution, removing cell debris, and filtering to obtain filtrate;
(3) filtering the filtrate with ultrafiltration membranes with the molecular weight cutoff of 12-15 KDa respectively, washing the concentrated solution with clear water at a ratio of 1:3 for 2-3 times, collecting the permeate containing the product, and concentrating with ultrafiltration membranes with the molecular weight cutoff of 3-5 KDa to obtain concentrated solution containing the product;
(4) spray drying the concentrated solution to obtain powder, i.e. phycocyanin.
2. The preparation method according to claim 1, wherein the step (1) comprises soaking the mixture of spirulina dry powder and purified water at a temperature of 20 ℃ or less for 8-12 h, and stirring at a rotation speed of 180-220 r/min for 80-120 min.
3. The preparation method according to claim 1 or 2, wherein in the step (2), the leachate is centrifugally separated at a rotating speed of 5500-6500 r/min.
4. The method according to any one of claims 1 to 3, wherein in the step (2), the filtration is performed by filtering the obtained centrifugate with a ceramic membrane of 200 to 400 nm.
5. The method according to claim 4, wherein the ceramic membrane is passed through the ceramic membrane at a flow rate of 40 to 60L/h at a pressure of 2 to 4MPa and a flow rate of 200 to 400 nm.
6. The preparation method according to any one of claims 1 to 5, wherein in the step (3), the ultrafiltration membrane with the molecular weight cut-off of 12 to 15KDa is firstly passed through at a flow rate of 18 to 22L/h under a pressure of 5 to 8MPa, and then the ultrafiltration membrane with the molecular weight cut-off of 3 to 5KDa is passed through at a flow rate of 18 to 22L/h under a pressure of 8 to 12 MPa.
7. The preparation method according to any one of claims 1 to 6, wherein in the step (4), the inlet air temperature of the spray drying is 180 to 190 ℃ and the outlet air temperature is 60 to 70 ℃.
8. Phycocyanin produced by the production method according to any one of claims 1 to 7.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607988A (en) * | 2009-07-03 | 2009-12-23 | 威海格瑞安生物工程有限公司 | From spirulina, extract the technology of Phycocyanins, C-, chlorophyll and spirulina polysaccharide |
| CN101942014A (en) * | 2010-08-23 | 2011-01-12 | 中国科学院烟台海岸带研究所 | Food grade phycocyanin and preparation method thereof |
| CN103554250A (en) * | 2013-11-22 | 2014-02-05 | 云南蓝钻生物科技有限公司 | Method for extracting phycocyanin |
| US20140371433A1 (en) * | 2012-01-12 | 2014-12-18 | Ezaki Glico Co., Ltd. | Method for preparing phycocyanin |
| WO2018172708A1 (en) * | 2017-03-24 | 2018-09-27 | Algosource | Method for preparing a liquid extract of phycobiliproteins, in particular phycocyanin, from cyanobacteria or microalgae and extract thus obtained |
| CN109021096A (en) * | 2018-08-27 | 2018-12-18 | 桐乡市博奥生物科技有限公司 | A kind of separation and Extraction purifying process of spirulina polysaccharide and phycocyanin |
-
2020
- 2020-01-13 CN CN202010032593.5A patent/CN111116737A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607988A (en) * | 2009-07-03 | 2009-12-23 | 威海格瑞安生物工程有限公司 | From spirulina, extract the technology of Phycocyanins, C-, chlorophyll and spirulina polysaccharide |
| CN101942014A (en) * | 2010-08-23 | 2011-01-12 | 中国科学院烟台海岸带研究所 | Food grade phycocyanin and preparation method thereof |
| US20140371433A1 (en) * | 2012-01-12 | 2014-12-18 | Ezaki Glico Co., Ltd. | Method for preparing phycocyanin |
| CN103554250A (en) * | 2013-11-22 | 2014-02-05 | 云南蓝钻生物科技有限公司 | Method for extracting phycocyanin |
| WO2018172708A1 (en) * | 2017-03-24 | 2018-09-27 | Algosource | Method for preparing a liquid extract of phycobiliproteins, in particular phycocyanin, from cyanobacteria or microalgae and extract thus obtained |
| CN109021096A (en) * | 2018-08-27 | 2018-12-18 | 桐乡市博奥生物科技有限公司 | A kind of separation and Extraction purifying process of spirulina polysaccharide and phycocyanin |
Non-Patent Citations (1)
| Title |
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| ILTER, I等: ""Optimization of phycocyanin extraction from Spirulina platensis using different techniques"" * |
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