CN109160947B - Preparation method and application of spirulina phycocyanin - Google Patents
Preparation method and application of spirulina phycocyanin Download PDFInfo
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- CN109160947B CN109160947B CN201811172799.7A CN201811172799A CN109160947B CN 109160947 B CN109160947 B CN 109160947B CN 201811172799 A CN201811172799 A CN 201811172799A CN 109160947 B CN109160947 B CN 109160947B
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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 belongs to a separation and extraction technology of natural product active ingredients of light industry and chemical industry biotechnology in biology and new medicine, relates to the field of extraction and application, and particularly relates to an extraction method and application of spirulina phycocyanin. The preparation method comprises the following steps: 1) pulverizing dried Spirulina, and sieving; 2) adding phosphate buffer solution into the spirulina powder, and uniformly mixing; 3) swelling the feed liquid in an environment of 4 ℃, and performing superfine shearing on the feed liquid after swelling; 4) centrifuging the extracting solution to obtain a supernatant; 5) slowly adding ammonium sulfate into the supernatant, standing, and centrifuging to obtain supernatant; 6) continuously and slowly adding ammonium sulfate into the supernatant, standing, and centrifuging to obtain blue precipitate; 7) freeze drying the precipitate to obtain phycocyanin. The invention adopts the swelling-superfine shearing method combined with the wall breaking technology, the time consumption is short, the phycocyanin yield is high, and the prepared spirulina phycocyanin extract has obvious inhibition effect on the proliferation of HeLa cells.
Description
Technical Field
The invention belongs to a separation and extraction technology of natural product active ingredients of light industry and chemical industry biotechnology in biology and new medicine, relates to the field of extraction and application, and particularly relates to a preparation method and application of spirulina phycocyanin.
Background
Spirulina is a microalgae plant growing in tropical and subtropical alkaline lakes, rich in various nutrients and active ingredients, and is called the "21 st century human ideal natural health food" by food and agricultural organizations of the United nations. With the improvement of living standard, the health and health preserving idea of people is gradually enhanced, the requirements of nutriments and health care products are gradually increased, and the spirulina health care products have great market potential. Spirulina contains a large amount of protein and various nutrient substances, wherein phycocyanin has the active functions of resisting cancer, improving immunity, resisting oxidation and the like, and has high nutritional value and economic value, so that the spirulina is increasingly developed into health-care products and functional foods. The traditional freeze-thawing extraction process of spirulina phycocyanin is complex and has higher cost. The invention combines the traditional process, adopts the swelling-superfine shearing method to carry out wall breaking, has short time consumption, saves the cost and improves the extraction rate. The antitumor effect of the spirulina phycocyanin extract is evaluated by performing an antitumor activity experiment on the extracted spirulina phycocyanin.
Disclosure of Invention
The invention provides a preparation method and application of spirulina phycocyanin, wherein the preparation method combines the traditional process with the modern process, refines the process parameters, adopts the wall breaking technology combining the swelling-superfine shearing method, and has the characteristics of short time consumption, cost saving, extraction rate improvement and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
a preparation method of spirulina phycocyanin comprises the following steps:
(1) drying Spirulina at 40 deg.C, pulverizing with universal pulverizer, and sieving with 30 mesh sieve;
(2) adding phosphate buffer solution into the spirulina powder, and uniformly mixing;
(3) swelling the feed liquid in an environment of 4 ℃, and performing superfine shearing on the feed liquid after swelling;
(4) centrifuging the superfine sheared feed liquid, and taking supernatant;
(5) slowly adding ammonium sulfate into the supernatant, standing, and centrifuging to obtain supernatant;
(6) continuously adding ammonium sulfate into the supernatant, standing, and centrifuging to obtain blue precipitate;
(7) freeze drying the precipitate to obtain phycocyanin extract.
Preferably, the volume ratio of the spirulina powder to the phosphate buffer solution added in the step (2) is 1: 15-1: 30 g/ml.
Preferably, the phosphate buffer added in the step (2) comprises 0.05-0.15 mol/L phosphate and 0.004mol/L sodium azide.
Preferably, the swelling temperature in the step (3) is 4 ℃, and the swelling time is 4-8 hours.
Preferably, the superfine shearing time of the feed liquid in the step (3) is 10-20 min.
Preferably, the ammonium sulfate added to the supernatant in the step (5) is added to a saturation degree of 10-30%.
Preferably, in the step (6), ammonium sulfate is added into the supernatant until the saturation degree is 35-70%.
Preferably, the step (7) is carried out by adopting a freeze drying method, and the drying time is 18-24 hours.
The preparation method and the application of the spirulina phycocyanin have the following beneficial effects compared with the prior art. The invention combines the traditional process and the modern process, adopts phosphate buffer solution as an extraction solvent, and adopts a swelling-superfine shearing method wall breaking technology during extraction, thereby saving the extraction time, improving the extraction rate and saving the cost. The spirulina phycocyanin extract prepared by the invention has stronger inhibition effect on tumor cells.
Detailed Description
The present invention is further described in detail with reference to the following examples, which are not intended to limit the technical scope of the present invention, and all changes and equivalents made based on the present invention shall fall within the scope of the present invention.
Example one
Accurately weighing 20g of dried and crushed spirulina powder, adding a phosphate buffer solution prepared from 0.05mol/L phosphate and 0.004mol/L sodium azide according to the volume ratio of the feed liquid of 1:15g/ml, and uniformly mixing; swelling for 4 hours at 4 ℃, shearing for 10min by using an ultra-fine shearing machine, centrifuging the sheared feed liquid, and taking supernatant; slowly adding ammonium sulfate into the supernatant until the saturation degree is 10%, removing other protein impurities, centrifuging, and taking the supernatant; continuously and slowly adding an ammonium sulfate solution into the supernatant until the saturation degree is 40%, and centrifuging to obtain a blue precipitate; freeze-drying the precipitate for 18 hr to obtain phycocyanin with yield of 9.23%.
Example two
Accurately weighing 20g of dried and crushed spirulina powder, adding phosphate buffer solution (with the concentration of 0.15mol/L and containing 0.004mol/L sodium azide) according to the volume ratio of 1:30g/ml of the feed liquid, and uniformly mixing; swelling for 8 hours at 4 ℃, shearing for 20min by using an ultra-fine shearing machine, centrifuging the sheared feed liquid, and taking supernatant; slowly adding ammonium sulfate into the supernatant until the saturation degree is 20%, removing other protein impurities, centrifuging, and taking the supernatant; continuously and slowly adding an ammonium sulfate solution into the supernatant until the saturation degree is 50%, and centrifuging to obtain a blue precipitate; freeze-drying the precipitate for 24 hr to obtain phycocyanin with yield of 10.08%.
EXAMPLE III
Accurately weighing 20g of dried and crushed spirulina powder, adding phosphate buffer solution (with the concentration of 0.15mol/L and containing 0.004mol/L sodium azide) according to the volume ratio of the feed liquid of 1:25g/ml, and uniformly mixing; swelling the mixture for 6 hours in an environment of 4 ℃, shearing the mixture for 10min by using an ultra-fine shearing machine, centrifuging the sheared feed liquid, and taking supernatant; slowly adding ammonium sulfate into the supernatant until the saturation degree is 30%, removing other protein impurities, centrifuging, and taking the supernatant; continuously and slowly adding an ammonium sulfate solution into the supernatant until the saturation degree is 60%, centrifuging, and taking a blue precipitate; vacuum freeze-drying the precipitate at-40 deg.C for 24 hr to obtain phycocyanin with yield of 9.83%.
Example four
Accurately weighing 20g of dried and crushed spirulina powder, adding phosphate buffer solution (with the concentration of 0.1mol/L and containing 0.004mol/L of sodium azide) according to the volume ratio of 1:20g/ml of the feed liquid, and uniformly mixing; swelling the mixture for 6 hours in an environment of 4 ℃, shearing the mixture for 15min by using an ultra-fine shearing machine, centrifuging the sheared feed liquid, and taking supernatant; slowly adding ammonium sulfate into the supernatant until the saturation degree is 30%, removing other protein impurities, centrifuging, and taking the supernatant; continuously and slowly adding an ammonium sulfate solution into the supernatant until the saturation degree is 70%, and centrifuging to obtain a blue precipitate; freeze-drying the precipitate for 18 hr to obtain phycocyanin in 10.54% yield.
Examples 1-4 Activity testing of the extracts obtained
Culturing HeLa cells, dividing into normal group and spirulina phycocyanin extract group, collecting HeLa cells in logarithmic growth phase, and mixing cell suspension at a ratio of 1 × 10 per well4The individual cells were seeded in 96-well plates at 37 ℃ with 5% CO2And culturing for 24h under saturated humidity, then changing the serum-free culture medium, and continuously culturing for 24h to enable the cells to enter a growth stationary phase. And (3) removing the culture medium of each well, adding a blank culture medium into a normal group, adding a culture medium containing a phycocyanin extract into a spirulina phycocyanin extract group, continuing to culture, taking out a 96-well plate after 48 hours, adding 20 mu L of CCK-8 solution into each well, and continuing to incubate for 30 min. The absorbance (A) of each well at 450nm was measured with a microplate reader, and the inhibition was calculated, the results of which are shown in Table 1.
TABLE 1 Effect of Spirulina phycocyanin extract on HeLa cell proliferation (n = 6)
As can be seen from Table 1, the spirulina phycocyanin extract has an obvious inhibitory effect on the proliferation of HeLa cells.
It is obvious to those skilled in the art that the present invention is not limited to the above embodiments, and it is within the scope of the present invention to adopt various insubstantial modifications of the method concept and technical scheme of the present invention, or to directly apply the concept and technical scheme of the present invention to other occasions without modification.
Claims (6)
1. A method for extracting phycocyanin from spirulina is characterized by comprising the following steps:
(1) drying Spirulina at 40 deg.C, pulverizing with universal pulverizer, and sieving with 30 mesh sieve;
(2) adding phosphate buffer solution into the spirulina powder, and uniformly mixing;
(3) swelling the feed liquid at 4 ℃, and performing superfine shearing on the feed liquid after swelling;
(4) centrifuging the superfine sheared feed liquid, and taking supernatant;
(5) slowly adding ammonium sulfate into the supernatant, standing, and centrifuging to obtain supernatant;
(6) continuously adding ammonium sulfate into the supernatant, standing, and centrifuging to obtain blue precipitate;
(7) freeze drying the precipitate to obtain phycocyanin extract;
the phosphate buffer solution added in the step (2) contains 0.004mol/L of sodium azide; in the step (5), ammonium sulfate is added into the supernatant until the saturation degree is 30%; and (3) adding ammonium sulfate into the supernatant in the step (6) until the saturation degree is 60-70%.
2. The method for extracting phycocyanin in spirulina of claim 1, wherein the volume ratio of the spirulina powder and the phosphate buffer added in the step (2) is 1: 15-1: 30 g/ml.
3. The method for extracting phycocyanin in spirulina of claim 1, wherein the concentration of the phosphate buffer added in the step (2) is 0.05-0.15 mol/L.
4. The method for extracting phycocyanin in spirulina of claim 1, wherein the swelling temperature in step (3) is 4 ℃, the swelling time is 4-8 hours,
5. the method for extracting phycocyanin in spirulina of claim 1, wherein the ultra-fine shearing time in step (3) is 10-20 min.
6. The method for extracting phycocyanin in spirulina of claim 1, wherein the step (7) is performed by freeze drying for 18-24 hours.
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Citations (3)
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| WO2011011174A1 (en) * | 2009-07-20 | 2011-01-27 | Desert Lake Technologies, Llc | Methods for removal of microcystins and isolation of phycocyanin from cyanobacteria |
| CN102993297A (en) * | 2012-11-28 | 2013-03-27 | 汕头大学 | Spirulina phycocyanin and extraction method thereof |
| CN104672325A (en) * | 2015-03-11 | 2015-06-03 | 福建农林大学 | Method for preparing phycocyanin from fresh spirulina |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011011174A1 (en) * | 2009-07-20 | 2011-01-27 | Desert Lake Technologies, Llc | Methods for removal of microcystins and isolation of phycocyanin from cyanobacteria |
| CN102993297A (en) * | 2012-11-28 | 2013-03-27 | 汕头大学 | Spirulina phycocyanin and extraction method thereof |
| CN104672325A (en) * | 2015-03-11 | 2015-06-03 | 福建农林大学 | Method for preparing phycocyanin from fresh spirulina |
Non-Patent Citations (3)
| Title |
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| 俞建峰等.基于超细剪切细胞破壁技术的藻蓝蛋白提取工艺.《食品与生物技术学报》.2017,第36卷(第10期),第1072页左栏第1.2.1节，第1073页左栏第2.2节，第1076页右栏第1段. * |
| 基于超细剪切细胞破壁技术的藻蓝蛋白提取工艺;俞建峰等;《食品与生物技术学报》;20171231;第36卷(第10期);第1072页左栏第1.2.1节，第1073页左栏第2.2节，第1076页右栏第1段 * |
| 钝顶螺旋藻中藻蓝蛋白提取纯化及稳定性研究;徐蕾然;《中国优秀硕士学位论文全文数据库 基础科学辑》;20160515;正文第16页第2.5.1.1节，第32页表3-4，第2段 * |
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