CN109160947A - A kind of preparation method and applications of spirulina phycocyanin - Google Patents
A kind of preparation method and applications of spirulina phycocyanin Download PDFInfo
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- CN109160947A CN109160947A CN201811172799.7A CN201811172799A CN109160947A CN 109160947 A CN109160947 A CN 109160947A CN 201811172799 A CN201811172799 A CN 201811172799A CN 109160947 A CN109160947 A CN 109160947A
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- spirulina
- phycocyanin
- supernatant
- extracting method
- ammonium sulfate
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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 the separation and extraction technologies of biology and the natural products effective ingredient of light industry and chemical industry biotechnology in new medicine, are related to extraction and application field, the extracting method and application of specially a kind of spirulina phycocyanin.The step of preparation method, is as follows: 1) crushing dry spirulina, sieving；2) phosphate buffer is added into spirulina powder, is uniformly mixed；3) feed liquid is placed in 4 DEG C of environment and is swollen, feed liquid is subjected to ultra-fine shearing after swelling；4) extracting solution is centrifuged, obtains supernatant；5) it is slowly added to ammonium sulfate into supernatant, stands, centrifugation obtains supernatant；6) it is slowly added to ammonium sulfate to supernatant relaying is continuous, stood, centrifugation obtains blue precipitate；7) sediment is freeze-dried to obtain phycocyanin.The present invention combines wall breaking technology using swelling-ultra-fine shearing method, and the used time is short, phycocyanin yield is high, and the spirulina phycocyanin extract of preparation significantly inhibits the proliferation of HeLa cell.
Description
Technical field
The invention belongs to the separation of biology and light industry in new medicine and the natural products effective ingredient of chemical industry biotechnology to mention
Technology is taken, extraction and application field more particularly to a kind of preparation method and applications of spirulina phycocyanin are related to.
Background technique
Spirulina is a kind of microalgae plant being grown in the torrid zone and subtropical zone alkaline lake, is rich in various nutriments
And active constituent, " the optimal natural health care of the 21 century mankind " is referred to as by United Nations Food and Agricultural Organization.With
Living standard improves, and people's health, health theory gradually increase, and the demand of nutriment and health care product is also stepped up, spiral
Algae health care product has very big market potential.Contain a large amount of protein and various nutriments in spirulina, wherein phycocyanin
Have the function of anticancer, improve immunity, anti-oxidant isoreactivity, has very high nutritive value and economic value, therefore increasingly
Mostly it is developed as health care product, functional food.Traditional spirulina phycocyanin freeze thawing abstraction process is complicated, higher cost.
Present invention combination traditional handicraft carries out broken wall using swelling-ultra-fine shearing method, and the used time is shorter, save the cost, improves extraction
Rate.By carrying out anti-tumor activity experiment to the spirulina phycocyanin of extraction, evaluation spirulina phycocyanin extract is anti-
Effect in terms of tumour.
Summary of the invention
The present invention provides the preparation method of spirulina phycocyanin and application, the present invention is by traditional handicraft and modern crafts
It combining, process for refining parameter, the wall breaking technology combined using swelling-ultra-fine shearing method has the used time short, save the cost,
The features such as improving recovery rate.
In order to achieve the above-mentioned object of the invention, the invention provides the following technical scheme:
A kind of preparation method of spirulina phycocyanin, comprising the following steps:
(1) spirulina is crushed in 40 DEG C of drying, using Universalpulverizer, crosses 30 mesh screens；
(2) phosphate buffer is added into spirulina powder, is uniformly mixed；
(3) feed liquid is placed in 4 DEG C of environment and is swollen, feed liquid is subjected to ultra-fine shearing after swelling；
(4) feed liquid after ultra-fine shearing is centrifuged, takes supernatant；
(5) it is slowly added to ammonium sulfate into supernatant, stands, centrifugation obtains supernatant；
(6) continuous addition ammonium sulfate is relayed to supernatant, stood, centrifugation obtains blue precipitate；
(7) by pellet frozen it is dry phycocyanin extract.
Preferably, the material liquid volume ratio that spirulina powder and phosphate buffer are added in the step (2) is 1:15 ~ 1:
30g/ml。
Preferably, include in the phosphate buffer being added in the step (2) 0.05 ~ 0.15mol/L phosphate and
The Sodium azide of 0.004mol/L.
Preferably, swelling temperature is 4 DEG C in the step (3), and swelling time is 4 ~ 8 hours.
It preferably, is 10 ~ 20min to the ultra-fine shear time of feed liquid in the step (3).
Preferably, the ammonium sulfate being added in the step (5) into supernatant to its saturation degree is 10% ~ 30%.
Preferably, ammonium sulfate to its saturation degree is added in the step (6) into supernatant is 35% ~ 70%.
Preferably, it is dried in the step (7) using freeze-drying, drying time is 18 ~ 24 hours.
Operation of the present invention is easily understood, compared with prior art, a kind of system of spirulina phycocyanin described in this patent
Preparation Method and its application have the advantages that.Present invention combination traditional handicraft and modern crafts, using phosphate buffer
Extraction time has been saved, recovery rate is improved in extraction using swelling-ultra-fine shearing method wall breaking technology as Extraction solvent,
Cost is saved.Spirulina phycocyanin extract prepared by the present invention has stronger inhibiting effect to tumour cell.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, but embodiment is not to the technology of the present invention side
The restriction of case, it is all based on variation made by the present invention or equivalent replacement, it is within the scope of protection of the invention.
Embodiment one
The spirulina powder 20g for accurately weighing drying and crushing is added according to material liquid volume ratio 1:15g/ml by 0.05mol/L phosphoric acid
The phosphate buffer that salt and 0.004mol/L Sodium azide are prepared, mixes；It places it in 4 DEG C of environment and is swollen 4 hours, using super
Thin cutter shears 10min, and the feed liquid after shearing is centrifuged, supernatant is taken；It is full to it that ammonium sulfate is slowly added into supernatant
It is 10% with degree, removes other protein impurities, is centrifuged, takes supernatant；Continue up be slowly added in clear liquid ammonium sulfate to its
Saturation degree is 40%, and centrifugation obtains blue precipitate；The sediment of precipitation is freeze-dried 18 hours, phycocyanin is obtained, algae is blue
Albumen yield is 9.23%.
Embodiment two
It is (dense that phosphate buffer is added according to material liquid volume ratio 1:30g/ml in the spirulina powder 20g for accurately weighing drying and crushing
Degree is 0.15mol/L, the Sodium azide containing 0.004mol/L), it mixes；It places it in 4 DEG C of environment and is swollen 8 hours, using super
Thin cutter shears 20min, and the feed liquid after shearing is centrifuged, supernatant is taken；It is full to it that ammonium sulfate is slowly added into supernatant
It is 20% with degree, removes other protein impurities, is centrifuged, takes supernatant；Continue up be slowly added in clear liquid ammonium sulfate to its
Saturation degree is 50%, and centrifugation obtains blue precipitate；The sediment of precipitation is freeze-dried 24 hours, phycocyanin is obtained, algae is blue
Albumen yield is 10.08%.
Embodiment three
It is (dense that phosphate buffer is added according to material liquid volume ratio 1:25g/ml in the spirulina powder 20g for accurately weighing drying and crushing
Degree is 0.15mol/L, the Sodium azide containing 0.004mol/L), it mixes；It places it in 4 DEG C of environment and is swollen 6 hours, using super
Thin cutter shears 10min, and the feed liquid after shearing is centrifuged, supernatant is taken；It is full to it that ammonium sulfate is slowly added into supernatant
It is 30% with degree, removes other protein impurities, is centrifuged, takes supernatant；Continue up be slowly added in clear liquid ammonium sulfate to its
Saturation degree is 60%, and centrifugation takes blue precipitate；By -40 DEG C of vacuum freeze dryings of sediment 24 hours of precipitation, algae indigo plant is obtained
Albumen, phycocyanin yield 9.83%.
Example IV
It is (dense that phosphate buffer is added according to material liquid volume ratio 1:20g/ml in the spirulina powder 20g for accurately weighing drying and crushing
Degree is 0.1mol/L, the Sodium azide containing 0.004mol/L), it mixes；It places it in 4 DEG C of environment and is swollen 6 hours, use is ultra-fine
Cutter shears 15min, and the feed liquid after shearing is centrifuged, supernatant is taken；Ammonium sulfate is slowly added into supernatant to its saturation
Degree is 30%, removes other protein impurities, is centrifuged, takes supernatant；Continue up that ammonium sulfate is slowly added in clear liquid is full to it
It is 70% with degree, centrifugation obtains blue precipitate；The sediment of precipitation is freeze-dried 18 hours, phycocyanin, algae indigo plant egg are obtained
White yield 10.54%.
Embodiment 1-4 extract obtained active testing
HeLa cell is cultivated, is classified as normally organizing and spirulina phycocyanin extract group, the HeLa of logarithmic growth phase is thin
Cell suspension is pressed every hole 1 × 10 by born of the same parents4A cell inoculation is in 96 orifice plates, 37 DEG C, 5%CO2And it is cultivated for 24 hours under saturated humidity
After change serum free medium, continue culture for 24 hours after, make cell enter growth resting stage.It inhales and abandons each hole culture medium, normal group is added
Blank cultures, spirulina phycocyanin extract group are added the culture medium containing phycocyanin extract, continue to cultivate, after 48h
96 orifice plates are taken out, 20 μ L of CCK-8 solution is added in every hole, continues to be incubated for 30min.Microplate reader measures the extinction in each hole at 450nm
It is worth (A), calculates inhibiting rate, the results are shown in Table 1.
Influence (n=6) of the 1 spirulina phycocyanin extract of table to HeLa cell Proliferation
As can be seen from Table 1, spirulina phycocyanin extract significantly inhibits the proliferation of HeLa cell.
For the ordinary skill in the art, specific embodiment is only exemplarily described the present invention,
Obviously the present invention specific implementation is not subject to the restrictions described above, as long as use the inventive concept and technical scheme of the present invention into
The improvement of capable various unsubstantialities, or not improved the conception and technical scheme of the invention are directly applied to other occasions
, it is within the scope of the present invention.
Claims (9)
1. a kind of extracting method of spirulina phycocyanin, it is characterised in that the following steps are included:
(1) spirulina is crushed in 40 DEG C of drying, using Universalpulverizer, crosses 30 mesh screens；
(2) phosphate buffer is added into spirulina powder, is uniformly mixed；
(3) feed liquid is placed under 4 DEG C of environment and is swollen, feed liquid is subjected to ultra-fine shearing after swelling；
(4) feed liquid after ultra-fine shearing is centrifuged, takes supernatant；
(5) it is slowly added to ammonium sulfate into supernatant, stands, centrifugation obtains supernatant；
(6) continuous addition ammonium sulfate is relayed to supernatant, stood, centrifugation obtains blue precipitate；
(7) by pellet frozen it is dry phycocyanin extract.
2. a kind of extracting method of spirulina phycocyanin as described in claim 1, which is characterized in that in the step (2)
The material liquid volume ratio that spirulina powder and phosphate buffer is added is 1:15 ~ 1:30g/ml.
3. a kind of extracting method of spirulina phycocyanin as described in claim 1, which is characterized in that in the step (2)
The phosphate buffering liquid concentration of addition is 0.05 ~ 0.15mol/L.
4. a kind of extracting method of spirulina phycocyanin as described in claim 1, which is characterized in that in the step (2)
Contain the Sodium azide of 0.004mol/L in the phosphate buffer of addition.
5. a kind of extracting method of spirulina phycocyanin as described in claim 1, which is characterized in that in the step (3)
Swelling temperature is 4 DEG C, and swelling time is 4 ~ 8 hours.
6. a kind of extracting method of spirulina phycocyanin as described in claim 1, which is characterized in that in the step (3)
Ultra-fine shear time is 10 ~ 20min.
7. a kind of extracting method of spirulina phycocyanin as described in claim 1, which is characterized in that in the step (5)
It is 10% ~ 30% that ammonium sulfate to saturation degree is added into supernatant.
8. a kind of extracting method of spirulina phycocyanin as described in claim 1, which is characterized in that in the step (6)
It is 35% ~ 70% that ammonium sulfate to saturation degree is added into supernatant.
9. a kind of extracting method of spirulina phycocyanin as described in claim 1, which is characterized in that in the step (7)
It is dried using freeze-drying, drying time is 18 ~ 24 hours.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023015792A1 (en) * | 2021-08-12 | 2023-02-16 | 海南绿藻世界生物科技有限公司 | Method for rapidly extracting phycocyanin |
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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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2018
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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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| 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》 * |
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| WO2023015792A1 (en) * | 2021-08-12 | 2023-02-16 | 海南绿藻世界生物科技有限公司 | Method for rapidly extracting phycocyanin |
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Effective date of registration: 20200428 Address after: 515000 Guangdong province Shantou Jinping District Science and Technology Park, G2 C2 rose three Applicant after: GUANGDONG UNITED FOODS Co.,Ltd. Applicant after: GUANGDONG YIJIAREN FOOD INDUSTRIAL TECHNOLOGY RESEARCH INSTITUTE Co.,Ltd. Address before: Jinyuan Road, Jimei Xinglin District of Xiamen City, Fujian Province, No. 239 361022 Applicant before: Lin Yan |
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