CN107312076A - A kind of method that phycoerythrin is extracted in the dry product from Porphyra yezoensis - Google Patents
A kind of method that phycoerythrin is extracted in the dry product from Porphyra yezoensis Download PDFInfo
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- CN107312076A CN107312076A CN201710646600.9A CN201710646600A CN107312076A CN 107312076 A CN107312076 A CN 107312076A CN 201710646600 A CN201710646600 A CN 201710646600A CN 107312076 A CN107312076 A CN 107312076A
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- phycoerythrin
- porphyra yezoensis
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- dry product
- supernatant
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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
- C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
Abstract
The invention belongs to marine biomaterial preparing technical field, and in particular to a kind of method that phycoerythrin is extracted in dry product from Porphyra yezoensis.Specifically：Porphyra yezoensis dry product is blended, 80 mesh sieves are crossed；Porphyra yezoensis powder is mixed by a certain percentage with phosphate buffer, Porphyra yezoensis clasmatosis is made using multigelation method and ultrasonic method；Crude extract is centrifuged, and is collected supernatant and is added ammonium sulfate precipitation, and centrifugation again produces phycoerythrin.The extracting method is simple to operate, and preparation condition is gentle, environment-friendly, and the phycoerythrin purity of gained is high, and stability is good.
Description
Technical field
The invention belongs to marine biomaterial preparing technical field, and in particular to one kind is carried from Porphyra yezoensis dry product
The method for taking phycoerythrin.
Background technology
Phycoerythrin (phycoerythrin) is that the important of many marine algas catches photochromic fibroin, exist mainly in red algae and
In the blue-green algae of part, formed by the tetrapyrrole chromophore covalent bond of apoenzyme oligomeric protein and open chain, composition oligomeric protein
Subunit has the phycoerythrin in α, β, γ etc., usual red algae to be with (α β)6γ form is present, with reference to residual in subunit cysteine
Pigment molecular on base has phycoerythrobilin and phycourobilin.Phycoerythrin has in 480~570nm wave-length coverages of visible region
Stronger absorption, can produce strong fluorescence.
Phycoerythrin is after extraction and purification, as with high value-added product, can be widely used for food, cosmetic
The fields such as product, biological medicine and detection agent, there are some researches show phycoerythrin can be made into sensitising agent applied to tumor photodynamic
Treatment.In addition, phycoerythrin is also a kind of important physiological activator, antiviral with antitumor, strengthen immunity etc. is more
Plant pharmacological action, great researching value.
Seaweed is a kind of algae being grown on the rock of shallow sea, have the title of " the tender son of cay ", and seaweed is China's weight
One of Economical cultivation marine alga wanted, annual sales amount is up to 700,000,000 yuan, and seaweed contains abundant phycoerythrin, therefore seaweed is to obtain
The preferable resource of phycoerythrin.New fresh laver water content is high, and storage difficulty is big, therefore its dried product is sale common at present
With the form of preservation.And for extracting phycoerythrin from fresh laver more than current research, for extracting algae from seaweed dry product
The research of Lactoferrin is less.Therefore, algae red egg will be greatly enhanced by making full use of seaweed dry product to extract source as phycoerythrin
White yield, the extracting method of Erecting and improving is very necessary.
The content of the invention
It is an object of the invention to provide a kind of method that phycoerythrin is extracted in dry product from Porphyra yezoensis.
To achieve the above object, the present invention use technical scheme for：
A kind of method that phycoerythrin is extracted in the dry product from Porphyra yezoensis：
(1) Porphyra yezoensis dry product is blended with pulverizer, crosses 80 mesh sieves；
(2) the Porphyra yezoensis powder of above-mentioned gained and phosphate buffer are pressed 1:10~1:The ratio of 40 (mass/volumes)
Example is mixed；
(3) by above-mentioned Porphyra yezoensis suspension multigelation 3~6 times, used under ice-water bath in ultrasonic cell disintegration machine
It is ultrasonically treated, obtain clasmatosis liquid；
(4) above-mentioned gained is crushed into liquid and 10min~20min is centrifuged under 8000g~10000g, collect supernatant；
(5) into above-mentioned gained supernatant add 40%~50% ammonium sulfate, place 12~24h, 8000g~
10min~20min is centrifuged under 10000g, supernatant is collected；
(6) gained supernatant is dialysed and is freeze-dried, produce phycoerythrin.
The pH of phosphate buffer in the step (2) is 7.0, and concentration is 0.01mol/L~0.1mol/L.
A freeze thawing is is placed in -20 DEG C of freeze overnights in the step (3), then then at thawing at room temperature.
Ultrasonically treated condition is in the step (3)：200W~600W, work 10s, suspends 10s, totally 2~10min.
The molecular cut off of dialysis procedure is 3.5kDa in the step (6).
Above-mentioned albumen is measured using ultraviolet specrophotometer, phycoerythrin extract solution 280nm, 617nm,
Absorbance under 560nm and 650nm, yield and purity are calculated according to following formula：
Wherein, ρPE=0.123A560-0.068A617+0.015A650, VPEFor extracting liquid volume (mL), MPEFor Porphyra yezoensis
Quality (g).
Beneficial effects of the present invention：
A kind of present invention extracts phycoerythrin method in providing dry product from seaweed, this method is simple and easy to apply, with low cost,
Extraction process is only with water as Extraction solvent, and environmentally friendly, prepared phycoerythrin purity and yield are high, and this method can
Exploitation for seaweed dry product provide thinking.
Brief description of the drawings
Absorbances of the Fig. 1 for phycoerythrin prepared in present example under different pH environment at 560nm.
Fig. 2 is the absorbance that phycoerythrin preserves after 1h at 560nm at different temperatures in present example.
Fig. 3 is the absorbance that phycoerythrin preserves after different time at 560nm under light illumination in present example.
Specific embodiment
With reference to Figure of description, the invention will be further described, and protection scope of the present invention is not only limited to
Following examples.
Embodiment 1
1) 10g Porphyra yezoensis dry products are taken, are crushed using pulverizer, 80 mesh sieves are crossed, it is standby.
2) 0.5g Porphyra yezoensis powder is taken, the phosphate buffer (pH 7.0) for adding 15mL 0.1M suspends, at -20 DEG C
Under the conditions of freeze overnight, then thaw at room temperature for frozen-thaw process, multigelation three times.Then under ice bath, with 600W's
Power smudge cells, with ultrasonic 10s, interval 10s mode carries out processing 2min.By obtained frond crush liquid with 8000g from
Heart 15min, centrifugation is repeated twice, and collects supernatant；
3) phycobniliprotein crude extract is stood overnight using 50% (w/v) ammonium sulfate progress precipitation.With 8000g at 4 DEG C
Centrifugation 15 minutes, takes precipitation to abandon supernatant.Be dissolved in water precipitation, dialyses (interception 3500Da), removes ammonium sulfate, and dialysis freezing is dry
It is dry, produce phycoerythrin.
The phycoerythrin purity of gained is 1.08, and yield is 0.83%.
The stability of phycoerythrin under various circumstances obtained by detection：
1) pH stability
6 parts of phycoerythrin extract solution is taken, every part of 2mL adjusts pH=with 0.1mol/L HCl or 0.1mol/L NaOH
2.0th, 5.0.6.0,7.0,7.0,9.0,11.0, and OD value of each solution at 560nm is determined within 10min, as a result see
Fig. 1.As a result stability highest of the phycoerythrin in the case where pH is 5.0 environment is shown.
2) metal ion stability
Take CaCl2、ZnSO4、MgSO4、Al(NO3)3、CuSO4, NaCl, KCl, be configured to concentration water-soluble for 0.01mol/L
Liquid, draws every kind of metal ion solution 0.5mL, is separately added into 0.5mL phycoerythrin extract solutions, shakes up, and places after 24h, dilute
Release, determine OD value of each solution at 560nm.
The metal ion stability result of table 1
It should be avoided and Cu when showing that phycoerythrin is preserved by the result of table 12+、Al3+、K+、Mg2+、Zn2+Plasma is contacted.
3) temperature stability
6 parts of phycoerythrin extract solution is taken, every part of 1mL places 1h at 4 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C respectively, treated
After cooling, dilution determines OD value of each solution at 560nm, as a result sees Fig. 2.As a result show phycoerythrin under the conditions of 4 DEG C most
It is stable.
4) oxidation-reduction stability
6 parts of phycoerythrin extract solution is taken, every part of 0.5mL is separately added into concentration 0.01mol/L K2Cr2O7、Na2SO3、
Na2S2O3、FeSO4, oxalic acid, KMnO4Solution 0.5mL, shakes up, and places after 24h, dilution, determines OD of each solution at 560nm
Value.
The oxidation-reduction stability result of table 2
Show that different reductant-oxidants have large effect, algae red egg to the stability of phycoerythrin by the result of table 2
White preservation process should avoid being in contact with reductant-oxidant.
5) light durability
5 parts of phycoerythrin extract solution is taken, every part of 1mL takes a avoid light place, and other samples are placed in illumination box
In 2h, 4h, 6h, 8h, dilution determines OD value of each solution at 560nm, as a result sees Fig. 3.As a result show phycoerythrin in illumination
Under it is unstable, should preserve in the dark.
Embodiment 2
1) 10g Porphyra yezoensis dry products are taken, are crushed using pulverizer, 80 mesh sieves are crossed, it is standby.
2) 0.5g Porphyra yezoensis powder is taken, the phosphate buffer (pH 7.0) for adding 15mL 0.1M suspends, at -20 DEG C
Under the conditions of freeze overnight, then thaw at room temperature for frozen-thaw process, multigelation three times.Under ice bath, with 600W power
Smudge cells, with ultrasonic 10s, interval 10s mode carries out processing 2min.Obtained frond is crushed into liquid to centrifuge with 8000g
15min, centrifugation is repeated twice, and collects supernatant；
3) phycobniliprotein crude extract is stood overnight using 50% (w/v) ammonium sulfate progress precipitation.With 8000g at 4 DEG C
Centrifugation 15 minutes, takes precipitation to abandon supernatant.Be dissolved in water precipitation, dialyses (interception 3500Da), removes ammonium sulfate, and dialysis freezing is dry
It is dry, produce phycoerythrin.
The phycoerythrin purity of gained is 1.09, and yield is 0.84%.
Embodiment 3
1) 10g Porphyra yezoensis dry products are taken, are crushed using pulverizer, 80 mesh sieves are crossed, it is standby.
2) 0.5g Porphyra yezoensis powder is taken, the phosphate buffer (pH 7.0) for adding 15mL 0.1M suspends, at -20 DEG C
Under the conditions of freeze overnight, then thaw at room temperature for frozen-thaw process, multigelation three times.Under ice bath, with 600W power
Smudge cells, with ultrasonic 10s, interval 10s mode carries out processing 2min.Obtained frond is crushed into liquid to centrifuge with 8000g
15min, centrifugation is repeated twice, and collects supernatant；
3) phycobniliprotein crude extract is stood overnight using 50% (w/v) ammonium sulfate progress precipitation.With 8000g at 4 DEG C
Centrifugation 15 minutes, takes precipitation to abandon supernatant.Be dissolved in water precipitation, dialyses (interception 3500Da), removes ammonium sulfate, and dialysis freezing is dry
It is dry, produce phycoerythrin.
The phycoerythrin purity of gained is 1.05, and yield is 1.04%.
Claims (5)
1. the method for phycoerythrin is extracted in a kind of dry product from Porphyra yezoensis, it is characterised in that：
(1) Porphyra yezoensis dry product is blended with pulverizer, crosses 80 mesh sieves；
(2) the Porphyra yezoensis powder of above-mentioned gained and phosphate buffer are pressed 1:10~1:The ratio of 40 (mass/volumes) is mixed
It is even；
(3) by above-mentioned Porphyra yezoensis suspension multigelation 3~6 times, ultrasound in ultrasonic cell disintegration machine is used under ice-water bath
Processing, obtains clasmatosis liquid；
(4) above-mentioned gained is crushed into liquid and 10min~20min is centrifuged under 8000g~10000g, collect supernatant；
(5) into above-mentioned gained supernatant add 40%~50% ammonium sulfate, place 12~24h, under 8000g~10000g
10min~20min is centrifuged, supernatant is collected；
(6) gained supernatant is dialysed and is freeze-dried, produce phycoerythrin.
2. a kind of method that phycoerythrin is extracted from Porphyra yezoensis as described in claim 1, it is characterised in that：The step
(2) pH of the phosphate buffer in is 7.0, and concentration is 0.01mol/L~0.1mol/L.
3. a kind of method that phycoerythrin is extracted from Porphyra yezoensis as described in claim 1, it is characterised in that：The step
(3) freeze thawing is is placed in -20 DEG C of freeze overnights in, then then at thawing at room temperature.
4. a kind of method that phycoerythrin is extracted from Porphyra yezoensis as described in claim 1, it is characterised in that：The step
(3) ultrasonically treated condition is in：200W~600W, work 10s, suspends 10s, totally 2~10min.
5. a kind of method that phycoerythrin is extracted from Porphyra yezoensis as described in claim 1, it is characterised in that：The step
(6) molecular cut off of dialysis procedure is 3.5kDa in.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113754745A (en) * | 2020-06-05 | 2021-12-07 | 哈尔滨工业大学（威海） | Novel method for extracting high-purity R-phycoerythrin in laver |
| CN115785196A (en) * | 2022-12-21 | 2023-03-14 | 台州科技职业学院 | Method for extracting porphyra haitanensis protein by ultrasonic-assisted wall breaking |
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| CN101624613A (en) * | 2009-08-06 | 2010-01-13 | 南京农业大学 | Bio-modification preparation method for improving activity of laver phycoerythrin |
| CN104130319A (en) * | 2014-08-19 | 2014-11-05 | 台州宾美生物科技有限公司 | Extraction method of phycoerythrin |
| CN104447967A (en) * | 2014-11-26 | 2015-03-25 | 南通中国科学院海洋研究所海洋科学与技术研究发展中心 | Method for simultaneously extracting phycoerythrin and sulphated porphyra polysaccharide from inferior nori |
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2017
- 2017-08-01 CN CN201710646600.9A patent/CN107312076A/en active Pending
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| CN101624613A (en) * | 2009-08-06 | 2010-01-13 | 南京农业大学 | Bio-modification preparation method for improving activity of laver phycoerythrin |
| CN104130319A (en) * | 2014-08-19 | 2014-11-05 | 台州宾美生物科技有限公司 | Extraction method of phycoerythrin |
| CN104130319B (en) * | 2014-08-19 | 2017-07-28 | 浙江宾美生物科技有限公司 | A kind of extracting method of phycoerythrin |
| CN104447967A (en) * | 2014-11-26 | 2015-03-25 | 南通中国科学院海洋研究所海洋科学与技术研究发展中心 | Method for simultaneously extracting phycoerythrin and sulphated porphyra polysaccharide from inferior nori |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113754745A (en) * | 2020-06-05 | 2021-12-07 | 哈尔滨工业大学（威海） | Novel method for extracting high-purity R-phycoerythrin in laver |
| CN115785196A (en) * | 2022-12-21 | 2023-03-14 | 台州科技职业学院 | Method for extracting porphyra haitanensis protein by ultrasonic-assisted wall breaking |
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