CN107298710A

CN107298710A - A kind of extracting method of spirulina phycocyanin

Info

Publication number: CN107298710A
Application number: CN201710544864.3A
Authority: CN
Inventors: 李冰
Original assignee: Pujiang County Biological Technology Co Ltd Europe
Current assignee: Pujiang County Biological Technology Co Ltd Europe
Priority date: 2017-07-06
Filing date: 2017-07-06
Publication date: 2017-10-27

Abstract

The present invention discloses a kind of extracting method of spirulina phycocyanin, including：Granular cell is crushed, saltoutd, aqueous two-phase extraction, ultrafiltration, it is characterised in that：The cell destruction step is：Functional protein peptide is added in algae solution, from sodium phosphate buffer as Extraction solvent, multigelation is centrifuged, supernatant is protein crude extract.Solve that sporoderm-broken rate in the prior art is not high to cause the problem of protein extracting ratio is low, the phycocyanin that the present invention is extracted can improve lymphocyte activity, body's immunity is improved by lymphatic system, the diseases prevention of enhancing body, resistance against diseases also have excellent anti-oxidant and radiation, anti-inflammatory, antibacterial function comprehensively, available for feed or field of beverage, the extraction process of spirulina phycocyanin of the present invention is simple, workable, production cost is low, can be mass spirulina phycocyanin.

Description

A kind of extracting method of spirulina phycocyanin

Technical field

The invention belongs to natural products protein extracting process field, more particularly to a kind of extraction of spirulina phycocyanin Method.

Background technology

It is how thin in tropical and subtropical region fresh water or saline and alkaline lake that spirulina (Spirulina) is that a class is distributed in Born of the same parents, without special-shaped born of the same parents, also known as thread spiral prokaryotes, cyanobacteria, the phycobniliprotein in spirulina is a kind of important Photochromic fibroin is caught, is made up of phycocyanin and allophycocyanin, the light energy of absorption passes to the blue egg of other algae by phycocyanin In vain, then by allophycocyanin photosynthesis reaction center is passed to, there are some researches show phycocyanin can improve lymphocyte work Property, body's immunity is improved by lymphatic system, comprehensively the diseases prevention of enhancing body, resistance against diseases.

Isolating and purifying for current spirulina phycocyanin and allophycocyanin generally carries out layer using hydroxyapetite media Analysis separation, but there are problems that weak adsorptivity, elution time length, applied sample amount, and also need to combine the operation of other more chromatographic enrichments Such as size exclusion chromatography and ion-exchange chromatography further improve phycocyanin purity.Also having been reported that can be by the behaviour that saltouts repeatedly Make or aqueous two phase extraction technique, improve the purity of phycobniliprotein, operated without more chromatographic enrichment, but multistep is extracted operation and equally existed Product yield is low, the problems such as operation cycle is long, especially to the phycocyanin with fluorescent characteristic, and its fluorescent characteristic is easily for a long time Extraction operating process in lose.

Prior art, such as Chinese invention granted patent document, the B of granted patent number CN 102993297, invention are related to The extracting method of spirulina phycocyanin, spirulina powder suspension is obtained using the processing of multigelation method combination mechanical crushing method Spirulina cells crush liquid；Liquid, which is crushed, using 30% and 50% ammonium sulfate salt fractionation precipitation spirulina cells obtains algae indigo plant Protein crude extract 1；Add the phycocyanin crude extract that PEG20000 and NaCl is further settled out higher degree；Use phosphate Buffer solution phycocyanin crude extract, is made phycocyanin crude extract 2；Phycocyanin crude extract 2 and then use PEG20000/ Na2SO4 double-aqueous phase systems are extracted, and the lower phase after extraction can obtain phycocyanin refining liquid by dialysis desalination, prepared by freeze-drying Phycocyanin finished product.The invention extraction process is simple, cost is low, suitable for intermittent and large-scale production processing high-purity, high receipts The spirulina phycocyanin finished product of rate.Phycocyanin crude extract can be long time stored made from the invention, and preparation is further purified Phycocyanin finished product there is good resisting oxidation free radical elimination effect and fluorescence intensity, but the phycocyanin extraction process In shell-broken effect it is not ideal, be unfavorable for improve albumen yield, the extracting method to the phycocyanin with fluorescent characteristic, Its fluorescent characteristic is easily lost in operating process is extracted for a long time.

The content of the invention

Phycocyanin purity and yield are improved by breaking-wall cell it is an object of the invention to provide one kind, fluorescence can be protected Property phycocyanin fluorescent characteristic, technique is simple, and production cost is low, a kind of extraction for the spirulina phycocyanin that can be mass Method.

To achieve the above object, the scheme taken is the present invention：A kind of extracting method of spirulina phycocyanin, bag Include：Granular cell is crushed, saltoutd, aqueous two-phase extraction, ultrafiltration, concrete operation step is as follows：

（1）Granular cell is crushed：Configure spirulina extract, solid-to-liquid ratio (m (g):V(mL) ) =1:150, add spirulina powder The 0.06%-0.12% functional protein peptides of weight, add 25mM sodium phosphate buffers (pH7.0), freeze repeatedly at -20 DEG C and 37 DEG C Melt 4-5 times, each freeze thawing time is 4h, by the shell-broken liquid after freeze thawing 4-5 time in centrifuging 30min, collection supernatant under 5000rpm The crude extract of phycocyanin is obtained, is placed in 4 DEG C of refrigerators and is in store for, the amino acid sequence of functional protein peptide is MKGLSFVLLVLLLMMPDGEGTDPEMQYWTCGRGLCRRFCYAEYFVGHHCPRRYRCCAIRA.The active peptide has hydrophilic Property and lipophilicity, hydrophily makes it be dissolved in distilled water, and lipophilicity makes itself and spirulina cells film combination, makes to be formed under cell membrane Aperture, causes intracellular content leaks, improves the operating efficiency that crude protein is extracted, also improves the yield of activated protein, utilizes Active peptide combination freeze-thaw method carries out clasmatosis, substantially reduces extraction time, extraction effect is good, protein content in crude protein liquid It is high, it is to avoid to need the protein denaturation caused by heating and mechanical force during break process, fluorescence phycocyanin can be protected Fluorescent characteristic；

（2）Salting-out step is：(NH is added into protein crude extract₄)₂SO₄Solution is stood at 3-5 DEG C to 75-84% saturation degrees 11-13h, centrifugation, produces salt precipitation thing；Substantial amounts of ammonium sulfate is added in the step, protein is obtained solubility reduction and is sunk Precipitation goes out, and this method advantage is that ammonium sulfate concentrations change has continuity, and effect of saltouing is good, saves energy consumption；

（3）Aqueous two-phase extraction step：Ethylene glycol and inorganic salts are made into pH for 5.8-6.5, Polyethylene glycol is 15-17%, nothing Machine salinity is 11-13%, adds salt precipitation thing, and magnetic agitation is then transferred in separatory funnel and is layered, upper phase is egg White solution, aqueous two phase extraction technique is a kind of new extraction distribution technique, and its reaction condition is gentle, easy technique amplification, point The phase time is short, and effect is good, and wherein polyethylene glycol is mutually upper phase, and inorganic salts are mutually lower phase, and phycocyanin is mainly distributed on poly- second two In alcohol phase；Inorganic salts are one or more of mixing in potassium phosphate, sodium ascorbyl phosphate, sodium sulphate, potassium sulfate, sodium potassium tartrate tetrahydrate；

（4）Ultrafiltration：Protein solution is placed in molecular cut off in 20-40KDa ultra-filtration centrifuge tubes, to centrifuge, is freeze-dried, is Spirulina phycocyanin, the step can effectively remove the poly- second in protein solution on the premise of activated protein is not destroyed Glycol, the concentration for improving albumen and the purity for ensureing albumen will not reduce or even increased, and the albumen can improve lymph Cytoactive, body's immunity is improved by lymphatic system, and the diseases prevention of enhancing body, resistance against diseases also have excellent comprehensively Anti-oxidant and radiation, anti-inflammatory, antibacterial function, the phycocyanin of extraction can be used for feed or field of beverage.

Compared with prior art, beneficial effects of the present invention are：1）It is thin that the phycocyanin that the present invention is extracted can improve lymph Cytoactive, body's immunity is improved by lymphatic system, and the diseases prevention of enhancing body, resistance against diseases also have excellent resist comprehensively Oxidation and radiation, anti-inflammatory, antibacterial function, available for feed or field of beverage；2）Carried out using active peptide combination freeze-thaw method thin Born of the same parents crush, and substantially reduce extraction time, extraction effect is good, protein content is high in crude protein liquid, it is to avoid break process process In need to the protein denaturation caused by heating and mechanical force, the fluorescent characteristic of fluorescence phycocyanin can be protected；3）Spiral shell of the present invention The extraction process for revolving algae phycocyanin is simple, and workable, production cost is low, can be mass spirulina phycocyanin.

Embodiment

Technical scheme is described in further detail with reference to embodiments：

Embodiment 1：

A kind of extracting method of spirulina phycocyanin, including：Granular cell is crushed, saltoutd, aqueous two-phase extraction, ultrafiltration, specific behaviour Make step as follows：

（1）Granular cell is crushed：Configure spirulina extract, solid-to-liquid ratio (m (g):V(mL) ) =1:150, add spirulina powder 0.08% functional protein peptide of weight, adds 25mM sodium phosphate buffers (pH7.0), in -20 DEG C and 37 DEG C of multigelations 5 times, Each freeze thawing time is 4h, by the shell-broken liquid after freeze thawing 5 times in centrifuging 30min under 5000rpm, collects supernatant and obtains the blue egg of algae White crude extract, is placed in 4 DEG C of refrigerators and is in store for, and the amino acid sequence of functional protein peptide is MKGLSFVLLVLLLMMPDGEGTDPEMQYWTCGRGLCRRFCYAEYFVGHHCPRRYRCCAIRA；

（2）Salting-out step is：(NH is added into protein crude extract₄)₂SO₄Solution stands 12h to 80% saturation degree at 3 DEG C, from The heart, produces salt precipitation thing；

（3）Aqueous two-phase extraction step：It is 6.0 that ethylene glycol and inorganic salts are made into pH, and Polyethylene glycol is 15%, and inorganic salts are dense Spend for 11%, inorganic salts are the mixture of potassium phosphate and sodium ascorbyl phosphate, add salt precipitation thing, and magnetic agitation is then transferred to It is layered in separatory funnel, upper phase is protein solution；

（4）Ultrafiltration：Protein solution is placed in molecular cut off in 20KDa ultra-filtration centrifuge tubes, to centrifuge, freeze-drying, as spiral shell Revolve algae phycocyanin.

Embodiment 2：

（1）Granular cell is crushed：Configure spirulina extract, solid-to-liquid ratio (m (g):V(mL) ) =1:150, add spirulina powder 0.10% functional protein peptide of weight, adds 25mM sodium phosphate buffers (pH7.0), in -20 DEG C and 37 DEG C of multigelations 4 times, Each freeze thawing time is 4h, by the shell-broken liquid after freeze thawing 4 times in centrifuging 30min under 5000rpm, collects supernatant and obtains the blue egg of algae White crude extract, is placed in 4 DEG C of refrigerators and is in store for, and the amino acid sequence of functional protein peptide is MKGLSFVLLVLLLMMPDGEGTDPEMQYWTCGRGLCRRFCYAEYFVGHHCPRRYRCCAIRA.The active peptide has hydrophilic Property and lipophilicity, hydrophily makes it be dissolved in distilled water, and lipophilicity makes itself and spirulina cells film combination, makes to be formed under cell membrane Aperture, causes intracellular content leaks, improves the operating efficiency that crude protein is extracted, also improves the yield of activated protein, utilizes Active peptide combination freeze-thaw method carries out clasmatosis, substantially reduces extraction time, extraction effect is good, protein content in crude protein liquid It is high, it is to avoid to need the protein denaturation caused by heating and mechanical force during break process, fluorescence phycocyanin can be protected Fluorescent characteristic；

（2）Salting-out step is：(NH is added into protein crude extract₄)₂SO₄Solution stands 11h to 75% saturation degree at 5 DEG C, from The heart, produces salt precipitation thing；, substantial amounts of ammonium sulfate is added in the step, protein is obtained solubility reduction and Precipitation, should Method advantage is that ammonium sulfate concentrations change has continuity, and effect of saltouing is good, saves energy consumption；

（3）Aqueous two-phase extraction step：It is 6.0 that ethylene glycol and inorganic salts are made into pH, and Polyethylene glycol is 15%, and inorganic salts are dense Spend for 11%, inorganic salts are the mixture in sodium ascorbyl phosphate, sodium sulphate, sodium potassium tartrate tetrahydrate；Addition salt precipitation thing, magnetic agitation, It is then transferred in separatory funnel and is layered, upper phase is protein solution, aqueous two phase extraction technique is a kind of new extraction distribution Technology, its reaction condition is gentle, and easy technique amplification, the split-phase time is short, and effect is good, and wherein polyethylene glycol is mutually upper phase, inorganic Salt is mutually lower phase, and phycocyanin is mainly distributed in polyethylene glycol phase；

（4）Ultrafiltration：Protein solution is placed in molecular cut off in 30KDa ultra-filtration centrifuge tubes, to centrifuge, freeze-drying, as spiral shell Algae phycocyanin is revolved, the step can effectively remove the poly- second two in protein solution on the premise of activated protein is not destroyed Alcohol, the concentration for improving albumen and the purity for ensureing albumen will not reduce or even increased, and it is thin that the albumen can improve lymph Cytoactive, body's immunity is improved by lymphatic system, and the diseases prevention of enhancing body, resistance against diseases also have excellent resist comprehensively Oxidation and radiation, anti-inflammatory, antibacterial function, available for feed or field of beverage.

Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.

Technical scheme is described in detail embodiment described above, it should be understood that it is described above only For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention, Supplement or similar fashion replacement etc., should be included in the scope of the protection.

SEQUENCE LISTING

<110>Pujiang County Ou Li Bioisystech Co., Ltd

<120>A kind of extracting method of spirulina phycocyanin

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 60

<212> PRT

<213>It is artificial synthesized

<400> 1

Met Lys Gly Leu Ser Phe Val Leu Leu Val Leu Leu Leu Met Met Pro

1 5 10 15

Asp Gly Glu Gly Thr Asp Pro Glu Met Gln Tyr Trp Thr Cys Gly Arg

20 25 30

Gly Leu Cys Arg Arg Phe Cys Tyr Ala Glu Tyr Phe Val Gly His His

35 40 45

Cys Pro Arg Arg Tyr Arg Cys Cys Ala Ile Arg Ala

50 55 60

Claims

1. a kind of extracting method of spirulina phycocyanin, including：Clasmatosis, saltout, aqueous two-phase extraction, ultrafiltration, its feature It is：The cell destruction step is：Functional protein peptide is added in algae solution, from sodium phosphate buffer as Extraction solvent, Multigelation, centrifugation, supernatant is protein crude extract.

2. a kind of extracting method of spirulina phycocyanin according to claim 1, it is characterised in that：The clasmatosis The solid-to-liquid ratio of spirulina powder and water in step in algae solution is 1:150, the pH value of sodium phosphate buffer is 7.0.

3. a kind of extracting method of spirulina phycocyanin according to claim 1, it is characterised in that：The functional protein The addition of peptide is the 0.06%-0.12% of spirulina powder weight, and the amino acid sequence of the functional protein peptide is MKGLSFVLLVLLLMMPDGEGTDPEMQYWTCGRGLCRRFCYAEYFVGHHCPRRYRCCAIRA。

4. a kind of extracting method of spirulina phycocyanin according to claim 1, it is characterised in that：The clasmatosis Multigelation in step is each 4h in -20 DEG C and 37 DEG C, multigelation 4-5 times.

5. a kind of extracting method of spirulina phycocyanin according to claim 1, it is characterised in that：The salting-out step For：(NH is added into protein crude extract₄)₂SO₄Solution stands 11-13h to 75-84% saturation degrees at 3-5 DEG C, centrifuges, produces Salt precipitation thing.

6. a kind of extracting method of spirulina phycocyanin according to claim 1, it is characterised in that：The aqueous two-phase extraction The pH for taking double-aqueous phase system in step is 5.8-6.5, and Polyethylene glycol is 15-17%, and inorganic salt concentration is 11-13%, described Inorganic salts are one or more of mixing in potassium phosphate, sodium ascorbyl phosphate, sodium sulphate, potassium sulfate, sodium potassium tartrate tetrahydrate.

7. a kind of extracting method of spirulina phycocyanin according to claim 1, it is characterised in that：The ultrafiltration step The molecular cut off of middle ultra-filtration centrifuge tube is 20-40KDa.