CN107216367A - The preparation method of Closterium activated protein - Google Patents
The preparation method of Closterium activated protein Download PDFInfo
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- CN107216367A CN107216367A CN201710482175.4A CN201710482175A CN107216367A CN 107216367 A CN107216367 A CN 107216367A CN 201710482175 A CN201710482175 A CN 201710482175A CN 107216367 A CN107216367 A CN 107216367A
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention discloses the preparation method of Closterium activated protein, concretely comprise the following steps：1）With PBS as Extraction solvent, high-pressure homogeneous carry out clasmatosis；2）(NH is added into protein crude extract4)2SO4Saltoutd；3）Salt precipitation thing is subjected to aqueous two-phase extraction with polyethylene glycol and inorganic salts double-aqueous phase system, upper phase is protein solution；4）Protein solution is placed in ultra-filtration centrifuge tube, centrifuged, freeze-drying, as Closterium activated protein.Have the beneficial effect that：The preparation method reaction condition of the present invention is gentle, and the property of activated protein is not destroyed, and activated protein purity is high, yield is high, technique is easy and effective, production cost is low, it is adaptable to industrialized production, and the activated protein has anti-inflammatory activity, active anticancer and improves immunity, the advantage of Scavenger of ROS.
Description
Technical field
The present invention relates to biological product technical field, more particularly, to the preparation method of Closterium activated protein.
Background technology
Microalgae is widely used in aquaculture as excellent natural bait, is a variety of mollusks, the shell-fish young
And zooplankter（Such as wheel animalcule）Main food source.The food value of microalgae is relevant with many factors, including cell size, cell membrane
Can digestible degree and cellular content biochemical composition, it is especially mostly important with the latter.Someone notices envirment factor very early
Influence of the change to micro algae growth and biochemical composition, follow-up study elaborates that various microalgaes are matched somebody with somebody in different culture mediums in succession
Intracellular biochemical under side, different training methods and different temperature, light intensity, throughput, salinity, QD, photoperiod and light quality
The change of composition.Closterium (Closlerium) belongs to Chlorophyta, engagement algae month, Gu Yan sections.Recorded more than 300 kinds, it is raw
Live in various what is said or talked about water water bodys.Closterium is slender cell space, and cell is crescent, and there is a core in center, and respectively there is a leaf on core both sides
Green body.There is vertical strip projection on the surface of chloroplaset, and cross section is in aristiform；There are 1 row to make powder core in chloroplaset.Cell two ends respectively have
One vacuole, wherein there is the gypsum crystallization of rotation.Similar with Closterium also has desmids, but cell shape is different.Desmids
It is slender cell space, is made up of 2 " semicells " and middle narrow algae waist.Mode of reproduction is identical with crescent algae.They are belonged to
Green alga.It is most of to make edible or feed.Green alga discharges substantial amounts of oxygen and containing substantial amounts of protein, green alga to the Nature
To the self-purification of water and industrially there is sizable effect.
Prior art such as Authorization Notice No. is the B of CN 102020705 Chinese invention patent, discloses a kind of spirulina point
From the preparation method of albumen, comprise the following steps：(1) spirulina cells crush the preparation of decoloration deodorization degreasing material；(2) dilute salt
Solution extracts albumen and centrifugal treating；(3) pH gradient segmentation acid adjustment protein precipitation and regulation are neutral；(4) ultra high temperature short time sterilization
And spray drying.The spirulina protein isolate produced using this method has that lighter color, fishy smell are light, protein content is high, molten
Solution property is good and digestible advantage, can be as a kind of high-quality complete protein nutritious supplementary pharmaceutical or additive application, but the system
Preparation Method carries out clasmatosis using ultramicro grinding processing, crushes not thorough.
The content of the invention
It is an object of the invention to provide a kind of activated protein purity height, yield height, technique is easy and effective, production cost
Low, yield is big, is adapted to the preparation method of Closterium activated protein prepared on a large scale.
The present invention is directed to the problem of being mentioned in above-mentioned technology, and the technical scheme taken is：The preparation of Closterium activated protein
Method, including granular cell is crushed, saltoutd, aqueous two-phase extraction, ultrafiltration, specifically include following steps：
Clasmatosis：The algae solution that moisture content is 96-98% is configured, functional protein peptide is added, is delayed from concentration for 0.5-1% PBS
Fliud flushing is as Extraction solvent, homogeneous 2-3 times under the conditions of homogenization pressure is 23-26MPa, it is ensured that the egg flowed out every time from homogenizer
Temperature≤40 DEG C of white mixed liquor, then the refrigerated centrifuge centrifugation 18-22min through 3-5 DEG C, 7000-9000rpm, supernatant
Effect of the PBS cushioning liquid to Cells of Blue-green Algae wall in as protein crude extract, the step, weakens the intensity of cell membrane, so that
Cell membrane is easier under homogenate broken, in addition, PBS system is faintly acid system, close to physiological environment, be more conducive to
The holding of algae protein active.Closterium in high pressure homogenizer, by adjustable high pressure homogeneous valve, under decompression state suddenly, production
Raw high speed shear impact phenomenon, strong shear action makes immiscible liquid-liquid or the material such as liquid-solid forms superfine equal
The dispersion of even emulsification states of matter, high-pressure homogenization crushes crescent frustule release algae albumen, being capable of the broken frustule of mass, profit
Use industrialized production；
Saltout：(NH is added into protein crude extract4)2SO4To 25-35% saturation degrees, static 11-13h at 3-5 DEG C, then in 3-5
DEG C, centrifuge 20-25min under 4000-6000rpm, produce salt precipitation thing, substantial amounts of ammonium sulfate added in the step, makes albumen
Matter obtains solubility and reduced and Precipitation, and this method advantage is that ammonium sulfate concentrations change has continuity, and effect of saltouing is good, section
About energy consumption；
Aqueous two-phase extraction：Polyethylene glycol and inorganic salts are made into pH for 5.8-6.5, Polyethylene glycol is 15-17%, inorganic salts
Concentration is 11-13% double-aqueous phase system, adds salt precipitation thing, and magnetic agitation is then transferred in separatory funnel and is layered, on
It is mutually protein solution, aqueous two phase extraction technique is a kind of new extraction distribution technique, and its reaction condition is gentle, easy technique
Amplification, the split-phase time is short, and effect is good, and wherein polyethylene glycol is mutually upper phase, and inorganic salts are mutually lower phase, and algae albumen is mainly distributed on poly-
In ethylene glycol phase；
Ultrafiltration：Protein solution is placed in molecular cut off in 20-40KDa ultra-filtration centrifuge tubes, to centrifuge, freeze-drying is new
Month algae activated protein, 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, the albumen have anti-inflammatory activity,
Active anticancer and improve immunity, can Scavenger of ROS, can by driving away free radical so as to showing anti-inflammatory activity, albumen
Hematopoietin activity is higher, has functions that to stimulate marrow hemopoiesis, feed or fish bait available for animal.
It is preferred that, the addition of functional protein peptide is the 0.008-0.01% of algae powder weight, amino acid in cell destruction step
Sequence is HSHSVCVSHRGRCYCRCLRCRVLHPGKLCVCVNCSR, the positive charge in the protein peptides and bacterium plasma membrane phospholipid point
Negative electrical charge on son is attracted each other and close, the destruction original ordered structure of lipid bilayer by electrostatic interaction, in cell
Ion channel is formed on film, changes the osmotic pressure of crescent frustule, intracellular content leaks are caused, improves what crude protein was extracted
Operating efficiency.
It is preferred that, inorganic salts are potassium phosphate, sodium ascorbyl phosphate, sodium sulphate, potassium sulfate, tartaric acid in aqueous two-phase extraction step
One or more of mixing in potassium sodium.
Compared with prior art, the advantage of the invention is that：
1. the extracting method for the Closterium activated protein that the present invention is provided, the activated protein purity extracted is high, yield is high, technique
Easy and effective, production cost is low, and yield is big, is adapted to prepare Closterium activated protein on a large scale.
2. carrying out clasmatosis using high pressure homogenizer, under decompression state suddenly, high speed shear impact phenomenon is produced, by force
Strong shear action makes immiscible liquid-liquid or the material such as liquid-solid forms the dispersion of superfine uniform emulsification states of matter,
High-pressure homogenization crushes crescent frustule release algae albumen, can mass crush frustule, it is adaptable to industrialized production.
3. aqueous two phase extraction technique is a kind of new extraction distribution technique, its reaction condition is gentle, easy technique amplification,
The split-phase time is short, and effect is good, and wherein polyethylene glycol is mutually upper phase, and inorganic salts are mutually lower phase, and algae albumen is mainly distributed on poly- second two
In alcohol phase.
4. the albumen have anti-inflammatory activity, active anticancer and improve immunity, can Scavenger of ROS, can by drive away from
By base so as to show anti-inflammatory activity, the hematopoietin activity of albumen is higher, has functions that to stimulate marrow hemopoiesis, can use
In the feed or fish bait of animal.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The preparation method of Closterium activated protein, including granular cell is crushed, saltoutd, aqueous two-phase extraction, ultrafiltration, specifically include following
Step：
1）Clasmatosis：The algae solution that moisture content is 98% is configured, functional protein peptide is added, from the PBS that concentration is 0.7%
As Extraction solvent, the homogeneous 3 times under the conditions of homogenization pressure is 25MPa, it is ensured that the mixed liquid of protein flowed out every time from homogenizer
Temperature≤40 DEG C, then through 4 DEG C, 8000rpm refrigerated centrifuge centrifugation 20min, supernatant is protein crude extract, the step
Effect of the PBS cushioning liquid to Cells of Blue-green Algae wall, weakens the intensity of cell membrane, so that cell membrane more holds under homogenate in rapid
It is broken, in addition, PBS system is faintly acid system, close to the holding of physiological environment, more conducively algae protein active.It is high
The Closterium in homogenizer is pressed, by adjustable high pressure homogeneous valve, under decompression state suddenly, high speed shear impact phenomenon is produced,
Strong shear action makes immiscible liquid-liquid or the material such as liquid-solid forms the scattered of superfine uniform emulsification states of matter
Thing, high-pressure homogenization crushes crescent frustule release algae albumen, can mass crush frustule, utilize industrialized production；
2）Saltout：(NH is added into protein crude extract4)2SO4To 32% saturation degree, static 12h at 4 DEG C, then 4 DEG C,
2min is centrifuged under 5000rpm, salt precipitation thing is produced, substantial amounts of ammonium sulfate is added in the step, protein is obtained solubility drop
Low and Precipitation, 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：It is 6 that polyethylene glycol and inorganic salts are made into pH, and Polyethylene glycol is 16%, and inorganic salt concentration is
12% double-aqueous phase system, adds salt precipitation thing, and magnetic agitation is then transferred in separatory funnel and is layered, upper phase is albumen
Solution, aqueous two phase extraction technique is a kind of new extraction distribution technique, and its reaction condition is gentle, easy technique amplification, split-phase
Time is short, and effect is good, and wherein polyethylene glycol is mutually upper phase, and inorganic salts are mutually lower phase, and algae albumen is mainly distributed on polyethylene glycol phase
In；
4）Ultrafiltration：Protein solution is placed in molecular cut off in 30KDa ultra-filtration centrifuge tubes, to centrifuge, freeze-drying, as crescent
Algae activated protein, the step can effectively remove the polyethylene glycol in protein solution on the premise of activated protein is not destroyed,
The concentration for the improving albumen and purity for ensureing albumen will not reduce or even increased, the albumen has anti-inflammatory activity, anti-
Cancer activity and improve immunity, can Scavenger of ROS, can be by driving away free radical so as to showing anti-inflammatory activity, the rush of albumen
Erythropoietin activity is higher, has functions that to stimulate marrow hemopoiesis, feed or fish bait available for animal.
The addition of functional protein peptide is the 0.01% of algae powder weight in above-mentioned cell destruction step, and amino acid sequence is
On positive charge and bacterium plasma membrane phospholipid molecule in HSHSVCVSHRGRCYCRCLRCRVLHPGKLCVCVNCSR, the protein peptides
Negative electrical charge is attracted each other and close, the destruction original ordered structure of lipid bilayer by electrostatic interaction, the shape on cell membrane
Into ion channel, change the osmotic pressure of crescent frustule, cause intracellular content leaks, improve the work effect that crude protein is extracted
Rate.
Inorganic salts are potassium phosphate, sodium ascorbyl phosphate, sodium sulphate, potassium sulfate, potassium tartrate in above-mentioned aqueous two-phase extraction step
One or more of mixing in sodium.
Embodiment 2：
The preparation method of Closterium activated protein, specifically includes following steps：
1）The algae solution that moisture content is 976-98% is configured, the 0.01% functional protein peptide that weight is algae powder weight is added, from dense
The PBS for 0.8% is spent as Extraction solvent, the homogeneous 2 times under the conditions of homogenization pressure is 26MPa, it is ensured that every time from homogeneous
Machine outflow mixed liquid of protein temperature≤40 DEG C, then through 5 DEG C, 9000rpm refrigerated centrifuge centrifugation 18min, supernatant
Effect of the PBS cushioning liquid to Cells of Blue-green Algae wall in as protein crude extract, the step, weakens the intensity of cell membrane, so that
Cell membrane is easier under homogenate broken, in addition, PBS system is faintly acid system, close to physiological environment, be more conducive to
The holding of algae protein active.Closterium in high pressure homogenizer, by adjustable high pressure homogeneous valve, under decompression state suddenly, production
Raw high speed shear impact phenomenon, strong shear action makes immiscible liquid-liquid or the material such as liquid-solid forms superfine equal
The dispersion of even emulsification states of matter, high-pressure homogenization crushes crescent frustule release algae albumen, being capable of the broken frustule of mass, profit
Use industrialized production；
2）(NH is added into protein crude extract4)2SO4To 35% saturation degree, static 11h at 5 DEG C is then centrifuged for, produces and saltout heavy
Substantial amounts of ammonium sulfate is added in starch, the step, protein is obtained solubility reduces and Precipitation, and this method advantage is sulphur
Sour ammonium concentration change has continuity, and effect of saltouing is good, saves energy consumption；
3）Aqueous two-phase extraction：It is 6.5 that polyethylene glycol and potassium phosphate are made into pH, and Polyethylene glycol is 15%, and no potassium phosphate is dense
The double-aqueous phase system for 13% is spent, salt precipitation thing is added, magnetic agitation is then transferred in separatory funnel and is layered, and upper phase is
Protein solution, aqueous two phase extraction technique is a kind of new extraction distribution technique, and its reaction condition is gentle, easy technique amplification,
The split-phase time is short, and effect is good, and wherein polyethylene glycol is mutually upper phase, and inorganic salts are mutually lower phase, and algae albumen is mainly distributed on poly- second two
In alcohol phase；
4）Protein solution is placed in molecular cut off in 40KDa ultra-filtration centrifuge tubes, to centrifuge, freeze-drying, as Closterium are lived
Property albumen, the step can effectively remove the polyethylene glycol in protein solution on the premise of activated protein is not destroyed, and improve
The concentration of albumen and ensure that the purity of albumen will not reduce or even increased, there is the albumen anti-inflammatory activity, anticancer to live
Property and improve immunity, can Scavenger of ROS, anti-inflammatory activity can be shown by driving away free radical, the rush of albumen is red thin
Born of the same parents' generation element activity is higher, has functions that to stimulate marrow hemopoiesis, feed or fish bait available for animal.
Embodiment 3：
The preparation method of Closterium activated protein, is concretely comprised the following steps：The algae solution that moisture content is 96-98% is configured, functional protein is added
Peptide, from the PBS that concentration is 1% as Extraction solvent, homogeneous 3 times, Ran Houjing under the conditions of homogenization pressure is 24MPa
Refrigerated centrifuge is centrifuged；(NH is added into supernatant4)2SO4Static to 30% saturation degree, centrifugation produces salt precipitation thing；Will
It is that 6.5, Polyethylene glycol is the aqueous two-phase that 17%, potassium tartrate na concn is 11% that polyethylene glycol and sodium potassium tartrate tetrahydrate, which are made into pH,
System, adds salt precipitation thing, and magnetic agitation is then transferred in separatory funnel and is layered, upper phase is protein solution；By albumen
Solution is placed in molecular cut off in 20-40KDa ultra-filtration centrifuge tubes, to centrifuge, freeze-drying, as Closterium activated protein.
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>Zhoushan Fu Sheng food science and technologies Co., Ltd
<120>The preparation method of Closterium activated protein
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> PRT
<213>It is artificial synthesized
<400> 1
His Ser His Ser Val Cys Val Ser His Arg Gly Arg Cys Tyr Cys Arg
1 5 10 15
Cys Leu Arg Cys Arg Val Leu His Pro Gly Lys Leu Cys Val Cys Val
20 25 30
Asn Cys Ser Arg
35
Claims (10)
1. the preparation method of Closterium activated protein, including granular cell is crushed, saltoutd, aqueous two-phase extraction, ultrafiltration, its feature exists
In：The cell destruction step is：Functional protein peptide is added in algae solution, from PBS as Extraction solvent, high pressure is equal
Matter, centrifugation, supernatant is protein crude extract.
2. the preparation method of Closterium activated protein according to claim 1, it is characterised in that：The cell destruction step
The moisture content of middle algae solution is 96-98%.
3. the preparation method of Closterium activated protein according to claim 1, it is characterised in that：The cell destruction step
The addition of middle functional protein peptide is the 0.008-0.01% of algae powder weight, and amino acid sequence is
HSHSVCVSHRGRCYCRCLRCRVLHPGKLCVCVNCSR。
4. the preparation method of Closterium activated protein according to claim 1, it is characterised in that：The cell destruction step
The concentration of middle PBS is 0.5-1%.
5. the preparation method of Closterium activated protein according to claim 1, it is characterised in that：The cell destruction step
Middle homogenization pressure is 23-26MPa, homogeneous 2-3 times.
6. the preparation method of Closterium activated protein according to claim 1, it is characterised in that：The cell destruction step
Middle centrifuging temperature is 3-5 DEG C, rotating speed is 7000-9000rpm, and the time is 18-22min.
7. the preparation method of Closterium activated protein according to claim 1, it is characterised in that：The salting-out step is：
(NH is added into protein crude extract4)2SO4To 78-82% saturation degrees, static 11-13h at 3-5 DEG C, centrifugation produces salt precipitation
Thing.
8. the preparation method of Closterium activated protein according to claim 1, it is characterised in that：The aqueous two-phase extraction step
The pH of double-aqueous phase system is 5.8-6.5 in rapid, and Polyethylene glycol is 15-17%, and inorganic salt concentration is 11-13%.
9. the preparation method of Closterium activated protein according to claim 8, it is characterised in that：The aqueous two-phase extraction step
Inorganic salts are one or more of mixing in potassium phosphate, sodium ascorbyl phosphate, sodium sulphate, potassium sulfate, sodium potassium tartrate tetrahydrate in rapid.
10. the preparation method of Closterium activated protein according to claim 1, it is characterised in that：In the ultrafiltration step
The molecular cut off of ultra-filtration centrifuge tube is 20-40KDa.
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| CN108060155A (en) * | 2017-12-29 | 2018-05-22 | 舟山富晟食品科技有限公司 | The protease extracted from squid viscera |
| WO2020133993A1 (en) * | 2018-12-26 | 2020-07-02 | 深圳翰宇药业股份有限公司 | Method for polypeptide purification |
| CN114621313A (en) * | 2020-12-10 | 2022-06-14 | 启迪禾美生物科技(嘉兴)有限公司 | Euglena protein extract and application thereof in cosmetics |
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| CN114621313A (en) * | 2020-12-10 | 2022-06-14 | 启迪禾美生物科技(嘉兴)有限公司 | Euglena protein extract and application thereof in cosmetics |
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