CN107312063A - The preparation method of blunt top spirulina activated protein - Google Patents
The preparation method of blunt top spirulina activated protein Download PDFInfo
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- CN107312063A CN107312063A CN201710482525.7A CN201710482525A CN107312063A CN 107312063 A CN107312063 A CN 107312063A CN 201710482525 A CN201710482525 A CN 201710482525A CN 107312063 A CN107312063 A CN 107312063A
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- activated protein
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- blunt top
- top spirulina
- crude protein
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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
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- 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
- C07K1/303—Extraction; Separation; Purification by precipitation by salting out
-
- 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
Abstract
The invention discloses the preparation method of blunt top spirulina activated protein, specific method is：Algae powder adds phosphate buffer and active peptides, and multigelation obtains crude protein liquid；Ammonium sulfate precipitation is added in crude protein liquid；Salt precipitation thing is loaded into bag filter, is placed in phosphate buffer and dialyses；Dialyzate is prepared into activated protein sterling using hydroxylapatite adsorption chromatography.Have the beneficial effect that：Preparation method of the present invention extracts activated protein, improves extraction efficiency and the yield of activated protein, simple to operate, it is not necessary to special instrument and equipment；Activated protein purity produced by the present invention is high, it is bright-colored, it is nutritious, lymphocyte activity can be improved, body's immunity is improved by lymphatic system, strengthen diseases prevention, the resistance against diseases of body, moreover it is possible to remove the free radical in organism and promote animal cell substitution, and with significant antitumor activity.
Description
Technical field
The present invention relates to biological product technical field, more particularly, to the preparation method of blunt top spirulina activated protein.
Background technology
Spirulina (Spirulina platensis) is a class rudimentary plant, belongs to Cyanophyta (Cyanophyta), quiver algae
Section (Oscillatoriaceae).They are intracellular without real nucleus as bacterium, so also known as cyanobacteria.Spiral shell
Rotation algae is usually the filamentous, cylindric that many cells are constituted, and in loose or close and rule spiral sigmoid, but shape is often
Influenceed and changed by growing environment, linear variation that is having or even producing stabilization.Spirulina is pyrophilous（25-36
℃）, it is high-alkali（pH8~12）, photoautotrophy is sought, but can also utilize the simultaneous health of some organic matters work in environment long, it is capable asexual numerous
Grow, usually rely on the generation of pseudo- ghost in the generation of tabula in filament or filament and form hormocystangium.Spirulina contains greatly
Measure protein, extremely enrich nutritional ingredient and various bioactivators in a balanced way, its cell membrane is practically free of cellulose, therefore
Easily it is digested, research shows the digestibility of spirulina more than 94%, and its protein digestibility, up to 75%, is one
Excellent natural food is planted, is recommended as " the optimal food of global human " by FAO (Food and Agriculture Organization of the United Nation).Hydrolysis of protein spirulina
Content is up to 50-70%, is made up of 18 kinds of amino acid, wherein the content of human body and 8 kinds of amino acid necessary to animal close to or
The standard of FAO recommendations is exceeded, has been the edible and bait protein resource with great Development volue in nature, with certain
Medical care effect.Spirulina is also containing seven kinds of vitamins, and wherein vitamin E is a kind of natural, beta carotene
It is active highest vitamin a source, vitamin A is a kind of free radical scavenger, to tumour (especially epithelial tissue canceration)
Preventing and treating has positive effect.Spirulina polysaccharide and phycobniliprotein more have significant physiologically active and many medical values, such as
Anti- curing oncoma and ulcer, raising body's immunity, regulation blood pressure, promotion animal cell substitution etc..In summary, spirulina
And its active component has broad application prospects in terms of functional food research and development.
Prior art such as Authorization Notice No. is the B of CN 103044520 Chinese invention patent, discloses a kind of arthrospira egg
White isolation and purification method, specific method is fully to soak algae powder in PBS, and avoid light place 24-72 is small under normal temperature after mixing
When centrifuge, collect supernatant；Supernatant is concentrated by ultrafiltration through filter membrane, chitosan is added in concentrate reaches pH of mixed
6.5-7.2, and be stirred；Water is added in the above-mentioned mixed liquor centrifuging and taking supernatant stirred, supernatant to carry out through filter membrane
It is concentrated by ultrafiltration, ultrafiltration displacement sodium ion obtains purifying concentrate；Concentrate is spray-dried, and produces pharmaceutical-grade arthrospira albumen.Should
Method production cost is low, easy and effective, is adapted to prepare pharmaceutical-grade arthrospira albumen, but preparation efficiency is low, and arthrospira albumen is obtained
Rate is low.
The content of the invention
It is an object of the invention to provide a kind of activated protein extraction efficiency is high, yield is high, purity is high, energy consumption is low, to ring
The preparation method of the friendly blunt top spirulina activated protein in border.
The present invention is directed to the problem of being mentioned in above-mentioned technology, and the technical scheme taken is：Blunt top spirulina activated protein
Preparation method, including crude protein extraction, saltout, dialyse, purifying, concretely comprise the following steps：
The extraction of crude protein：It is 1 by solid-liquid ratio:2 ~ 3 add distilled water in algae powder, and suction filtration, deionized water is washed 2 ~ 3 times, plus
It is the phosphate buffer and active peptides that 6.5 ~ 7, concentration is 0.008 ~ 0.012mol/L, algae powder and phosphate buffer to enter pH
W/v be 1:14 ~ 16,110 ~ 130min is stirred, in -22 ~ -20 DEG C of snap frozens, is then thawed at 3 ~ 5 DEG C, instead
Freeze thawing 3 ~ 4 times again, 28 ~ 33min of high speed refrigerated centrifuge under 3 ~ 5 DEG C, 10000 ~ 12000rpm, supernatant is crude protein liquid,
Activated protein belongs to intracellular protein, and first having to the cell membrane of smudge cells, cell membrane makes its dissolution, and clasmatosis degree is got over
Height, the yield of activated protein is also higher, and multigelation method is increased using the formation and cell liquid salinity of intracellular ice pellets to be caused
Being swelled makes clasmatosis, simple to operate, it is not necessary to special instrument and equipment, and the step is mutually tied using protein peptides with multigelation method
Close, improve the operating efficiency of crude protein extraction and the yield of activated protein；
Saltout：The weight of the chitosan solution that concentration is 1.8 ~ 2.2wt%, algae powder and chitosan solution is added into crude protein liquid
Volume ratio is 1:4 ~ 6, pH to 6.5 ~ 7 is adjusted, 20 ~ 25min, the high speed refrigerated centrifuge under 3 ~ 5 DEG C, 10000 ~ 12000rpm is stirred
28 ~ 33min, supernatant is put into ice-water bath, and adding ammonium sulfate makes its concentration be 50 ~ 55wt%, stirs completely molten to ammonium sulfate
Solution, stands overnight rear low-temperature centrifugation, produces salt precipitation thing, substantial amounts of ammonium sulfate is added in the step, protein is dissolved
Degree is reduced and Precipitation, and this method advantage is that ammonium sulfate concentrations change has continuity, and effect of saltouing is good, saves energy consumption；
Dialysis：Salt precipitation thing is dissolved in phosphate buffer, bag filter is then charged into, bubble is excluded, sealing is placed in phosphoric acid
Dialysed in salt buffer, change buffer solution 4 ~ 6 times, solution is dialyzate in bag, and this method is using small-molecule substance in solution
In can be by bag filter, and macromolecular substances by the property of bag filter, can not reach the purpose of separation small molecular weight impurity, operation
Simply, effect is good, saves energy consumption；
Purifying：The hydroxyapatite that 10 ~ 12h will be soaked with buffer solution is fitted into the pillar containing pre-add buffer solution sum, adds saturating
Liquid is analysed, after cylinder, buffer solution elution is added, freeze-drying produces blunt top spirulina activated protein, the activated protein
It is bright-colored, it is nutritious, lymphocyte activity can be improved, body's immunity is improved by lymphatic system, enhancing body
Diseases prevention, resistance against diseases, moreover it is possible to remove the free radical in organism and promote animal cell substitution, and with significant anti-swollen
Tumor activity.
Preferably, the addition of active peptides is the 0.5 ~ 0.8% of algae powder, the amino acid sequence of active peptides is
HSHACASYSRCYCRCLRCRPGCVNCYRCSR, the active peptide has hydrophily and lipophilicity, and hydrophily makes it be dissolved in distillation
In water, lipophilicity makes itself and spirulina cells film combination, makes to form aperture under cell membrane, causes intracellular content leaks, carry
The operating efficiency that high crude protein is extracted, also improves the yield of activated protein.
Preferably, chromatographic column specification is 2 ~ 3cm of diameter, long 48 ~ 52cm, buffer solution is that 6.5 ~ 7, concentration is from pH
0.008 ~ 0.012mol/L phosphate buffer and 0.1 ~ 0.12mol/L KCl buffer solutions, flow velocity are 0.6 ~ 0.8ml/min.
Compared with prior art, the advantage of the invention is that：The present invention is extracted using active protein peptide and multigelation method
The method that crude protein is combined extracts activated protein, improves extraction efficiency and the yield of activated protein, simple to operate, it is not necessary to
Special instrument and equipment；Activated protein purity produced by the present invention is high, bright-colored, nutritious, can improve lymphocyte work
Property, body's immunity is improved by lymphatic system, strengthens the diseases prevention of body, resistance against diseases, moreover it is possible to remove in organism from
By base and promotion animal cell substitution, and with significant antitumor activity.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The preparation method of blunt top spirulina activated protein, including crude protein extraction, saltout, dialyse, purifying, concretely comprise the following steps：
1）The extraction of crude protein：It is 1 by solid-liquid ratio:3 add distilled water in algae powder, use Buchner funnel suction filtration, deionization washing
Wash 3 times, it is the phosphate buffer and active peptides that 6.7, concentration is 0.008mol/L, algae powder and phosphate buffer to add pH
W/v be 1:15,120min is stirred, in -20 DEG C of snap frozens, is then thawed at 4 DEG C, multigelation 3 times, 4
DEG C, high speed refrigerated centrifuge 30min under 12000rpm, supernatant is crude protein liquid, and activated protein belongs to intracellular protein, first
The cell membrane of smudge cells, cell membrane is wanted to make its dissolution, clasmatosis degree is higher, and the yield of activated protein is also higher, repeatedly
Freeze-thaw method increases to cause to be swelled using the formation and cell liquid salinity of intracellular ice pellets makes clasmatosis, simple to operate, is not required to
Special instrument and equipment is wanted, the step is combined using protein peptides and multigelation method, improve the operating efficiency that crude protein is extracted
With the yield of activated protein；
2）Saltout：The bulking value of the chitosan solution that concentration is 2wt%, algae powder and chitosan solution is added into crude protein liquid
Than for 1:5, pH to 6.7 is adjusted, 20min is stirred, high speed refrigerated centrifuge 30min, supernatant is put under 4 DEG C, 12000rpm
In ice-water bath, add ammonium sulfate make its concentration be 52wt%, stirring be completely dissolved to ammonium sulfate, stand overnight, 4 DEG C,
High speed refrigerated centrifuge 30min under 12000rpm, produces salt precipitation thing, and substantial amounts of ammonium sulfate is added in the step, makes protein
Obtaining solubility reduces and Precipitation, and this method advantage is that ammonium sulfate concentrations change has continuity, and effect of saltouing is good, saves
Energy consumption；
3）Dialysis：Salt precipitation thing is dissolved in phosphate buffer, bag filter is then charged into, bubble is excluded, sealing is placed in phosphorus
Dialysed in phthalate buffer, change buffer solution 5 times, solution is dialyzate in bag, and this method is using small-molecule substance in solution
In can be by bag filter, and macromolecular substances by the property of bag filter, can not reach the purpose of separation small molecular weight impurity, operation
Simply, effect is good, saves energy consumption；
4）Purifying：The hydroxyapatite that 12h is soaked with buffer solution is fitted into the pillar containing pre-add buffer solution sum, dialysis is added
Liquid, after cylinder, adds buffer solution elution, freeze-drying produces blunt top spirulina activated protein, the activated protein face
Color is bright-coloured, nutritious, can improve lymphocyte activity, and body's immunity is improved by lymphatic system, strengthens the anti-of body
Disease, resistance against diseases, moreover it is possible to remove the free radical in organism and promote animal cell substitution, and with significant antitumor
Activity.
The addition of above-mentioned active peptides is the 0.6% of algae powder, and the amino acid sequence of active peptides is
HSHACASYSRCYCRCLRCRPGCVNCYRCSR, the active peptide has hydrophily and lipophilicity, and hydrophily makes it be dissolved in distillation
In water, lipophilicity makes itself and spirulina cells film combination, makes to form aperture under cell membrane, causes intracellular content leaks, carry
The operating efficiency that high crude protein is extracted, also improves the yield of activated protein.
Above-mentioned chromatographic column specification is diameter 2.5cm, and long 50cm, buffer solution is that 6.7, concentration is 0.008mol/L's from pH
The KCl buffer solutions of phosphate buffer and 0.1mol/L, flow velocity is 0.8ml/min.
Embodiment 2：
The preparation method of blunt top spirulina activated protein, is concretely comprised the following steps：
1）It is 1 by solid-liquid ratio:2.5 add distilled water in algae powder, and suction filtration, deionized water is washed 3 times, and it is that 7, concentration is to add pH
The w/v of 0.012mol/L phosphate buffer and active peptides, algae powder and phosphate buffer is 1:14, stirring
110min, in -22 DEG C of snap frozens, then thaws at 5 DEG C, multigelation 4 times, and high speed refrigerated centrifuge, supernatant is thick
Protein liquid, activated protein belongs to intracellular protein, and first having to the cell membrane of smudge cells, cell membrane makes its dissolution, clasmatosis
Degree is higher, and the yield of activated protein is also higher, and formation and cell liquid salinity of the multigelation method using intracellular ice pellets increase
Height, which causes to be swelled, makes clasmatosis, simple to operate, it is not necessary to special instrument and equipment, and the step uses protein peptides and multigelation
Method is combined, and improves the operating efficiency of crude protein extraction and the yield of activated protein；
2）The chitosan solution that concentration is 2.2wt% is added into crude protein liquid, the w/v of algae powder and chitosan solution is
1:6, pH to 7 is adjusted, 20min is stirred, supernatant is put into ice-water bath by high speed refrigerated centrifuge, adding ammonium sulfate makes its concentration
For 55wt%, stirring is completely dissolved to ammonium sulfate, stands overnight rear low-temperature centrifugation, produce salt precipitation thing, is added in the step big
The ammonium sulfate of amount, protein is obtained solubility reduces and Precipitation, and this method advantage is that ammonium sulfate concentrations change has and connected
Continuous property, effect of saltouing is good, saves energy consumption；
3）Salt precipitation thing is dissolved in phosphate buffer, bag filter is then charged into, bubble is excluded, sealing is placed in phosphate
Dialysed in buffer solution, change buffer solution 5 times, solution is dialyzate in bag, and this method in the solution may be used using small-molecule substance
By bag filter, and macromolecular substances can not reach the purpose of separation small molecular weight impurity, operation letter by the property of bag filter
Single, effect is good, saves energy consumption；
4）The hydroxyapatite that 11h is soaked with buffer solution is fitted into the pillar containing pre-add buffer solution sum, dialyzate is added, it is complete
It is complete to enter after cylinder, buffer solution elution is added, freeze-drying produces blunt top spirulina activated protein, the activated protein color is fresh
It is gorgeous, it is nutritious, lymphocyte activity can be improved, body's immunity is improved by lymphatic system, strengthens the diseases prevention of body, resist
Sick ability, moreover it is possible to remove the free radical in organism and promote animal cell substitution, and with significant antitumor activity.
The addition of above-mentioned active peptides is the 0.6% of algae powder, and the amino acid sequence of active peptides is
HSHACASYSRCYCRCLRCRPGCVNCYRCSR, the active peptide has hydrophily and lipophilicity, and hydrophily makes it be dissolved in distillation
In water, lipophilicity makes itself and spirulina cells film combination, makes to form aperture under cell membrane, causes intracellular content leaks, carry
The operating efficiency that high crude protein is extracted, also improves the yield of activated protein.
Above-mentioned chromatographic column specification is diameter 3cm, long 52cm, and buffer solution is from the phosphoric acid that pH is that 7, concentration is 0.012mol/L
The KCl buffer solutions of salt buffer and 0.1mol/L, flow velocity is 0.6ml/min.
Embodiment 3：
The preparation method of blunt top spirulina activated protein is concretely comprised the following steps：It is 1 by solid-liquid ratio:2.8 add distillation in algae powder
Water, suction filtration, deionized water is washed 2 ~ 3 times, and addition pH is that the phosphate buffer that 6.5, concentration is 0.01mol/L and activity are more
Peptide, the wherein w/v of algae powder and phosphate buffer are 1:14 ~ 16, the addition of active peptides is the 0.5% of algae powder,
130min is stirred, in -20 DEG C of snap frozens, is then thawed at 3 DEG C, multigelation 3 times, centrifugation；Added into supernatant dense
Spend the chitosan solution for 1.8wt%, the w/v of algae powder and chitosan solution is 1:4, adjust pH to 6.5, stirring
Centrifuged after 25min, supernatant is put into ice-water bath, adding ammonium sulfate makes its concentration be 53wt%, stirs completely molten to ammonium sulfate
Solution, is centrifuged after standing overnight；Sediment is dissolved in phosphate buffer, bag filter is then charged into, phosphate buffer is placed in
Middle dialysis, changes buffer solution 6 times；Dialyzate is prepared into activated protein sterling, chromatographic column using hydroxylapatite adsorption chromatography
Specification is diameter 3cm, long 52cm, buffer solution from pH be phosphate buffer that 6.5, concentration is 0.01mol/L and
0.12mol/L KCl buffer solutions, flow velocity is 0.7ml/min, freeze-drying, produces blunt top spirulina 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>Leqing Rui Yao food technologies Co., Ltd
<120>The preparation method of blunt top spirulina activated protein
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> PRT
<213>It is artificial synthesized
<400> 1
His Ser His Ala Cys Ala Ser Tyr Ser Arg Cys Tyr Cys Arg Cys Leu
1 5 10 15
Arg Cys Arg Pro Gly Cys Val Asn Cys Tyr Arg Cys Ser Arg
20 25 30
Claims (10)
1. the preparation method of blunt top spirulina activated protein, including crude protein extraction, saltout, dialyse, purifying, its feature exists
In：The extraction step of the crude protein is：Distilled water is added in algae powder, suction filtration, washing adds phosphate buffer and activity
Polypeptide, stirs, multigelation, centrifugation, and supernatant is crude protein liquid.
2. the preparation method of blunt top spirulina activated protein according to claim 1, it is characterised in that：The crude protein
The solid-liquid ratio of algae powder and distilled water is 1 in extraction step:2~3.
3. the preparation method of blunt top spirulina activated protein according to claim 1, it is characterised in that：The crude protein
The pH of phosphate buffer is that 6.5 ~ 7, concentration is 0.008 ~ 0.012mol/L in extraction step, algae powder and phosphate buffer
W/v is 1:14~16.
4. the preparation method of blunt top spirulina activated protein according to claim 1, it is characterised in that：The crude protein
The addition of active peptides is the 0.5 ~ 0.8% of algae powder in extraction step, and the amino acid sequence of active peptides is
HSHACASYSRCYCRCLRCRPGCVNCYRCSR。
5. the preparation method of blunt top spirulina activated protein according to claim 1, it is characterised in that：The crude protein
The temperature of snap frozen is -22 ~ -20 DEG C in extraction step, and thaw point is 3 ~ 5 DEG C, multigelation 3 ~ 4 times.
6. the preparation method of blunt top spirulina activated protein according to claim 1, it is characterised in that：The crude protein
The temperature centrifuged in extraction step is 3 ~ 5 DEG C, and speed is 10000 ~ 12000rpm, and the time is 28 ~ 33min.
7. the preparation method of blunt top spirulina activated protein according to claim 1, it is characterised in that：The salting-out step
For：Chitosan solution is added, pH is adjusted, stirred, supernatant is put into ice-water bath by centrifugation, adding ammonium sulfate makes its concentration be
50 ~ 55wt%, stirring to ammonium sulfate is completely dissolved, and is centrifuged after standing overnight, is produced salt precipitation thing.
8. the preparation method of blunt top spirulina activated protein according to claim 7, it is characterised in that：The salting-out step
The concentration of middle chitosan solution is 1.8 ~ 2.2wt%, and the w/v of algae powder and chitosan solution is 1:4~6.
9. the preparation method of blunt top spirulina activated protein according to claim 1, it is characterised in that：The purification step
Middle hydroxyapatite soaks 10 ~ 12h with buffer solution.
10. the preparation method of blunt top spirulina activated protein according to claim 1, it is characterised in that：The purifying step
Chromatographic column specification is 2 ~ 3cm of diameter in rapid, long 48 ~ 52cm, and buffer solution is that 6.5 ~ 7, concentration is 0.008 ~ 0.012mol/ from pH
L phosphate buffer and 0.1 ~ 0.12mol/L KCl buffer solutions, flow velocity are 0.6 ~ 0.8ml/min.
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| CN108752445A (en) * | 2018-06-20 | 2018-11-06 | 广东药科大学 | A kind of preparation process and its neuroprotection purposes of spirulina protein |
| CN111004415A (en) * | 2019-12-16 | 2020-04-14 | 晋中学院 | Spirulina platensis algae protein-chitosan composite membrane, preparation method and application |
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| CN111004415B (en) * | 2019-12-16 | 2022-03-04 | 晋中学院 | Spirulina platensis algae protein-chitosan composite membrane, preparation method and application |
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