CN107325175A - A kind of method of methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin - Google Patents
A kind of method of methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin Download PDFInfo
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- CN107325175A CN107325175A CN201710080079.7A CN201710080079A CN107325175A CN 107325175 A CN107325175 A CN 107325175A CN 201710080079 A CN201710080079 A CN 201710080079A CN 107325175 A CN107325175 A CN 107325175A
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- phycocyanin
- methylcellulose
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- 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
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Abstract
The invention discloses a kind of method of methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin, comprise the following steps：(1) phycocyanin crude extract is prepared：Fresh spirulina is cleaned with deionized water 24 times, be 210 420kg/cm through pressure2High pressure cell refiner, crush the time be 5 6min, ultrasonic treatment time be 12 14min；(2) aqueous two-phase extraction：Methylcellulose and trisodium citrate are added into the crude extract containing phycocyanin obtained by step (1), concussion incorporation time is 1 2h, stands split-phase；(3) membrane aperture is used to carry out coarse filtration to phycocyanin crude extract for 3 5um filter membrane, processing time is 20 26 hours, is freeze-dried 10 12 hours then at 10 12 DEG C, aqueous two-phase extraction phycocyanin is made.Good separating effect of the present invention, is separated by filtration rate of recovery height, and product quality is high, and operating cost is low.
Description
Technical field
This area is related to natural products deep process technology field, more particularly, to a kind of methylcellulose and trisodium citrate
The method of Bi-aqueous extraction phycocyanin.
Background technology
Phycocyanin (also known as algocyan, abbreviation PC) is a kind of dark blue powder separated from spirulina, color and luster
It is attractive in appearance.It is the rare Photosynthesis Pigment albumen of a class, and amino acid composition is complete, is distinctive in algae to catch photopigment egg
In vain.PC has radioresistance, anti-oxidant, the antitumor and advantage such as have no toxic side effect, in health care of food, drug therapy, cosmetics
And had a wide range of applications in terms of fluorescence probe.
Phycocyanin is a kind of natural blue pigment, and as food pigment addition in soft drink, aqueous two-phase extraction is raw
Thing material isolates and purifies that middle one kind is effective and valuable method, and people have been obtained in recent years and have widely been paid close attention to, have been applied to
In the purge process of protein, enzyme, natural materials etc., conventional is PEG/ inorganic salt systems.Its content of water in system it is high, gentle and
It is nontoxic, it can effectively ensure the activity of biomolecule.With other separation method (such as ion-exchange chromatographies, gel permeation chromatography
Deng) compare, aqueous two-phase extraction has the advantages such as disengaging time is short, extraction efficiency is high, pollution is few.
At present, a kind of methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin of good separating effect are lacked
Method.
The content of the invention
To solve the above problems, it is an object of the invention to provide a kind of good separating effect, being separated by filtration the high first of the rate of recovery
The method of base cellulose and trisodium citrate Bi-aqueous extraction phycocyanin.
To realize above-mentioned technical purpose, the technical solution adopted by the present invention is as follows：The present invention a kind of methylcellulose and
The method of trisodium citrate Bi-aqueous extraction phycocyanin, comprises the following steps：
(1) phycocyanin crude extract is prepared：Fresh spirulina is cleaned with deionized water 2-4 times, the spiral shell of washes clean
Algae is revolved, is 210-420kg/cm through pressure2High pressure cell refiner, it is 5-6min to crush the time, and ultrasonic treatment time is
12-14min, by broken spirulina suspension in centrifuge, centrifugation time is 10-18min, and supernatant is collected by centrifugation, system
The crude extract of phycocyanin must be contained in storage at 4-5 DEG C；
(2) aqueous two-phase extraction：Methylcellulose and citric acid are added into the crude extract containing phycocyanin obtained by step (1)
Trisodium, concussion incorporation time is 1-2h, stands split-phase, and phycocyanin is enriched in phase；
(3) membrane aperture is used to carry out coarse filtration to phycocyanin crude extract for 3-5um filter membrane, processing time is 20-26
Hour, it is freeze-dried 10-12 hours then at -10--12 DEG C, aqueous two-phase extraction phycocyanin is made.
Further, in step (1), the centrifugation rate is 800-1000r/min.
Further, in step (2), the quality of the methylcellulose is the 9-13% of double-aqueous phase system gross mass.
Further, in step (2), the trisodium citrate is the 16-25% of double-aqueous phase system gross mass.
Further, in step (2), the quality of the crude extract containing phycocyanin is the 70- of double-aqueous phase system gross mass
75%.
Further, in step (3), the flow velocity of the filter membrane is 150mL/min, and permeation flux is 60L/ (h
m2)。
Beneficial effect：Good separating effect of the present invention, system viscosity is relatively low, and short the time required to split-phase, the time of having saved subtracts again
Small energy consumption.It is separated by filtration the rate of recovery high, product quality is high, operating cost is low；It is molten that process need not add chemicals, solvent
Agent, does not bring secondary pollution matter into；Equipment can automatic running, stability is good, easily realizes industrialization demand.
Compared with prior art, the invention has the advantages that：
(1) double-aqueous phase system of the invention is used to extract phycocyanin in the broken liquid of spirulina cells, through single extraction algae
Azurin yield is up to 90.6%, and distribution coefficient reaches 8.03, and separation factor reaches 6.5.As a result spirulina phycocyanin is confirmed
Distribution coefficient and separation factor in the system is higher.
(2) repeatedly aqueous two-phase extraction is conducive to C-PC purity to improve, and after three double water extractions, C-PC purity is 3.92, is returned
Yield is 87.7%.Continue to increase the number of times of extraction, purity is continuously increased.
Embodiment
The present invention is further illustrated by the following examples.It should be understood that these embodiments are the explainations of the present invention
And citing, the scope of the present invention is not limited in any form.
Embodiment 1
A kind of methylcellulose of the present invention and the method for trisodium citrate Bi-aqueous extraction phycocyanin, including such as
Lower step：
(1) phycocyanin crude extract is prepared：Fresh spirulina is cleaned with deionized water 2 times, the spiral of washes clean
Algae, is 210kg/cm through pressure2High pressure cell homogenate crusher machine 5min, ultrasonic treatment time is 14min, spiral shell that will be broken
It is 15min to revolve algae suspension centrifugation time in centrifuge, and supernatant is collected by centrifugation, and the crude extract containing phycocyanin is made in 4
Stored at DEG C；The speed of agitator is 800r/min.
(2) aqueous two-phase extraction：Methylcellulose and citric acid are added into the crude extract containing phycocyanin obtained by step (1)
Trisodium, thoroughly stirs 1h, stands split-phase, and phycocyanin is enriched in phase；The quality of the methylcellulose is double-aqueous phase system
The 9% of gross mass.Trisodium citrate is the 16% of double-aqueous phase system gross mass.The quality of crude extract containing phycocyanin is double water
The 68% of phase system gross mass.
(3) membrane aperture is used to carry out coarse filtration to phycocyanin crude extract for 3um filter membrane, processing time is 20 hours,
Phycocyanin powder is obtained then at -12 DEG C of freeze-dryings within 10 hours.
Phycocyanin solution has maximum absorption wavelength at 280nm and 620nm, and spirulina protein has maximum at 280nm
Absworption peak, phycocyanin has phycocyanin purity in maximum absorption band, freeze thawing broken wall rear screw algae solution to be 0.4 at 620nm,
Wherein spirulina foreign protein content is more.
Embodiment 2
The difference of embodiment 2 and embodiment 1 is：A kind of methylcellulose and trisodium citrate the aqueous two-phase extraction of the present invention
The method for taking separation phycocyanin, comprises the following steps：
(1) phycocyanin crude extract is prepared：Fresh spirulina is cleaned with deionized water 3 times, the spiral of washes clean
Algae is 300kg/cm through pressure2High pressure cell homogenate crusher machine 5.5min, ultrasonic treatment time is 12min, will be broken
Spirulina suspension in centrifuge centrifugation time be 10min, supernatant is collected by centrifugation, be made the crude extract containing phycocyanin in
Stored at 5 DEG C；The speed of agitator is 900r/min.
(2) aqueous two-phase extraction：Methylcellulose and citric acid are added into the crude extract containing phycocyanin obtained by step (1)
Trisodium, thoroughly stirs 2h, stands split-phase, and phycocyanin is enriched in phase；The quality of the methylcellulose is double-aqueous phase system
The 10% of gross mass.Trisodium citrate is the 21% of double-aqueous phase system gross mass.The quality of crude extract containing phycocyanin is double
The 70% of aqueous phase system gross mass.
(3) membrane aperture is used to carry out coarse filtration to phycocyanin crude extract for 4um filter membrane, processing time is 24 hours,
Phycocyanin powder is obtained then at -11 DEG C of freeze-dryings within 11 hours.
Embodiment 3
The difference of embodiment 3 and embodiment 1 is：A kind of methylcellulose and trisodium citrate the aqueous two-phase extraction of the present invention
The method for taking separation phycocyanin, comprises the following steps：
(1) phycocyanin crude extract is prepared：Fresh spirulina is cleaned with deionized water 4 times, the spiral of washes clean
Algae is 420kg/cm through pressure2High pressure cell homogenate crusher machine 6min, ultrasonic treatment time is 14min, spiral shell that will be broken
Revolve algae suspension in centrifuge centrifugation time be 18min, supernatant is collected by centrifugation, be made the crude extract containing phycocyanin in
Stored at 4.5 DEG C；The speed of agitator is 1000r/min.
(2) aqueous two-phase extraction：Methylcellulose and citric acid are added into the crude extract containing phycocyanin obtained by step (1)
Trisodium, thoroughly stirs 1.5h, stands split-phase, and phycocyanin is enriched in phase；The quality of the methylcellulose is aqueous two-phase body
It is the 13% of gross mass.Trisodium citrate is the 25% of double-aqueous phase system gross mass.The quality of crude extract containing phycocyanin is
The 65% of double-aqueous phase system gross mass.
(3) membrane aperture is used to carry out coarse filtration to phycocyanin crude extract for 5um filter membrane, processing time is 26 hours,
Phycocyanin powder is obtained then at -10 DEG C of freeze-dryings within 12 hours.The flow velocity of the filter membrane is 150 mL/min, and permeation flux is
60L/(h·m2)。
Experiment 1
Trisodium citrate mass fraction, which is distributed C-PC, to be influenceed
Trisodium citrate mass fraction is as shown in table 1 on C-PC distribution influences,
Table 1
As shown in Table 1, when the timing of mass fraction one of methylcellulose, trisodium citrate mass fraction is 16%, pH value
For 6.2, under room temperature condition, double-aqueous phase system can not be formed when trisodium citrate is less than a certain consumption, with trisodium citrate
Mass fraction increase, is gradually reduced, distribution coefficient, purity and the rate of recovery gradually increase compared to value.When trisodium citrate quality point
When number is 21%, then C-PC distribution coefficient, purity and yield highest are gradually reduced.The reason for causing this phenomenon may
It is, the mass fraction increase of trisodium citrate that the salting out increase of lower phase makes C-PC mutually be migrated upwards by lower phase.But lemon
When the mass fraction of sour trisodium is too high, lower phase salting out is too strong, the hydrated sheath on destruction C-PC surfaces, C-PC by part or
All separate out.Furthermore, in the case where the mass fraction of methylcellulose is constant, as trisodium citrate mass fraction is certain
In the range of raising, double-aqueous phase system is gradually distance from critical point, and material increases in two-phase median surface tension force, is conducive to phycocyanin
In the enrichment of upper phase, polysaccharide is enriched with lower phase；When trisodium citrate mass fraction is bigger, upper phase volume is gradually reduced, upper phase body
Product is gradually reduced, while viscosity gradually increases, is unfavorable for the enrichment in upper phase of phycocyanin.C-PC transfer ability by
Limitation, is unfavorable for C-PC enrichment.
As shown in Table 1, the phycocyanin purity that the analysis of 16%-21% trisodium citrates is obtained is relatively low, 21%-25% citric acids
Trisodium separates out phycocyanin, and phycocyanin purity gradually increases, pure more than 25% saturation degree trisodium citrate phycocyanin of saltouing
Degree change is little.It is determined that：Trisodium citrate saturation degree between 16%-25% can for removing a large amount of spirulina foreign proteins,
Trisodium citrate more than 25% saturation degree is used for the enriching and purifying for precipitating phycocyanin and phycocyanin.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, the present invention
Claimed scope is by appended claims, specification and its equivalent thereof.
Claims (6)
1. a kind of method of methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin, it is characterised in that including such as
Lower step：
(1) phycocyanin crude extract is prepared：Fresh spirulina is cleaned with deionized water 2-4 times, the spirulina of washes clean,
It is 210-420kg/cm through pressure2High pressure cell refiner, crush the time be 5-6min, ultrasonic treatment time is 12-
14min, by broken spirulina suspension in centrifuge, centrifugation time is 10-18min, and supernatant is collected by centrifugation, and is made and contains
The crude extract of phycocyanin is in storage at 4-5 DEG C；
(2) aqueous two-phase extraction：Methylcellulose and citric acid three are added into the crude extract containing phycocyanin obtained by step (1)
Sodium, concussion incorporation time is 1-2h, stands split-phase, and phycocyanin is enriched in phase；
(3) membrane aperture is used to carry out coarse filtration to phycocyanin crude extract for 3-5um filter membrane, processing time is 20-26 hours,
It is freeze-dried 10-12 hours then at -10--12 DEG C, aqueous two-phase extraction phycocyanin is made.
2. the method for methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin according to claim 1, its
It is characterised by：In step (1), the centrifugation rate is 800-1000r/min.
3. the method for methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin according to claim 1, its
It is characterised by：In step (2), the quality of the methylcellulose is the 9-13% of double-aqueous phase system gross mass.
4. the method for methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin according to claim 1, its
It is characterised by：In step (2), the trisodium citrate is the 17-25% of double-aqueous phase system gross mass.
5. the method for methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin according to claim 1, its
It is characterised by：In step (2), the quality of the crude extract containing phycocyanin is the 65-70% of double-aqueous phase system gross mass.
6. the method for methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin according to claim 1, its
It is characterised by：In step (3), the flow velocity of the filter membrane is 150mL/min, and permeation flux is 60L/ (hm2)。
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| CN201710080079.7A CN107325175A (en) | 2017-02-15 | 2017-02-15 | A kind of method of methylcellulose and trisodium citrate Bi-aqueous extraction phycocyanin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024108780A1 (en) * | 2022-11-24 | 2024-05-30 | 盐池县怡健生物工程有限公司 | Method for extracting and purifying phycocyanin from spirulina platensis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009112149A8 (en) * | 2008-03-10 | 2010-09-10 | Bayer Technology Services Gmbh | Method for purifying therapeutic proteins by means of multi-stage extraction |
| CN105820237A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for two aqueous phase extraction separation of phycocyanin through dextran and sodium potassium tartrate |
| CN105820238A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for separating phycocyanin through aqueous two-phase extraction of methoxyl group polyethylene glycol/potassium sodium tartrate |
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2017
- 2017-02-15 CN CN201710080079.7A patent/CN107325175A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009112149A8 (en) * | 2008-03-10 | 2010-09-10 | Bayer Technology Services Gmbh | Method for purifying therapeutic proteins by means of multi-stage extraction |
| CN105820237A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for two aqueous phase extraction separation of phycocyanin through dextran and sodium potassium tartrate |
| CN105820238A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for separating phycocyanin through aqueous two-phase extraction of methoxyl group polyethylene glycol/potassium sodium tartrate |
Non-Patent Citations (3)
| Title |
|---|
| S. CHETHANA·CHETAN A. ET AL.: "Single step aqueous two-phase extraction for downstream processing of C-phycocyanin from Spirulina platensis", 《J FOOD SCI TECHNOL》 * |
| 付丽丽等: "双水相萃取纯化钝顶螺旋藻藻蓝蛋白技术探讨", 《食品科技》 * |
| 张莉等: "《分离检测实训》", 31 January 2013, 中国科学技术大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024108780A1 (en) * | 2022-11-24 | 2024-05-30 | 盐池县怡健生物工程有限公司 | Method for extracting and purifying phycocyanin from spirulina platensis |
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Application publication date: 20171107 |