CN105820238A - Method for separating phycocyanin through aqueous two-phase extraction of methoxyl group polyethylene glycol/potassium sodium tartrate - Google Patents
Method for separating phycocyanin through aqueous two-phase extraction of methoxyl group polyethylene glycol/potassium sodium tartrate Download PDFInfo
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Abstract
The invention discloses a method for separating phycocyanin through aqueous two-phase extraction of methoxyl group polyethylene glycol/potassium sodium tartrate. The method comprises the following steps: (1) preparing a phycocyanin crude extract: cleaning fresh spirulina for 2-4 times with distilled water, crushing the cleaned spirulina for 5-7min through a high-pressure cell homogenating machine of which the pressure is 200-400kg/cm<2>, performing ultrasonic treatment for 12-15 min, performing centrifugation in crushed spirulina suspending liquid for 10min, collecting a supernatant, preparing the crude extract containing phycocyanin, and storing the prepared crude extract containing the phycocyanin under 4-5 DEG C; (2) performing aqueous two-phase extraction: adding methoxyl group polyethylene glycol and potassium sodium tartrate to the crude extract containing the phycocyanin, obtained in the step (1), performing uniform velocity stirring for 1-2h, performing standing for 24h, so that the two phases are separated, and collecting a top phase; (3) preparing finished products: exchanging a chromatography column for the phycocyanin fluid obtained in the step (2) through DE-AE-Sephadex, performing linear eluting with sodium chloride of 0-0.35mol/L, collecting an eluent in which the sodium chloride concentration is 0.25-0.3mol/L, and performing dialysis freeze-drying so as to prepare the phycocyanin powder. The method disclosed by the invention is good in separation effect, high in filtering separation recovery rate and product quality, and low in operating cost.
Description
Technical field
This area relates to natural product deep process technology field, especially relates to a kind of methoxy poly (ethylene glycol)/wine
The method of stone acid potassium sodium Bi-aqueous extraction phycocyanin.
Background technology
Spirulina (Spirulina) is the many cells thread microalgae (M of a kind of helical form, not branch
Icroalga), it is Cyanophyta (Cyanophyta), hormogon mesh (Hormogonales), quivers
Algae section (Oscilatoriaceae), the prokaryote such as ancient low of Spirullina (Spirulina).The most
Knowing that spirulina has kind more than 30, most have Development volue is spirulina plalensis, spirulina maxim and salt pool spiral
Algae, wherein the nutritive value of spirulina plalensis is the highest.Spirulina plalensis has that protein content is high, breeding is fast
Feature, in algae powder, phycocyanin (Phycocyanin, PC) content is up to 20%, is a kind of content of phycocyanin
Higher algae species.Phycocyanin has anticancer, antioxidation, treatment cerebral ischemia, carries body height and exempt from
The different physiological roles such as epidemic disease power.The purification technique of phycocyanin also becomes one of Important Problems of people's research.
At present, the separating and purifying technology that phycocyanin is conventional is saltoutd, aqueous two-phase extraction, reverse micelle extraction, post layer
Analysis etc. are several.The isolated and purified most employing precipitation of Spirulina phycocyanin reported, centrifugal, dialysis and color
, in actual production process, there are complex steps, energy consumption height, low, the operation cycle length of productivity etc. in the techniques such as spectrum
Shortcoming.
Methoxy poly (ethylene glycol) have good water solublity, wettability, lubricity, physiological inertia, to human body
Non-stimulated, gentle, it is widely used in cosmetics and pharmaceuticals industry.Different molecular quality fraction can be chosen
Product changes the viscosity of goods, hygroscopicity and organizational structure.(molecular weight is little for the low product of relative molecular weight
In 2000) be suitable to make wetting agent and consistency modifiers, for cream, emulsion, toothpaste with pick must cream etc..Phase
The product that molecular weight is high is applicable to lip pomade, deodorant stick, fancy soap, picks palpus soap, foundation cream and cosmetics etc..
In abluent, also it is used as suspending agent and thickening agent.In pharmaceuticals industry, as ointment, Emulsion, ointment,
Lotion and the substrate of suppository.Also have and this product and acrylic acid etc. built reaction containing polymerizable is double, synthesizing ester with
Increase its reactivity, be the principal monomer class raw material preparing polycarboxylate high range water reducing agent for cement.Molecule
The hydroxyl activity of chain end is farthest retained, and has beautiful and charming hydrophilic and hydroxyl reaction activity.
At present, lack a kind of high methoxy poly (ethylene glycol) of separation and recovery rate/sodium potassium tartrate tetrahydrate aqueous two-phase that filters to extract
Take the method separating phycocyanin.
Summary of the invention
For solving the problems referred to above, it is an object of the invention to provide a kind of high methoxyl group of separation and recovery rate that filters and gather
The method of ethylene glycol/sodium potassium tartrate tetrahydrate Bi-aqueous extraction phycocyanin.
For realizing above-mentioned technical purpose, the technical solution used in the present invention is as follows: a kind of methoxyl group of the present invention
The method of Polyethylene Glycol/sodium potassium tartrate tetrahydrate Bi-aqueous extraction phycocyanin, comprises the steps:
(1) preparing phycocyanin crude extract: clean fresh spirulina 2～4 times with distilled water, washing is dry
Clean spirulina is 200～400kg/cm through pressure2High pressure cell refiner crush 5～7min, ultrasonic Treatment
12～15min, by broken spirulina suspension in centrifugal 10min, collect supernatant, prepare the indigo plant egg in algae
White crude extract stores at 4～5 DEG C；
(2) aqueous two-phase extraction: contain addition methoxyl group in the crude extract of phycocyanin to step (1) gained and gather
Ethylene glycol and sodium potassium tartrate tetrahydrate, at the uniform velocity stirring 1～2h, stand 24h and make biphase separating, phase in collection；
(3) prepared by finished product: step (2) gained phycocyanin liquid is crossed DE-AE-Sephadex anion and hands over
Changing chromatographic column, use the sodium chloride of 0～0.35mol/L to carry out linear elution, collecting sodium chloride concentration is
Eluent when 0.25～0.3mol/L, dialysis lyophilizing prepares phycocyanin powder.
Further, in step (1), the centrifugal equipment of described broken spirulina suspension is centrifugal
Machine, centrifugation rate is 8000～10000r/min.
Further, in step (2), described methoxy poly (ethylene glycol) quality is the total matter of double-aqueous phase system
The 12～20% of amount.
Further, in step (2), described sodium potassium tartrate tetrahydrate quality is double-aqueous phase system gross mass
17～23%.
Further, in step (2), the quality of the described crude extract containing phycocyanin is aqueous two-phase body
It is the 65～71% of gross mass.
Beneficial effect: good separating effect of the present invention, filters separation and recovery rate high, and product quality is high, runs into
This is low；Process, without adding chemical drugs, solvent solvent, does not bring secondary pollution matter into；Equipment can be automatic
Run, good stability.
Compared with prior art, present invention have the advantage that
(1) aqueous two-phase extraction of the present invention is compared with other separation methods, have easily amplification, operating time short,
Become phase material to bioactive substance to be separated without advantages such as toxic side effects.In double-aqueous phase system, treat point
From compositional selecting be allocated in a wherein phase, and other impurity compositions are allocated in another phase, therefore make to treat
Concentration and the purity of separation component are all improved.The factor wherein affecting aqueous two-phase extraction is a lot, mainly has:
Type of polymer, polymer average molecular mass and concentration, become phase Inorganic Salts and concentration, ionic strength,
PH value etc..
(2) double-aqueous phase system of the present invention is used for extracting spirulina cells and crushes phycocyanin in liquid, through once
Extraction phycocyanin yield is up to 94.11%, and partition coefficient reaches 8.03, and separation factor reaches 6.5.
Result confirms that Spirulina phycocyanin partition coefficient in this system and separation factor are the highest.
Accompanying drawing explanation
Fig. 1 is the ultraviolet-ray visible absorbing figure of breaking cellular wall rear screw algae solution；
Fig. 2 is that sodium potassium tartrate tetrahydrate saturation affects figure to phycocyanin purity；
Fig. 3 is that pH value affects figure to what double-aqueous phase system and phycocyanin extracted.
Detailed description of the invention
The present invention is further illustrated by the following examples with Figure of description.It should be understood that these are real
Execute explaination and citing that example is the present invention, limit the scope of the present invention the most in any form.
Embodiment 1
A kind of side of the methoxy poly (ethylene glycol) of the present invention/sodium potassium tartrate tetrahydrate Bi-aqueous extraction phycocyanin
Method, comprises the steps:
(1) phycocyanin crude extract is prepared: clean fresh spirulina 2 times with distilled water, washes clean
Spirulina through pressure be 200kg/cm2High pressure cell refiner crush 5min, ultrasonic Treatment 12min,
By broken spirulina suspension in centrifugal 10min, collect supernatant, prepare the crude extract containing phycocyanin
Store at 4.5 DEG C；The centrifugal equipment of described broken spirulina suspension is centrifuge, and centrifugation rate is
9000r/min。
(2) aqueous two-phase extraction: contain addition methoxyl group in the crude extract of phycocyanin to step (1) gained and gather
Ethylene glycol and sodium potassium tartrate tetrahydrate, at the uniform velocity stir 1h, stands 24h and makes biphase separating, phase in collection；Described first
Epoxide Polyethylene Glycol quality is the 15% of double-aqueous phase system gross mass.Described sodium potassium tartrate tetrahydrate quality is aqueous two-phase body
It is the 19% of gross mass.Quality is double-aqueous phase system gross mass the 71% of the described crude extract containing phycocyanin.
(3) prepared by finished product: step (2) gained phycocyanin liquid is crossed DE-AE-Sephadex anion and hands over
Changing chromatographic column, use the sodium chloride of 0～0.35mol/L to carry out linear elution, collecting sodium chloride concentration is
Eluent when 0.25～0.3mol/L, dialysis lyophilizing prepares phycocyanin powder.
As it is shown in figure 1, the ultraviolet-ray visible absorbing figure that Fig. 1 is breaking cellular wall rear screw algae solution, phycocyanin solution
Having maximum absorption wavelength at 280nm and 620nm, spirulina protein has maximum absorption band at 280nm,
Phycocyanin has maximum absorption band at 620nm, and in freeze thawing breaking cellular wall rear screw algae solution, phycocyanin purity is
0.4, wherein spirulina foreign protein content is more.
Embodiment 2
A kind of side of the methoxy poly (ethylene glycol) of the present invention/sodium potassium tartrate tetrahydrate Bi-aqueous extraction phycocyanin
Method, comprises the steps:
(1) phycocyanin crude extract is prepared: clean fresh spirulina 3 times with distilled water, washes clean
Spirulina through pressure be 400kg/cm2High pressure cell refiner crush 7min, ultrasonic Treatment 13min,
By broken spirulina suspension in centrifugal 10min, collect supernatant, prepare the crude extract containing phycocyanin
Store at 5 DEG C；The centrifugal equipment of described broken spirulina suspension is centrifuge, and centrifugation rate is
8000r/min。
(2) aqueous two-phase extraction: contain addition methoxyl group in the crude extract of phycocyanin to step (1) gained and gather
Ethylene glycol and sodium potassium tartrate tetrahydrate, at the uniform velocity stir 2h, stands 24h and makes biphase separating, phase in collection；Described first
Epoxide Polyethylene Glycol quality is the 18% of double-aqueous phase system gross mass.Described sodium potassium tartrate tetrahydrate quality is aqueous two-phase body
It is the 23% of gross mass.Quality is double-aqueous phase system gross mass the 67% of the described crude extract containing phycocyanin.
(3) prepared by finished product: step (2) gained phycocyanin liquid is crossed DE-AE-Sephadex anion and hands over
Changing chromatographic column, use the sodium chloride of 0～0.35mol/L to carry out linear elution, collecting sodium chloride concentration is
Eluent when 0.25～0.3mol/L, dialysis lyophilizing prepares phycocyanin powder.
Embodiment 3
A kind of side of the methoxy poly (ethylene glycol) of the present invention/sodium potassium tartrate tetrahydrate Bi-aqueous extraction phycocyanin
Method, comprises the steps:
(1) phycocyanin crude extract is prepared: clean fresh spirulina 4 times with distilled water, washes clean
Spirulina through pressure be 300kg/cm2High pressure cell refiner crush 6min, ultrasonic Treatment 15min,
By broken spirulina suspension in centrifugal 10min, collect supernatant, prepare the crude extract containing phycocyanin
Store at 4 DEG C；The centrifugal equipment of described broken spirulina suspension is centrifuge, and centrifugation rate is
10000r/min。
(2) aqueous two-phase extraction: contain addition methoxyl group in the crude extract of phycocyanin to step (1) gained and gather
Ethylene glycol and sodium potassium tartrate tetrahydrate, at the uniform velocity stir 1.6h, stands 24h and makes biphase separating, phase in collection；Described
Methoxy poly (ethylene glycol) quality is the 17% of double-aqueous phase system gross mass.Described sodium potassium tartrate tetrahydrate quality is aqueous two-phase
The 20% of system gross mass.Quality is double-aqueous phase system gross mass the 65% of the described crude extract containing phycocyanin.
Test 1
The impact that aqueous two-phase is formed by the mass fraction of sodium potassium tartrate tetrahydrate
The impact that aqueous two-phase is formed by the mass fraction of sodium potassium tartrate tetrahydrate is as shown in table 1,
Table 1
| Soluble tartar. na concn (%, w/w) | Split-phase situation |
| 15 | Not split-phase |
| 17 | Split-phase is complete |
| 19 | Split-phase is complete |
| 21 | Split-phase is complete |
| 23 | Split-phase is complete |
| 25 | Split-phase is complete |
| 27 | Sodium potassium tartrate tetrahydrate is not completely dissolved |
As shown in Table 1, when mass fraction one timing of methoxy poly (ethylene glycol), along with sodium potassium tartrate tetrahydrate is dense
The increase of degree, compares and is gradually lowered, and the partition coefficient of phycocyanin first raises and reduces afterwards, phycocyanin pure
Degree should present first to raise and reduces afterwards, and in upper phase, the extraction of phycocyanin reduces after taking the lead in raising.Work as methoxyl group
Mass fraction one timing of Polyethylene Glycol, when Soluble tartar. na concn is 21%, phycocyanin purity in upper phase,
Extraction rate reached is to maximum.Along with the raising within the specific limits of Soluble tartar. na concn, double-aqueous phase system by
Gradually away from critical point, material increases at biphase median surface tension force, and beneficially phycocyanin is enriched with in upper phase, many
Sugar is enriched with in lower phase；When Soluble tartar. na concn is bigger, upper phase volume is gradually reduced, and viscosity is gradually simultaneously
Increase, be unfavorable for the enrichment in upper phase of phycocyanin.
As in figure 2 it is shown, the 10%～30% phycocyanin purity that obtains of sodium potassium tartrate tetrahydrate analysis is relatively low, 30%～
45% sodium potassium tartrate tetrahydrate separates out phycocyanin, and phycocyanin purity is gradually increased, more than 45% saturation wine
The analysis phycocyanin purity change of stone acid k-na salt is little.Determine: the sodium potassium tartrate tetrahydrate between 10%～30% is satisfied
Can be used to remove a large amount of spirulina foreign protein with degree, be used for precipitating more than the sodium potassium tartrate tetrahydrate of 40% saturation
Phycocyanin and the enriching and purifying of phycocyanin.
Test 2
The impact that phycocyanin is extracted by methoxy poly (ethylene glycol) mass fraction
The impact that phycocyanin is extracted by methoxy poly (ethylene glycol) mass fraction is as shown in table 2,
Table 2
From table 2 it can be seen that the change of methoxy poly (ethylene glycol) mass fraction is to phycocyanin extraction yield and pure
Degree all has an impact.When the mass fraction of methoxy poly (ethylene glycol) is 15%, phycocyanin extraction yield reaches
94.11%, purity has reached 0.76.
From test 2, in the double-aqueous phase system that methoxy poly (ethylene glycol) and sodium potassium tartrate tetrahydrate are formed, upper phase
For methoxy poly (ethylene glycol) phase, lower phase is Soluble tartar. sodium salt phase.When the concentration of methoxy poly (ethylene glycol) is more than
When it becomes the concentration of phase critical point, system is away from critical point, and anchor line (string) length increases, and biphase property qualitative difference is also
Increasing, protein interfacial tension difference in biphase increases simultaneously so that it is tend to distributing to side.In first
In epoxide Polyethylene Glycol-sodium potassium tartrate tetrahydrate double-aqueous phase system, increasing of Soluble tartar. na concn can make lower phase saltout
Effect is strengthened, the phycocyanin upwards phase transfer in lower phase, and is enriched in phase.When the algae being enriched to upper phase
When azurin concentration exceedes its dissolubility, phycocyanin will be precipitated and be distributed in up and down mutually between, cause
Extraction yield declines.Meanwhile, when methoxy poly (ethylene glycol) relative molecular mass is bigger, due to the suction that it is extremely strong
Outlet capacity, can cause lower phase Soluble tartar. na concn to increase, and upper phase free water content reduces, and will also result in portion
Protein is divided to separate out between up and down mutually.When the relative molecular mass of methoxy poly (ethylene glycol) be 4000u or
During 6000u, lower phase is not detected by phycocyanin, but up and down mutually between have phycocyanin to separate out.Select
Suitable methoxy poly (ethylene glycol) relative molecular mass and Soluble tartar. na concn are for improving the extraction of phycocyanin
Take rate extremely important.
Test 3
The impact that double-aqueous phase system and phycocyanin are extracted by ionic strength
Double-aqueous phase system is the most alternate exists potential difference, by add variety classes and concentration salt so that
State of charge and the hydrophobic interaction of system change, and then material distribution in biphase is produced notable shadow
Ring.Configuration methoxy poly (ethylene glycol) mass fraction is 15%, and the mass fraction of sodium potassium tartrate tetrahydrate is the system of 17%,
And the impact in the case of investigating the NaCl adding variable concentrations respectively, phycocyanin extracted.Ionic strength is to double
The impact of aqueous phase system and phycocyanin extraction is as shown in table 3,
Table 3
As shown in Table 3, along with the rising of NaCl concentration, in upper phase, phycocyanin purity the most first rises with extraction ratio
Reduce after height.When the mass fraction of methoxy poly (ethylene glycol) is 15%, and the mass fraction of sodium potassium tartrate tetrahydrate is 17%
Time, compared with the system being not added with NaCl, in the case of adding the NaCl that mass fraction is 1.0%, in upper phase
Phycocyanin purity is the highest with extraction ratio.
Test 4
The impact that double-aqueous phase system and phycocyanin are extracted by pH value
The change of double-aqueous phase system pH value can change biphase some position, and affects protein belt point character, thus shadow
Ring phycocyanin distribution in biphase.The mass fraction of preparation sodium potassium tartrate tetrahydrate is 18%, the poly-second of methoxyl group two
The mass fraction of alcohol is the double-aqueous phase system of 12%, and system pH is 5.8～6.3.Owing to phycocyanin is more stable
PH scope be 4.5～8.5, therefore regulation system pH is respectively 4.0,5.0,6.0,7.0 and 8.0.
Impact that double-aqueous phase system and phycocyanin are extracted by pH value is as it is shown on figure 3, along with the liter of double-aqueous phase system pH
Height, in upper phase, the purity of phycocyanin and extraction ratio are all in reducing trend.And the original pH of system is about 6.0.
The ultimate principle of the present invention, principal character and advantages of the present invention have more than been shown and described.The industry
Skilled person will appreciate that, the present invention is not restricted to the described embodiments, in above-described embodiment and description
The principle that the present invention is simply described described, without departing from the spirit and scope of the present invention, the present invention
Also have various changes and modifications, claimed scope by appending claims, description and
Its equivalent defines.
Claims (5)
1. a method for methoxy poly (ethylene glycol)/sodium potassium tartrate tetrahydrate Bi-aqueous extraction phycocyanin, it is special
Levy and be to comprise the steps:
(1) preparing phycocyanin crude extract: clean fresh spirulina 2～4 times with distilled water, washing is dry
Clean spirulina is 200～400kg/cm through pressure2High pressure cell refiner crush 5～7min, ultrasonic Treatment
12～15min, by broken spirulina suspension in centrifugal 10min, collect supernatant, prepare the indigo plant egg in algae
White crude extract stores at 4～5 DEG C；
(2) aqueous two-phase extraction: contain addition methoxyl group in the crude extract of phycocyanin to step (1) gained and gather
Ethylene glycol and sodium potassium tartrate tetrahydrate, at the uniform velocity stirring 1～2h, stand 24h and make biphase separating, phase in collection；
(3) prepared by finished product: step (2) gained phycocyanin liquid is crossed DE-AE-Sephadex anion and hands over
Changing chromatographic column, use the sodium chloride of 0～0.35mol/L to carry out linear elution, collecting sodium chloride concentration is
Eluent when 0.25～0.3mol/L, dialysis lyophilizing prepares phycocyanin powder.
Methoxy poly (ethylene glycol) the most according to claim 1/sodium potassium tartrate tetrahydrate Bi-aqueous extraction algae is blue
The method of albumen, it is characterised in that: in step (1), described broken spirulina suspension centrifugal
Equipment is centrifuge, and centrifugation rate is 8000～10000r/min.
Methoxy poly (ethylene glycol) the most according to claim 1/sodium potassium tartrate tetrahydrate Bi-aqueous extraction algae is blue
The method of albumen, it is characterised in that: in step (2), described methoxy poly (ethylene glycol) quality is aqueous two-phase
The 12～20% of system gross mass.
4. according to the methoxy poly (ethylene glycol) described in claim 1 or 3/sodium potassium tartrate tetrahydrate Bi-aqueous extraction
The method of phycocyanin, it is characterised in that: in step (2), described sodium potassium tartrate tetrahydrate quality is aqueous two-phase
The 17～23% of system gross mass.
5. according to the methoxy poly (ethylene glycol)/sodium potassium tartrate tetrahydrate aqueous two-phase extraction described in claim 1 or 3 or 4
The method separating phycocyanin, it is characterised in that: in step (2), the described crude extract containing phycocyanin
Quality is double-aqueous phase system gross mass 65～71%.
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| CN107216367A (en) * | 2017-06-22 | 2017-09-29 | 舟山富晟食品科技有限公司 | The preparation method of Closterium activated protein |
| CN109206504A (en) * | 2018-10-17 | 2019-01-15 | 云南师范大学 | A kind of isolation and purification method of high purity phycocyanin |
| 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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