CN104861082B - Method for separating polysaccharide and protein by using choline ionic liquid two-phase aqueous system - Google Patents
Method for separating polysaccharide and protein by using choline ionic liquid two-phase aqueous system Download PDFInfo
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Abstract
The invention relates to a method for separating polysaccharide and protein by using a choline ionic liquid two-phase aqueous system, belongs to the technical field of bioactive substance separation and purification, and particularly relates to application of the ionic liquid two-phase aqueous system composed of choline chloride [Ch]Cl and inorganic phosphate K3PO4 in selective separation of fungal polysaccharide and protein. The method is mainly characterized by comprising the following steps: after degreasing crushed fungi mycelium or sporocarp with petroleum ether, using hot water with the temperature of 95 DEG C for reflux extraction for 8 hours, centrifuging, concentrating, dialyzing, and freeze-drying, so as to obtain a fungus aqueous extract; carrying out extraction separation by using the ionic liquid two-phase aqueous system composed of choline chloride[Ch]C1 and inorganic phosphate K3P04; carrying out lower-layer inorganic salt phase dialysis, ethanol precipitation and freeze-drying so as to obtain fungal polysaccharide; carrying out extraction on an upper-layer ionic liquid phase by using dichloromethane, concentrating and recycling an ionic liquid. The ionic liquid two-phase aqueous system provided by the invention is suitable for industrial production of active polysaccharides, has the effects of being green, quick and efficient, the ionic liquid can be recycled for use, the production cost is lowered, and the ionic liquid two-phase aqueous system can be commonly used for the selective separation of various fungal polysaccharide and protein.
Description
Technical field
The present invention relates to a kind of utilize cholinium ion liquid double-aqueous phase system separating polyose and method of protein, refer in particular to ion
Liquid chlorine choline [ch] cl and inorganic phosphate k3po4The ionic liquid double-aqueous phase system of composition is used for fungus polysaccharide and protein
Selective Separation, belong to bioactive substance separating and purifying technology field.
Background technology
The isolation and purification method of fungus polysaccharide has a lot, such as ethanol precipitation, ion exchange chromatography, gel filtration chromatography
With membrane filtration etc., researcher must select correct isolation and purification method according to the property of polysaccharide, can obtain homogeneous work
Property composition.Wherein, ethanol precipitation is a kind of simple and effective method, but protein while extracting polysaccharide
Precipitate therewith, and protein content increases with concentration of alcohol and increases, this brings certain difficulty for follow-up polysaccharide purification.
Additionally, ion exchange chromatography and gel filtration chromatography are to apply a kind of most common method in separation of polysaccharides purge process, but pure
Change process has the shortcomings that time-consuming, costly, product recovery rate is low, solvent load is big and complex operation, thus limits true
The industrial applications of granulose and exploitation.
In recent years, as a kind of efficient and gentle novel green separation system, ionic liquid double-aqueous phase system combine from
Sub- liquid and the advantage of aqueous two-phase extraction, are widely used in protein, antibiotic, alkaloid, medicine, small molecule organic compound
The separation of thing and purification, and maintain activity and the conformation of original biological substance in separation process.Especially, domestic surgery
Skilled worker author passes through glyoxaline ion liquid such as l- butyl -3- methyl imidazolium tetrafluoroborate ([bmim] bf4) and inorganic salt group
The double-aqueous phase system becoming is used for the Selective Separation of polysaccharide and protein.However, studying progressively to ionic liquid with people
Deeply, increasing report finds that the imidazoles being widely used and pyridine ionic liquid have to organism in various degree
Murder by poisoning.Research finds, choline-like ionic liquid is a kind of ionic liquid of good biocompatibility, and wide material sources, cheaply easy
?.Therefore, the cholinium ion liquid with biocompatibility is utilized to form the separation that double-aqueous phase system is used for bioactive substance
Tool is of great significance.
Through retrieval, yet there are no using choline chloride [ch] cl and inorganic phosphate k3po4The ionic liquid aqueous two-phase constituting
System is used for polysaccharide and the detached report of protein selective.
Content of the invention
It is an object of the invention to provide the side of a kind of utilization cholinium ion liquid double-aqueous phase system separating polyose and protein
Method, to realize " green ", efficient, Selective Separation fungus polysaccharide and protein.
In order to solve above technical problem, the present invention utilizes cholinium ion liquid double-aqueous phase system for fungus polysaccharide and albumen
The Selective Separation of matter, in protein and inorganic salt phase in above layer ionic liquid phase, polysaccharide extract rate is index, optimizes lightning strip
Part.Concrete technical scheme is as follows:
A kind of using cholinium ion liquid double-aqueous phase system separating polyose and method of protein it is characterised in that include with
Lower step:
Step one, defat: be placed in Soxhlet extractor after fungal mycelium or sporophore are pulverized, add petroleum ether, boiling
30 ~ 60 DEG C of journey, 35 DEG C of backflow 6 h；So after defat twice, naturally dry to obtain fungal mycelium after defat or sporophore；
Step 2, prepares funguses water extract: the fungal mycelium after defat described in step one or sporophore are pressed 1:10
The solid-liquid ratio of g/ml adds in distilled water, 95 DEG C of reflux, extract, 8 h, then is centrifuged 10 min under the conditions of rotating speed is 5000 rpm,
Concentrate, lyophilization after distilled water dialysis 48 h obtains funguses water extract；
Step 3, using ionic liquid double-aqueous phase system Selective Separation fungus polysaccharide and protein: described in step 2
Funguses water extract be made into the funguses water extract aqueous solution that mass concentration is 10 mg/ml, take 1.0 ml funguses water extract aqueous solutions
It is added in double-aqueous phase system, then be placed in 20 DEG C of waters bath with thermostatic control and vibrate 30 min, centrifugation, separate and biphase up and down contains funguses
Lower floor's inorganic salt phase of polysaccharide and the upper strata ionic liquid phase containing impurity such as protein；
Described double-aqueous phase system is by ionic liquid choline chloride [ch] cl, inorganic phosphate k3po4Form with distilled water,
Described ionic liquid choline chloride [ch] cl, inorganic phosphate k3po4Mass ratio with distilled water is: 0.25-0.5: 0.7-
1.0 :1.0；
Step 4, prepares fungus polysaccharide: by the lower floor's inorganic salt phase distilled water containing fungus polysaccharide described in step 3
Dialyse 48 h, using the ethanol that concentration is 95%, fungus polysaccharide dialysis solution is precipitated；The volume of described ethanol is fungus polysaccharide
4 times of dialysis solution；4 DEG C stand overnight, then are centrifuged 10 min under conditions of rotating speed is 5000 rpm, cold after collection precipitate
Lyophilizing is dry, obtains fungus polysaccharide；
Step 5, reclaims ionic liquid: the upper strata ionic liquid phase containing impurity such as protein described in step 3 is used two
Chloromethanes extract 3 ~ 5 times, temperature≤45 DEG C when dichloromethane phase concentrates, and reclaim ionic liquid [ch] cl.
Described funguses are Phellinus igniarius (L. ex Fr.) Quel., Lentinus Edodess, Ganoderma, Grifola frondosa, Cordyceps, Flammulina velutiper (Fr.) Sing, any one in Pleurotus eryngii.
The present invention has beneficial effect.The present invention passes through will be by [ch] cl/k3po4The ionic liquid double-aqueous phase system of composition is used
In protein and inorganic salt phase in the Selective Separation of fungus polysaccharide and protein, above layer ionic liquid phase, polysaccharide extract rate is
Index, thus optimizing separation condition, reaches the purpose of " green ", efficient, Selective Separation phellin polysaccharides and protein.This
Bright cholinium ion liquid double-aqueous phase system is suitable for large-scale industrial production active polysaccharide, and ionic liquid can be recycled, fall
Low production cost, and funguses such as Phellinus igniarius (L. ex Fr.) Quel., Lentinus Edodess, Ganoderma, Grifola frondosa, Cordyceps, Flammulina velutiper (Fr.) Sing and Pleurotus eryngii can be widely used in
Isoreactivity polysaccharide and the Selective Separation of protein.
Specific embodiment
Below by embodiment, the present invention is described in further detail it should be pointed out that embodiment described below purport
Being easy to the understanding of the present invention, and any restriction effect is not played to it.In the present invention above layer ionic liquid phase, protein carries
The extraction ratio taking polysaccharide in rate and lower floor's inorganic salt phase carrys out overall merit [ch] cl/k3po4The ionic liquid double-aqueous phase system choosing of composition
The separation fungus polysaccharide of selecting property and the effect of protein.Extraction rate of protein is defined as upper strata ionic liquid phase protein quality and funguses
The ratio of protein quality in water extract, protein content determination adopts dying method with coomassie brilliant blue.Polysaccharide extract rate is defined as
The ratio of polysaccharide quality in lower floor's inorganic salt phase polysaccharide quality and funguses water extract, determination of polysaccharide adopts phenolsulfuric acid ratio
Color method.
Embodiment one: with phellinus igniarius mycelium as object of study
Step one, defat: phellinus igniarius mycelium is pulverized, defat cotton wraps up, and is placed in 500 ml Soxhlet extractors, adds
200 ~ 300 ml petroleum ether (boiling range: 30 ~ 60 DEG C), 35 DEG C of backflow 6 h, such ungrease treatment 2 times, naturally dry, obtain defat
Phellinus igniarius mycelium.
Step 2, prepares Phellinus igniarius (L. ex Fr.) Quel. water extract: weighs after defat in step one phellinus igniarius mycelium 100 g by the material of 1:10g/ml
Liquor ratio adds distilled water 1000 ml, is placed in 2000 ml round-bottomed flasks, after stirring, in 95 DEG C of reflux, extract, 8 h.
Mixture 300 ml plastic centrifuge tubes, 5000 rpm, 10 min centrifugations, supernatant is incorporated in 45 DEG C of rotary evaporator decompressions and steams
Send out, be concentrated into 1.5 times of original volume, distilled water dialysis 48 h, vacuum lyophilization, obtain Phellinus igniarius (L. ex Fr.) Quel. water extract.
Step 3, using ionic liquid double-aqueous phase system Selective Separation phellin polysaccharides and protein:
The Phellinus igniarius (L. ex Fr.) Quel. water extract solution that 1.0 ml mass concentrations are 10 mg/ml is added to by 1.0 g ionic liquids [ch]
cl、2,8 g k3po4In the ionic liquid double-aqueous phase system of 4.0 ml distilled water compositions, the mass ratio of three is 0.25:0.7:
1.0.Fully vibration makes ionic liquid and inorganic salt be completely dissolved, and is placed in 20 DEG C of waters bath with thermostatic control and vibrates 30 min, 3000 rpm,
10 min centrifugations, separate biphase up and down.The extraction ratio recording protein in the ionic liquid phase of upper strata is 75.1%, lower floor's inorganic salt phase
The extraction ratio of middle phellin polysaccharides is 78.8%.
Step 4, prepares phellin polysaccharides: lower floor contains inorganic salt phase distilled water dialysis 48 h of phellin polysaccharides, phellin polysaccharides are saturating
Analysis liquid 95% ethanol precipitation of 4 times of volumes, 4 DEG C stand overnight, centrifugation, and lyophilization obtains phellin polysaccharides；
Step 5, reclaims ionic liquid: ionic liquid phase dichloromethane in upper strata extracts 3 ~ 5 times, the concentration of dichloromethane phase (≤
45 DEG C), reclaim ionic liquid [ch] cl.
Embodiment two: with mushroom fruiting body as object of study
With embodiment one, its difference is wherein ionic liquid [ch] cl 2.0 g, k to test process process3po4
4.0 g and distilled water 4.0 ml, the mass ratio of three records the extraction ratio of protein in the ionic liquid phase of upper strata for 0.5:1.0:1.0
For 86.3%, in lower floor's inorganic salt phase, the extraction ratio of lentinan is 55.5%.
Embodiment three: with Cordyceps mycelium as object of study
With embodiment one, its difference is wherein ionic liquid [ch] cl 1.2 g, k to test process process3po4
3.2 g and distilled water 4.0 ml, the mass ratio of three is 0.3:0.8:1.0, records the extraction of protein in the ionic liquid phase of upper strata
Rate is 85.7%, and in lower floor's inorganic salt phase, the extraction ratio of Cordyceps polysaccharides is 71.9%.
By above example as can be seen that [ch] cl/k3po4Composition ionic liquid double-aqueous phase system can be used in Phellinus igniarius (L. ex Fr.) Quel.,
The Selective Separation of Lentinus Edodess, Cordyceps polysaccharides and protein, and there is green, effect quickly and efficiently.
Claims (2)
1. one kind utilizes cholinium ion liquid double-aqueous phase system separating polyose and method of protein it is characterised in that including following
Step:
Step one, defat: it is placed in Soxhlet extractor after fungal mycelium or sporophore are pulverized, add petroleum ether, boiling range 30 ~
60 DEG C, 35 DEG C of backflow 6 h；So after defat twice, naturally dry to obtain fungal mycelium after defat or sporophore；
Step 2, prepares funguses water extract: the fungal mycelium after defat described in step one or sporophore are pressed 1:10 g/ml
Solid-liquid ratio add distilled water in, 95 DEG C of reflux, extract, 8 h, then rotating speed be 5000 rpm under the conditions of be centrifuged 10 min, concentrate,
Lyophilization after distilled water dialysis 48 h obtains funguses water extract；
Step 3, using ionic liquid double-aqueous phase system Selective Separation fungus polysaccharide and protein: will be true described in step 2
Bacterium water extract is made into the funguses water extract aqueous solution that mass concentration is 10 mg/ml, takes 1.0 ml funguses water extract aqueous solutions to add
To in double-aqueous phase system, then it is placed in 20 DEG C of waters bath with thermostatic control and vibrates 30 min, centrifugation, separate and biphase up and down contains fungus polysaccharide
Lower floor's inorganic salt phase and the upper strata ionic liquid phase containing impurity such as protein；
Described double-aqueous phase system is by ionic liquid choline chloride [ch] cl, inorganic phosphate k3po4Form with distilled water, described
Ionic liquid choline chloride [ch] cl, inorganic phosphate k3po4Mass ratio with distilled water is: 0.25-0.5: 0.7-1.0:
1.0；
Step 4, prepares fungus polysaccharide: by the lower floor's inorganic salt phase distilled water dialysis containing fungus polysaccharide described in step 3
48 h, are precipitated to fungus polysaccharide dialysis solution using the ethanol that concentration is 95%；The volume of described ethanol is fungus polysaccharide dialysis
4 times of liquid；4 DEG C stand overnight, then are centrifuged 10 min under conditions of rotating speed is 5000 rpm, and after collecting precipitate, freezing is dry
Dry, obtain fungus polysaccharide；
Step 5, reclaims ionic liquid: by the upper strata ionic liquid phase dichloromethane containing impurity such as protein described in step 3
Alkane extracts 3 ~ 5 times, temperature≤45 DEG C when dichloromethane phase concentrates, and reclaims ionic liquid [ch] cl.
2. one kind according to claim 1 utilizes cholinium ion liquid double-aqueous phase system separating polyose and method of protein,
It is characterized in that: described funguses are Phellinus igniarius (L. ex Fr.) Quel., Lentinus Edodess, Ganoderma, Grifola frondosa, Cordyceps, Flammulina velutiper (Fr.) Sing, any one in Pleurotus eryngii.
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| CN103113487A (en) * | 2013-02-21 | 2013-05-22 | 嘉兴职业技术学院 | Method for preparing flammulina velutipes polysaccharides and protein efficiently synchronously |
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Effective date of registration: 20201225 Address after: 224200 No.88 Gangcheng Avenue, Dongtai coastal economic zone, Zhenjiang City, Jiangsu Province Patentee after: Dongtai Haibin science and Technology Pioneer Park Management Co.,Ltd. Address before: No. 101, Nanxu Avenue, Zhenjiang City, Jiangsu Province, 212000 Patentee before: JIANGSU HUIZHI INTELLECTUAL PROPERTY SERVICES Co.,Ltd. Effective date of registration: 20201225 Address after: No. 101, Nanxu Avenue, Zhenjiang City, Jiangsu Province, 212000 Patentee after: JIANGSU HUIZHI INTELLECTUAL PROPERTY SERVICES Co.,Ltd. Address before: 212013 No. 301, Xuefu Road, Zhenjiang, Jiangsu Patentee before: JIANGSU University |
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Address after: 224200 No.88 Gangcheng Avenue, Dongtai coastal economic zone, Yancheng City, Jiangsu Province Patentee after: Dongtai Haibin science and Technology Pioneer Park Management Co.,Ltd. Address before: 224200 No.88 Gangcheng Avenue, Dongtai coastal economic zone, Zhenjiang City, Jiangsu Province Patentee before: Dongtai Haibin science and Technology Pioneer Park Management Co.,Ltd. |