CN109369816A - The extracting method of sargassum fusifome anti-aging polysaccharide - Google Patents
The extracting method of sargassum fusifome anti-aging polysaccharide Download PDFInfo
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Abstract
The invention discloses the extracting methods of sargassum fusifome anti-aging polysaccharide, including the preparation of brown alga Thick many candies extracting solution, macroporous resin purification, ion-exchange chromatography, dialysis and gel chromatography column purification, macroporous resin purification step are as follows: loading after anti-aging Thick many candies preparing solution to macroreticular resin D3520-Cl column is purified, is eluted with deionized water, the eluent of collection is concentrated under reduced pressure into viscous pasty state, add ethanol solution into concentrate again, it is uniformly mixed, precipitates overnight, centrifugation, it is dry, it is spare.Have the beneficial effect that the extracting method simple possible of sargassum fusifome anti-aging polysaccharide of the present invention, the extraction efficiency of polysaccharide, yield and purity are higher, and pigment removal is high.
Description
Technical field
The present invention relates to technical field of polysaccharide extraction, more particularly, to the extracting method of sargassum fusifome anti-aging polysaccharide.
Background technique
Polysaccharide (polysaccharide) is widely present in the organisms such as animals and plants and microorganism, is single by 10 or more
For sugar by polymer made of glucosides key connection, it is the important producing high-molecular of another class outside isolating protein and nucleic acid in organism
Close object.General plant polyose is made of hundreds and thousands of a monosaccharide, due to the type and mode of connection of monosaccharide and the structure of anomeric carbon
Type can all influence the structure of polysaccharide, therefore structure of the polysaccharide acquired from nature in structure more than protein and nucleic acid is wanted
It is complicated very much.Into after the 1990s, the isolating and purifying of polysaccharide and compound, compound mensuration and structural analysis etc.
Research has rapid progress, simultaneously because the development of Celluar and Molecular Biology, research has found saccharide compound spy
Be not the adjusting that sugar chain on saccharide complex takes part in various biological phenomenas, as cell recognition, adhesion with merge, signal is conveyed,
The immune participation that sugar chain is all be unable to do without with response, cell transformation and differentiation.Due to various effects of saccharide complex, research also at
For the new forward position of life field of scientific study.A large number of studies show that polysaccharide have antitumor, anticoagulation, it is hypoglycemic, anti-oxidant and
Immune isoreactivity, and there is no cytotoxicity, it is small to be applied to organism toxic side effect, therefore obtain on clinical and functional food
It is widely used, the research and development of related polysaccharides living resources becomes natural using the growing interest by scientist at home and abroad
The research hotspot in the fields such as medicament research and development, biochemistry.
Sargassum fusifome system Phaeophyta Sargassaceae Sargassum plant, alias jade grass, sea grass, hexagonal dish, as dual-purpose of drug and food
Economical alga has " longevity greens/mustard green " laudatory title, is praised highly as the food that lengthens one's life.Polysaccharide is the characteristic ingredient that content is most in sargassum fusifome, about
Account for the 45%-65% of its dry weight.Studies have shown that except as dietary fiber regulating gastointestinal function, body is adjusted to lipid and carbohydrate etc.
Metabolism outside, Hijiki polysaccharide also has the bioactivity such as anti-oxidant, antitumor and enhancing is immune.These effects can be straight
Human health status is connect or improved indirectly, damage of the destructive stimulus to body is reduced, slows down the process with diseases associated with senescence.
Thus speculate, polysaccharide is the effective component that sargassum fusifome plays anti-aging effects.Currently, Hijiki polysaccharide extractive technique mainly has enzyme
Solution, alkaline hydrolysiss, acid hydrolyzation, water extraction method, ultrasonic wave auxiliary, but in the extracting method of existing Hijiki polysaccharide, all have not
Foot, including cost are too high, and complicated for operation, energy-consuming consumptive material is more, and polyoses content and purity is not high.Most importantly: polysaccharide is thick
Extract is often complicated mixture, has not only included the polysaccharide fragment that molecular weight is different, structure is different, but also including protein, color
The substances such as element.Therefore it needs to carry out separation and purification treatment to it.
The prior art such as Authorization Notice No. is the Chinese invention patent of 105601762 B of CN, discloses a kind of operating procedure
Simply, energy-consuming consumptive material is few, extracting method of polyoses content and the higher Hijiki polysaccharide of purity, includes the following steps: that (1) is dried
Sargassum fusifome is crushed into powder, and crosses 40-200 mesh screen；(2) sargassum fusifome algae powder obtained in step (1) and 95% ethyl alcohol is sufficiently mixed
It closes, carries out reflux degreasing；(3) the algae powder in recycling step (2) after degreasing is added distilled water, is filtered repeatedly by bolting silk
Filtrate is collected, merges above-mentioned filtrate, is concentrated under reduced pressure, obtain concentrate；(4) concentrate in step (3), according to concentrate,
It is added 95% ethyl alcohol, after alcohol precipitation, precipitating is collected in centrifugation, is added distilled water, and stirring is to being completely dissolved；It (5) will be more in step (4)
Sugar juice, vacuum freezedrying obtain polysaccharide product.But the purity of the Hijiki polysaccharide is lower, color is poor.
Summary of the invention
The purpose of the present invention is to provide a kind of extracting method simple possible, the extraction efficiency of polysaccharide, yield and purity are equal
It is higher, the extracting method of the high sargassum fusifome anti-aging polysaccharide of pigment removal.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken are as follows:
The extracting method of sargassum fusifome anti-aging polysaccharide, including the preparation of brown alga Thick many candies extracting solution, macroporous resin purification, ion exchange
Chromatography, dialysis and gel chromatography column purification, specific steps are as follows:
The preparation of anti-aging Thick many candies: it by Sangassivm fuciforime (Harv) Setch powder using the ethanol solution reflux 1.5-2.5h that concentration is 93-97%, repeats back to
Stream 2-4 times, except depigmentaton, degreasing and removes monosaccharide, is then added 28-33 times and measures distilled water and Sangassivm fuciforime (Harv) Setch powder weight 0.03-
0.05 ‰ calcium lactates, ultrasound assisted extraction 2.5-3.5h under being 120-170W in 80-90 DEG C, power, filtering, filtrate are as anti-ageing
Uneducated person's polysaccharide solution is acted on using ultrasonic wave and generates cavitation effect, forms high pressure in histocyte, the release of high pressure is in solution
It is middle to form powerful shock wave, it can effectively reduce, eliminate resistance between water and sargassum fusifome, be imitated to increase mass transfer
Rate, while cell tissue deformation fracture by the physical shear power that shock wave generates, release intracellular organic matter, greatly accelerate
Extraction process, and due to anti-aging polysaccharide be it is polar, do not dissolve in ethanol solution, therefore make anti-aging polysaccharide using ethanol solution
Precipitating is formed, so that other substances such as polysaccharide and water-soluble monosaccharide be made to separate；
Macroporous resin purification: by anti-aging Thick many candies preparing solution at loading after the solution of concentration 3-5mg/mL to macroreticular resin
D3520-Cl column is purified, and is eluted with deionized water, and the eluent of collection is concentrated under reduced pressure into viscous pasty state, then to dense
Add ethanol solution in contracting liquid, be uniformly mixed, precipitates overnight, be centrifuged, dry, spare, which is introduced wherein with D3520-Cl
Chloromethyl (CH with greater activity2Cl), the polarity of non-polar macroporous resin D3520 is improved, so that anti-aging polysaccharide
In pigment and protein easily enter the inside of D3520-Cl, suction-operated is generated with its internal active site, to mention
Its high adsorbance, can effectively remove the pigment and protein in anti-aging polysaccharide, effectively improve the purity of polysaccharide, simultaneously
It can effectively protect the structure and activity of polysaccharide；
Polysaccharide after macroporous resin purification: being made into the solution of concentration 18-22mg/mL by ion-exchange chromatography with deionized water, on
Sample to DEAE Sepharose CL-6B column carries out cation exchange chromatography, uses 0mol/L, 0.1mol/L, 0.3mol/ respectively
L, the sodium chloride solution of 0.5mol/L, 0.7mol/L carry out gradient elution, and the sodium chloride solution elution volume of various concentration is 3-
The flow velocity of 4BV, elution are arranged to 0.7-0.8mL/min, and using phend-sulphuric acid tracing detection polyoses content, sample is not until having
There is SFPS that can detect, draws elution curve, the eluent in each eluting peak section is collected respectively, through macroporous resin purification
Polysaccharide afterwards still contains a certain amount of impurity, which has three-dimensional space net structure with DEAE Sepharose CL-6B,
Not only there is ion exchange, and have the function of molecular sieve, the range of separating polyose relative molecular weight is located at
Between 10kDa-4000kDa, so that the dual separating effect that the step can reach according to the difference of charge and molecular weight, makes polysaccharide
Solution realizes concentration and preliminary purification, has extraordinary separation efficiency；
Dialysis: with molecular cut off being that 3000-4000Da bag filter is dialysed to molten outside bag by the eluent after ion-exchange chromatography
The conductivity of liquid and the conductivity of deionized water are identical, dialyzate freeze-drying, spare, contain in the polysaccharide after salting liquid elutes
There are the impurity such as inorganic salts, oligosaccharide and partial pigment, if recovered liquid is directly carried out ethanol precipitation, a large amount of salt branch is simultaneously
It precipitates with glycocalix, influences the purity and activity research of subsequent polysaccharide, therefore it is more to select simple and effective bag filter to carry out
The desalination of sugar；
Gel chromatography column purification: the polysaccharide crude that dialysis obtains is made into the solution of concentration 38-42mg/mL, difference loading is extremely
Sephadex G-200 gel column is further purified, and is eluted with distilled water, is tracked and is examined by ultraviolet-visible spectrophotometer
Its absorbance at 480nm is tested, elution curve is drawn using the volume of eluent and absorbance as transverse and longitudinal coordinate respectively, collects each
The eluent in eluting peak region, the component for selecting antiageing effect best are concentrated into small size, are freeze-dried to get anti-aging
Polysaccharide, spent ion exchange resin separate polysaccharide at different levels foundation be its electrically charged amount difference, so each fraction polysaccharide very may be used
It can be the mixture of the identical several polysaccharide of electrically charged amount, and the principle of gel chromatography is according to the big of molecular weight of material to be separated
It is small to be separated, it can obtain the sargassum fusifome anti-aging polysaccharide of high-purity.
Preferably, the preparation step of macroporous resin purification step D3520-Cl are as follows: by macroreticular resin D3520 concentration
It is swollen 5-6h for the ethanol solution of 93-97%, then cleans 2-4 all over rear natural air drying, so with the ethanol solution that concentration is 93-97%
It is afterwards 1:3.5-4.0(g/mL by solid-to-liquid ratio) D3520 is placed in 60-70 DEG C of CCl4Middle swelling 30-40h, adds ZnCl2, lemon
Lemon acid iron, NaCl and chloromethyl ether, under conditions of temperature is 45-55 DEG C, ultrasonic power is 70-80W react 21-26h to get
D3520-Cl.The step takes Ultrasonic Heating effect to D3520 chloromethylation, can increase the swellbility of resin, make
Partial collapse hole reopens in D3520, increases the number of active site in D3520, and Ultrasonic Heating effect increases reaction
The energy of system, this will increase bioactive molecule number and reduce thickness of diffusion layer, the final speed for increasing chloromethylation and
Degree, the chloromethylation degree 12.3% of the macroreticular resin made.
Further preferably, D3520, ZnCl2, NaCl and chloromethyl ether weight ratio be 1:1.3-1.5:0.40-0.42:
0.15-0.17, ZnCl2It is 1:0.12-0.13 with ironic citrate molar ratio.Since the high-crosslinking-degree of nonpolar D3520 can make it
Serious space steric effect is generated, modifying agent is difficult to enter inside D3520 and reacts, this is largely reduced
The modification degree of D3520, and ironic citrate it is special exist can synergistic supersonic wave effect reduce D3520 diffusion into the surface thickness
Degree promotes ZnCl2, NaCl, iron powder and chloromethyl ether be diffused inside D3520, improve chloromethylation reagents and D3520
Touch opportunity carries out so that reaction is more easy, and in addition there are the generating rates for being conducive to raising intermediate product, so that intermediate product
It rapidly enters inside D3520, accelerates intermediate product and δ+CH2The mutual conversion of Cl, so that the chloromethylation degree of D3520 is improved, into
And the pigment and protein in anti-aging polysaccharide can be removed effectively during improving macroporous resin purification, effectively improve polysaccharide
Purity, the sensory effects for improving polysaccharide.
Preferably, L-calcium lactate and the ratio of D-ALPHA-Hydroxypropionic acid calcium are 100 in calcium lactate in anti-aging Thick many candies preparation step:
6.3-7.5.The special of the calcium lactate exists and can combine with the anti-aging polysaccharide of dissolution, and it is more to change anti-aging not soluble in water
The hydrophily of sugar, while it is quickly dissolved in water-soluble anti-aging polysaccharide in water, the extraction rate and yield of polysaccharide are improved, simultaneously
The viscosity that can reduce anti-aging Thick many candies solution is conducive to pigment molecular diffusion and reduces liquid-film resistance, and then is conducive to color
Element is adsorbed by D3520-Cl, and can enhance the binding force of functional group on pigment and D3520-Cl, avoids pigment from D3520-
It is desorbed on Cl, improves the removal rate of pigment.
Compared with the prior art, the advantages of the present invention are as follows: the extracting method simple possible of anti-aging polysaccharide of the present invention is more
The extraction efficiency of sugar, yield and purity are higher, and easy to industrialized production, safety is higher, greatly improves the additional of sargassum fusifome
Value；Macroporous resin purification step of the present invention has the chloromethyl (CH of greater activity with macroreticular resin2Cl), so that anti-aging is more
Pigment and protein in sugar easily enter the inside of D3520-Cl, generate suction-operated with its internal active site, thus
Its adsorbance is improved, the purity of polysaccharide is effectively improved, improves the sensory effects of polysaccharide；Extracting method of the present invention can improve polysaccharide
Extraction rate and yield reduce the viscosity of anti-aging Thick many candies solution, are conducive to pigment and adsorb by D3520-Cl, and can enhance
The binding force of functional group, avoids pigment from desorbing from D3520-Cl, improves the removal rate of pigment on pigment and D3520-Cl.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
The extracting method of sargassum fusifome anti-aging polysaccharide, including the preparation of brown alga Thick many candies extracting solution, macroporous resin purification, ion exchange
Chromatography, dialysis and gel chromatography column purification, specific steps are as follows:
1) prepared by anti-aging Thick many candies: the ethanol solution reflux 1.5h for being 93% using concentration by Sangassivm fuciforime (Harv) Setch powder repeats reflux 2 times,
Except depigmentaton, degreasing and monosaccharide is removed, then 28- times is added and measures 0.03 ‰ calcium lactate of distilled water and Sangassivm fuciforime (Harv) Setch powder weight, 80
DEG C, power be 120-170W under ultrasound assisted extraction 2.5h, filtering, filtrate is anti-aging Thick many candies solution, utilizes ultrasonic wave
Effect generates cavitation effect, high pressure is formed in histocyte, the release of high pressure forms powerful shock wave in the solution, can
It effectively reduces, eliminate resistance between water and sargassum fusifome, to increase mass-transfer efficiency, while cell tissue is by shock wave
The physical shear power of generation and deformation fracture, release intracellular organic matter, have greatly accelerated extraction process, and due to anti-aging polysaccharide
Be it is polar, do not dissolve in ethanol solution, therefore using ethanol solution make anti-aging polysaccharide formed precipitating, thus make polysaccharide with it is water-soluble
Property other substances separation such as monosaccharide；
2) macroporous resin purification: by anti-aging Thick many candies preparing solution at loading after the solution of concentration 3mg/mL to macroreticular resin
D3520-Cl column is purified, and is eluted with deionized water, and the eluent of collection is concentrated under reduced pressure into viscous pasty state, then to dense
Add ethanol solution in contracting liquid, be uniformly mixed, precipitates overnight, be centrifuged, dry, spare, which is introduced wherein with D3520-Cl
Chloromethyl (CH with greater activity2Cl), the polarity of non-polar macroporous resin D3520 is improved, so that anti-aging polysaccharide
In pigment and protein easily enter the inside of D3520-Cl, suction-operated is generated with its internal active site, to mention
Its high adsorbance, can effectively remove the pigment and protein in anti-aging polysaccharide, effectively improve the purity of polysaccharide, simultaneously
It can effectively protect the structure and activity of polysaccharide；
3) polysaccharide after macroporous resin purification ion-exchange chromatography: is made into the solution of concentration 18mg/mL, loading with deionized water
To DEAE Sepharose CL-6B column carry out cation exchange chromatography, respectively with 0mol/L, 0.1mol/L, 0.3mol/L,
The sodium chloride solution of 0.5mol/L, 0.7mol/L carry out gradient elution, and the sodium chloride solution elution volume of various concentration is 3BV,
The flow velocity of elution is arranged to 0.7mL/min, and using phend-sulphuric acid tracing detection polyoses content, sample is not until having SFPS can
With detection, elution curve is drawn, collects the eluent in each eluting peak section, the polysaccharide after macroporous resin purification respectively
Still contain a certain amount of impurity, which has three-dimensional space net structure with DEAE Sepharose CL-6B, not only have
Ion exchange, and have the function of molecular sieve, the range of separating polyose relative molecular weight is located at 10kDa-4000kDa
Between, so that the dual separating effect that the step can reach according to the difference of charge and molecular weight, makes polysaccharide solution realize concentration
And preliminary purification, there is extraordinary separation efficiency；
4) it dialyses: with molecular cut off being that 3000Da bag filter is dialysed to solution outside bag by the eluent after ion-exchange chromatography
Conductivity is identical as the conductivity of deionized water, dialyzate freeze-drying, it is spare, in the polysaccharide after salting liquid elutes containing whether there is or not
The impurity such as machine salt, oligosaccharide and partial pigment, if recovered liquid is directly carried out ethanol precipitation, a large amount of salt branch is simultaneously with more
Sugar is deposited, and influences the purity and activity research of subsequent polysaccharide, therefore simple and effective bag filter is selected to carry out polysaccharide
Desalination；
5) gel chromatography column purification: the polysaccharide crude that dialysis obtains is made into the solution of concentration 38mg/mL, difference loading is extremely
Sephadex G-200 gel column is further purified, and is eluted with distilled water, is tracked and is examined by ultraviolet-visible spectrophotometer
Its absorbance at 480nm is tested, elution curve is drawn using the volume of eluent and absorbance as transverse and longitudinal coordinate respectively, collects each
The eluent in eluting peak region, the component for selecting antiageing effect best are concentrated into small size, are freeze-dried to get anti-aging
Polysaccharide, spent ion exchange resin separate polysaccharide at different levels foundation be its electrically charged amount difference, so each fraction polysaccharide very may be used
It can be the mixture of the identical several polysaccharide of electrically charged amount, and the principle of gel chromatography is according to the big of molecular weight of material to be separated
It is small to be separated, it can obtain the sargassum fusifome anti-aging polysaccharide of high-purity.
The preparation step of macroporous resin purification step D3520-Cl are as follows: the second for being 93% by macroreticular resin D3520 concentration
Alcoholic solution is swollen 5h, then natural air drying after then cleaning 2 times with the ethanol solution that concentration is 93% is 1:3.5 by solid-to-liquid ratio
(g/mL) D3520 is placed in 60 DEG C of CCl4Middle swelling 30h, adds ZnCl2, ironic citrate, NaCl and chloromethyl ether, in temperature
21h is reacted under conditions of degree is 45 DEG C, ultrasonic power is 70W to get D3520-Cl.Above-mentioned D3520, ZnCl2, NaCl and chloromethane
The weight ratio of ether is 1:1.3:0.40:0.15, ZnCl2It is 1:0.12 with ironic citrate molar ratio.Due to the height of nonpolar D3520
The degree of cross linking can make it generate serious space steric effect, be difficult to enter modifying agent inside D3520 and react, this is very
The modification degree of D3520 is reduced in big degree, and the special presence of ironic citrate being capable of synergistic supersonic wave effect reduction D3520
Surface diffusion layer thickness promotes ZnCl2, NaCl, iron powder and chloromethyl ether be diffused inside D3520, improve chloromethylation
The touch opportunity of reagent and D3520 carries out so that reaction is more easy, and in addition there are be conducive to improve the generation speed of intermediate product
Rate accelerates intermediate product and δ so that intermediate product rapidly enters inside D3520+CH2The mutual conversion of Cl, to improve D3520
Chloromethylation degree, and then improve and can remove effectively pigment and albumen in anti-aging polysaccharide during macroporous resin purification
Matter effectively improves the purity of polysaccharide, improves the sensory effects of polysaccharide.The step takes Ultrasonic Heating effect to D3520 chloromethyl
Change and be modified, the swellbility of resin can be increased, reopen partial collapse hole in D3520, increases active site in D3520
Number, and Ultrasonic Heating effect increases the energy of reaction system, this will increase bioactive molecule number and reduce thickness of diffusion layer,
The final speed and degree for increasing chloromethylation, the chloromethylation degree 12.3% of the macroreticular resin made.
L-calcium lactate and the ratio of D-ALPHA-Hydroxypropionic acid calcium are 100:6.3 in calcium lactate in anti-aging Thick many candies preparation step.The cream
The special of sour calcium exists and can combine with the anti-aging polysaccharide of dissolution, changes the hydrophily of anti-aging polysaccharide not soluble in water,
It is quickly dissolved in water-soluble anti-aging polysaccharide in water simultaneously, improves the extraction rate and yield of polysaccharide, while can reduce anti-
The viscosity of aging Thick many candies solution is conducive to pigment molecular diffusion and reduces liquid-film resistance, and then is conducive to pigment in quilt
D3520-Cl absorption, and the binding force of functional group on pigment and D3520-Cl can be enhanced, avoid pigment from solving from D3520-Cl
It inhales, improves the removal rate of pigment.
Embodiment 2:
The extracting method of sargassum fusifome anti-aging polysaccharide, including the preparation of brown alga Thick many candies extracting solution, macroporous resin purification, ion exchange
Chromatography, dialysis and gel chromatography column purification, specific steps are as follows:
1) prepared by anti-aging Thick many candies: the ethanol solution reflux 2.0h for being 95% using concentration by Sangassivm fuciforime (Harv) Setch powder repeats reflux 3 times,
Except depigmentaton, degreasing and monosaccharide is removed, 30 times of amount distilled water and 0.04 ‰ calcium lactate of Sangassivm fuciforime (Harv) Setch powder weight is then added, 85
DEG C, power be 150W under ultrasound assisted extraction 3.0h, filtering, filtrate is anti-aging Thick many candies solution；
2) macroporous resin purification: by anti-aging Thick many candies preparing solution at loading after the solution of concentration 4mg/mL to macroreticular resin
D3520-Cl column is purified, and is eluted with deionized water, and the eluent of collection is concentrated under reduced pressure into viscous pasty state, then to dense
In contracting liquid plus ethanol solution, uniformly mixed, precipitates overnight, centrifugation, drying are spare；
3) polysaccharide after macroporous resin purification ion-exchange chromatography: is made into the solution of concentration 20mg/mL, loading with deionized water
To DEAE Sepharose CL-6B column carry out cation exchange chromatography, respectively with 0mol/L, 0.1mol/L, 0.3mol/L,
The sodium chloride solution of 0.5mol/L, 0.7mol/L carry out gradient elution, and the sodium chloride solution elution volume of various concentration is 3BV,
The flow velocity of elution is arranged to 0.75mL/min, and using phend-sulphuric acid tracing detection polyoses content, sample is not until having SFPS can
With detection, elution curve is drawn, collects the eluent in each eluting peak section respectively；
4) it dialyses: with molecular cut off being that 3500Da bag filter is dialysed to solution outside bag by the eluent after ion-exchange chromatography
Conductivity is identical as the conductivity of deionized water, dialyzate freeze-drying, spare；
5) gel chromatography column purification: the polysaccharide crude that dialysis obtains is made into the solution of concentration 40mg/mL, difference loading is extremely
Sephadex G-200 gel column is further purified, and is eluted with distilled water, is tracked and is examined by ultraviolet-visible spectrophotometer
Its absorbance at 480nm is tested, elution curve is drawn using the volume of eluent and absorbance as transverse and longitudinal coordinate respectively, collects each
The eluent in eluting peak region, the component for selecting antiageing effect best are concentrated into small size, are freeze-dried to get anti-aging
Polysaccharide.
The preparation step of macroporous resin purification step D3520-Cl are as follows: the second for being 95% by macroreticular resin D3520 concentration
Alcoholic solution is swollen 5.5h, then natural air drying after then cleaning 3 times with the ethanol solution that concentration is 95% is 1:3.7 by solid-to-liquid ratio
(g/mL) D3520 is placed in 65 DEG C of CCl4Middle swelling 36h, adds ZnCl2, ironic citrate, NaCl and chloromethyl ether, in temperature
Degree is 50 DEG C, ultrasonic power is reacted for 24 hours under conditions of being 75W to get D3520-Cl.Above-mentioned D3520, ZnCl2, NaCl and chloromethane
The weight ratio of ether is 1:1.4:0.41:0.16, ZnCl2It is 1:0.125 with ironic citrate molar ratio.
L-calcium lactate and the ratio of D-ALPHA-Hydroxypropionic acid calcium are 100:7.0 in calcium lactate in anti-aging Thick many candies preparation step.
Embodiment 3:
The extracting method of sargassum fusifome anti-aging polysaccharide, including the preparation of brown alga Thick many candies extracting solution, macroporous resin purification, ion exchange
Chromatography, dialysis and gel chromatography column purification, specific steps are as follows:
1) prepared by anti-aging Thick many candies: the ethanol solution reflux 2.5h for being 97% using concentration by Sangassivm fuciforime (Harv) Setch powder repeats reflux 4 times,
Except depigmentaton, degreasing and monosaccharide is removed, 33 times of amount distilled water and 0.05 ‰ calcium lactate of Sangassivm fuciforime (Harv) Setch powder weight is then added, 90
DEG C, power be 170W under ultrasound assisted extraction 3.5h, filtering, filtrate is anti-aging Thick many candies solution；
2) macroporous resin purification: by anti-aging Thick many candies preparing solution at loading after the solution of concentration 5mg/mL to macroreticular resin
D3520-Cl column is purified, and is eluted with deionized water, and the eluent of collection is concentrated under reduced pressure into viscous pasty state, then to dense
In contracting liquid plus ethanol solution, uniformly mixed, precipitates overnight, centrifugation, drying are spare；
3) polysaccharide after macroporous resin purification ion-exchange chromatography: is made into the solution of concentration 22mg/mL, loading with deionized water
To DEAE Sepharose CL-6B column carry out cation exchange chromatography, respectively with 0mol/L, 0.1mol/L, 0.3mol/L,
The sodium chloride solution of 0.5mol/L, 0.7mol/L carry out gradient elution, and the sodium chloride solution elution volume of various concentration is 4BV,
The flow velocity of elution is arranged to 0.8mL/min, and using phend-sulphuric acid tracing detection polyoses content, sample is not until having SFPS can
With detection, elution curve is drawn, collects the eluent in each eluting peak section respectively；
4) it dialyses: with molecular cut off being that 4000Da bag filter is dialysed to solution outside bag by the eluent after ion-exchange chromatography
Conductivity is identical as the conductivity of deionized water, dialyzate freeze-drying, spare；
5) gel chromatography column purification: the polysaccharide crude that dialysis obtains is made into the solution of concentration 42mg/mL, difference loading is extremely
Sephadex G-200 gel column is further purified, and is eluted with distilled water, is tracked and is examined by ultraviolet-visible spectrophotometer
Its absorbance at 480nm is tested, elution curve is drawn using the volume of eluent and absorbance as transverse and longitudinal coordinate respectively, collects each
The eluent in eluting peak region, the component for selecting antiageing effect best are concentrated into small size, are freeze-dried to get anti-aging
Polysaccharide.
The preparation step of macroporous resin purification step D3520-Cl are as follows: the second for being 97% by macroreticular resin D3520 concentration
Alcoholic solution is swollen 6h, then natural air drying after then cleaning 4 times with the ethanol solution that concentration is 97% is 1:4.0 by solid-to-liquid ratio
(g/mL) D3520 is placed in 70 DEG C of CCl4Middle swelling 40h, adds ZnCl2, ironic citrate, NaCl and chloromethyl ether, in temperature
26h is reacted under conditions of degree is 55 DEG C, ultrasonic power is 80W to get D3520-Cl.Above-mentioned D3520, ZnCl2, NaCl and chloromethane
The weight ratio of ether is 1:1.5:0.42:0.17, ZnCl2It is 1:0.13 with ironic citrate molar ratio.
L-calcium lactate and the ratio of D-ALPHA-Hydroxypropionic acid calcium are 100:7.5 in calcium lactate in anti-aging Thick many candies preparation step.
Comparative example 1:
Ironic citrate is not added in the preparation step of D3520-Cl, rest part and embodiment 2 are completely the same.
Comparative example 2:
Macroporous resin purification step macroreticular resin is the D3520 without chloromethylation, and purification process and embodiment 2 are complete
It is complete consistent.
Embodiment 4:
Embodiment 2 is set as test group, comparative example 1, comparative example 2 and is set to control group 1, control group 2 and control group 3.
The measurement of chloromethylation degree: the D3520-Cl, 0.300g that precision weighs 0.100g test group and control group 1 obtains
KNO3With 0.300g NaOH, be placed in nickel crucible, mixed repeatedly to uniform, nickel crucible is placed on mud triangle, with alcolhol burner plus
Heat makes resin burning ashing, calcination 3h in 700 DEG C of Muffle furnaces is put into after crucible is cooled to room temperature, stops heating.Crucible is moved
Enter after drier is cooled to room temperature, is transferred in the beaker of 250mL, suitable distilled water, which is added, makes crucible just be immersed in distilled water
In, sealed beaker takes out crucible after impregnating 15h, with 10 times inside and outside distilled water repeated flushing crucible, will impregnate and rinse crucible
Distilled water be merged into 500mL volumetric flask, be added 100mL 0.1mol/L KNO3, with 10mol/L NaOH and HNO3By solution
PH value adjust in the range of 2.95 ± 0.02, use distilled water constant volume later, measure the current potential of solution, utilize standard curve meter
Solution concentration is calculated, and calculates the chloromethylation degree of D3520-Cl with following formula, the results are shown in Table 1.
C in formulaCAnd C1(mol/L) chlorine ion concentration in D3520-Cl and D3520 solution, m are respectively representedrAnd m1(g) divide
The weight of D3520-Cl and D3520 are not represented, and 0.5 represents the volume (L) of solution.
The measurement of polysaccharide recovery, pigment removal and albumen removal rate: Phenol sulfuric acid procedure, Bradford are used first
Method, colorimetric method measure the polysaccharide concentration, protein concentration and pigment content of anti-aging Thick many candies mother liquor respectively.Then it collects and closes
And the eluent containing polysaccharide, polyoses content, albumen and the pigment content of the macroporous resin purification eluent of collection are measured, is calculated
Polysaccharide recovery, pigment removal and albumen removal rate, the results are shown in Table 1.
1 measurement result of table
| Project | Chloromethylation degree (%) | Polysaccharide recovery (%) | Pigment removal (%) | The removal rate (%) of albumen |
| Test group | 12.3 | 67.33 | 89.97 | 79.03 |
| Control group 1 | 6.8 | 63.09 | 85.21 | 70.35 |
| Control group 2 | - | 60.35 | 81.55 | 63.87 |
As shown in Table 1, the chloromethylation degree of test group will be much higher than control group 1, illustrate that the addition of ironic citrate can be improved
The chloromethylation degree of D3520；Test group and the polysaccharide recovery of control group 1, pigment removal and albumen removal rate are higher than control
Group 2, and the polysaccharide recovery of test group, pigment removal and albumen removal rate are above control group 1 and control group 2, explanation
The chloromethylation of D3520 is conducive to the recycling of polysaccharide, good to the removal effect of pigment and albumen, and the chloromethane of D3520-Cl
Base degree is high, better effect.
Routine operation in operating procedure of the invention is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention,
Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.
Claims (9)
1. the extracting method of sargassum fusifome anti-aging polysaccharide, including the preparation of brown alga Thick many candies extracting solution, macroporous resin purification, ion friendship
Change chromatography, dialysis and gel chromatography column purification, it is characterised in that: the macroporous resin purification step are as follows: by anti-aging Thick many candies
Loading to macroreticular resin D3520-Cl column is purified after preparing solution, is eluted with deionized water, by the eluent of collection
It is concentrated under reduced pressure into viscous pasty state, then adds ethanol solution into concentrate, is uniformly mixed, precipitates overnight, is centrifuged, it is dry, it is spare.
2. the extracting method of sargassum fusifome anti-aging polysaccharide according to claim 1, it is characterised in that: the anti-ageing uneducated person is more
Sugar juice is deployed into the solution of concentration 3-5mg/mL.
3. the extracting method of sargassum fusifome anti-aging polysaccharide according to claim 1, it is characterised in that: the D3520-Cl's
Preparation step are as follows: macroreticular resin D3520 ethanol solution is swollen 5-6h, then cleans 2-4 all over rear natural wind with ethanol solution
It is dry, be then 1:3.5-4.0(g/mL by solid-to-liquid ratio) D3520 is placed in 60-70 DEG C of CCl4Middle swelling 30-40h, adds
ZnCl2, ironic citrate, NaCl and chloromethyl ether, react 21- under conditions of temperature is 45-55 DEG C, ultrasonic power is 70-80W
26h is to get D3520-Cl.
4. the extracting method of sargassum fusifome anti-aging polysaccharide according to claim 3, it is characterised in that: the D3520,
ZnCl2, NaCl and chloromethyl ether weight ratio be 1:1.3-1.5:0.40-0.42:0.15-0.17, the ZnCl2And ironic citrate
Molar ratio is 1:0.12-0.13.
5. the extracting method of sargassum fusifome anti-aging polysaccharide according to claim 1, it is characterised in that: the anti-ageing uneducated person is more
Sugared preparation step are as follows: by Sangassivm fuciforime (Harv) Setch powder using the ethanol solution reflux 1.5-2.5h that concentration is 93-97%, reflux 2-4 times is repeated,
Then it is added 28-33 times and measures ‰ calcium lactate of distilled water and Sangassivm fuciforime (Harv) Setch powder weight 0.03-0.05, in 80-90 DEG C, power 120-
Ultrasound assisted extraction 2.5-3.5h under 170W, filtering, filtrate is anti-aging Thick many candies solution.
6. the extracting method of sargassum fusifome anti-aging polysaccharide according to claim 5, it is characterised in that: L- in the calcium lactate
The ratio of calcium lactate and D-ALPHA-Hydroxypropionic acid calcium is 100:6.3-7.5.
7. the extracting method of sargassum fusifome anti-aging polysaccharide according to claim 1, it is characterised in that: the ion exchange layer
Analyse step are as follows: the polysaccharide solution loading after macroporous resin purification to DEAE Sepharose CL-6B column is subjected to ion exchange layer
Analysis purifying, carries out gradient with the sodium chloride solution of 0mol/L, 0.1mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L respectively and washes
De-, the sodium chloride solution elution volume of various concentration is 3-4BV, and the flow velocity of elution is arranged to 0.7-0.8mL/min, using benzene
Phenol-sulfuric acid method tracing detection polyoses content, sample are drawn elution curve, are received respectively until not having SFPS that can detect
Collect the eluent in each eluting peak section.
8. the extracting method of sargassum fusifome anti-aging polysaccharide according to claim 1, it is characterised in that: the dialysis step
Are as follows: it by the eluent after ion-exchange chromatography with molecular cut off is the dialysis of 3000-4000Da bag filter, dialyzate freezing is dry
It is dry.
9. the extracting method of sargassum fusifome anti-aging polysaccharide according to claim 1, it is characterised in that: the gel chromatographic columns
Purification step are as follows: the polysaccharide crude that dialysis obtains is made into the solution of concentration 38-42mg/mL, respectively loading to Sephadex G-
200 gel columns are further purified, and are eluted with distilled water, by ultraviolet-visible spectrophotometer follow-up inspection its in 480nm
The absorbance at place draws elution curve using the volume of eluent and absorbance as transverse and longitudinal coordinate respectively, collects each eluting peak region
Eluent, select the best component of antiageing effect, be concentrated into small size, be freeze-dried to get anti-aging polysaccharide.
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|---|---|---|---|---|
| CN109369817A (en) * | 2018-09-11 | 2019-02-22 | 浦江县昂宝生物技术有限公司 | A kind of purification process of sargassum fusifome anti-aging polysaccharide |
| CN113519702A (en) * | 2021-07-17 | 2021-10-22 | 杭州吾尾科技有限公司 | Preparation of carrageen fucoidan and application of carrageen fucoidan in dog and cat foods |
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2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109369817A (en) * | 2018-09-11 | 2019-02-22 | 浦江县昂宝生物技术有限公司 | A kind of purification process of sargassum fusifome anti-aging polysaccharide |
| CN113519702A (en) * | 2021-07-17 | 2021-10-22 | 杭州吾尾科技有限公司 | Preparation of carrageen fucoidan and application of carrageen fucoidan in dog and cat foods |
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Application publication date: 20190222 |