CN110357983A - A kind of preparation method of sea cucumber fucoidan and chondroitin sulfate oligosaccharide - Google Patents
A kind of preparation method of sea cucumber fucoidan and chondroitin sulfate oligosaccharide Download PDFInfo
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- CN110357983A CN110357983A CN201910750448.8A CN201910750448A CN110357983A CN 110357983 A CN110357983 A CN 110357983A CN 201910750448 A CN201910750448 A CN 201910750448A CN 110357983 A CN110357983 A CN 110357983A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
Abstract
The present invention provides preparation and the isolation and purification methods of a kind of sea cucumber fucoidan oligosaccharide and chondroitin sulfate oligosaccharide, the sea cucumber sulfated polysaccharide mixture extracted from sea cucumber is raw material, adjusting sea cucumber sulfated polysaccharide solution ph is 5.0-6.0, use 110-115 DEG C of high-pressure steam heating degradation 60-90min, so that fucoidan is first degraded to oligosaccharide, go out fucoidan oligosaccharide using ultra-filtration and separation.Then it is 3.0-4.0 that the ultra-filter retentate by molecular weight greater than 5kDa, which adjusts pH value, and using 120-130 DEG C of thermal degradation 90-120min, concentrated frozen is dry, and sea cucumber fucosido chondroitin sulfate oligosaccharide is made.The method of the present invention saves the ion chromatographic separation of fucoidan and chondroitin sulfate in sea cucumber, gel chromatography purifying and desalination, it realizes the degradation of sea cucumber fucoidan and chondroitin sulfate and isolates and purifies step completion, enormously simplify preparation process.
Description
Technical field
The invention belongs to small molecule compound preparation technical fields, and in particular to a kind of sea cucumber fucoidan and sulphur
The preparation method of aching and limp ossein oligosaccharide.
Background technique
Sea cucumber belongs to Echinodermata Holothuroidea, is a kind of precious marine product with important nutrition and economic value.Sea cucumber
There are two kinds of sulfated polysaccharides of fucoidan and fucosido chondroitin sulfate in body wall, wherein fucoidan is
The straight chain sulfated polysaccharide that fucose is formed with α 1 → 3 or α 1 → 4 key connection, has a small amount of fucose side chain.Sea cucumber fucosido
Chondroitin sulfate is glucuronic acid and acetylamino galactosamine is the glycosaminoglycan for repeating disaccharide unit, with β-Isosorbide-5-Nitrae-and β -1,
3 glucosides key connections form, and have the fucose branch of sulphation on the acetylamino galactosamine and glucuronic acid of chondroitin sulfate,
This is the unexistent structure of the chondroitin sulfate of other animals.Root according to research reports, fucoidan and sulphur in sea cucumber
Aching and limp ossein has good antithrombotic, antiviral and antitumor, antithrombotic, anti-oxidant, raising immunity, protection intestinal mucosa screen
The bioactivity such as barrier, are active materials important in sea cucumber.
But the fucoidan and chondroitin sulfate extracted from sea cucumber has high molecular weight and high charge structure,
Anticoagulant active is high, and bioavilability is low when taking orally, therefore limits it in the application of field of medicaments.And the biology benefit of oligosaccharide
Expenditure is higher, and anticoagulant active is low, has better application prospect.Therefore, preparation sea cucumber sulfate oligosaccharide is to improve sea cucumber sulphur
The important way of acid polysaccharide bioactivity.
The main biodegrading process of sea cucumber sulfated polysaccharide has enzymatic hydrolysis, acid degradation, free radical cracking, nuclear radiation, ultrasound polyethylene-reducing at present
Solution etc..Because lacking the single-minded degrading enzyme of commercialization, at present mainly using acid degradation and free-radical oxidation degradation preparation low molecular weight
Sea cucumber sulfated polysaccharide, but it is generally existing more using soda acid and mantoquita, and chemical contamination is serious, imurity-removal, complex steps.And
Nuclear radiation, sonication the high requirements on the equipment, product desulfurization phenomenon are serious.And high-pressure steam method is in 100-140 DEG C of high temperature
Under can degrade seaweed, sea cucumber sulphur fucoidan and sea cucumber chondroitin sulfate.When by control degradation temperature and degradation
Between, the sulfated polysaccharide of different molecular weight can be prepared.This method does not add any chemical reagents, does not need desalination and removing heavy-metal
Processing, therefore there is green and controllable advantage.But sea cucumber sulfuric acid fucosan and chondroitin sulfate need at 126 DEG C of high temperature
480min is managed, oligosaccharide could be obtained.The pyroreaction time is too long, influences greatly on the sulfate radical content of sulfated polysaccharide and structure,
And the rate of recovery is relatively low.And these methods are to two kinds of sulfated polysaccharides of fucoidan in sea cucumber and chondroitin sulfate
Degradation without selectivity, cannot achieve the separation of two kinds of sulfated polysaccharides.
Fucoidan and chondroitin sulfate are two kinds of structures sulfated polysaccharides different with bioactivity in sea cucumber, because
This needs isolates and purifies.Isolating and purifying mainly for sea cucumber sulfated polysaccharide has alcohol grading separation, the separation of salinity grade, ion to hand at present
Change chromatographic isolation, gel chromatography separation.Alcohol grading separation separates sea cucumber sulfated polysaccharide with salinity grade, and separative efficiency is relatively low,
Product purity is low.And exclusion chromatography it is per treatment sample size it is too low, cannot achieve large-scale production.Ion-exchange chromatography
Sea cucumber fucoidan and chondroitin sulfate can be separated efficiently, in large quantities, and product purity is high.But ion-exchange chromatography
Column is at high cost, and product desalting steps are cumbersome.
Summary of the invention
For the deficiency for the technology that sea cucumber sulfated polysaccharide isolates and purifies and degrades in the prior art, the present invention passes through control pH
Value and degradation temperature, successively degrade in two steps sea cucumber fucoidan and chondroitin sulfate, in combination with Ultra filtration membrane
Technology realizes isolating and purifying for fucoidan and chondroitin sulfate oligosaccharide, obtain respectively fucoidan and
Chondroitin sulfate oligosaccharide sterling.
The preparation method of a kind of sea cucumber fucoidan and chondroitin sulfate oligosaccharide provided by the present invention, including
Following step:
1) sea cucumber sulfated polysaccharide mixture is configured to the aqueous solution that concentration is 0.05-2%, stirred evenly；Then it uses
The pH value that acetum adjusts sea cucumber polysaccharide aqueous solution is 5.0-6.0；
2) by the sea cucumber polysaccharide aqueous solution of step 1) preparation in 110-115 DEG C of compressed steam degradation 60-90min, cooling
Solution is to room temperature；
A kind of its concrete operations is in high-pressure sterilizing pot or reaction kettle carry out, and heating rate is in 10min by room temperature
It is warming up to 110-115 DEG C；Then 110-115 DEG C of degradation 60-90min is kept；After degradation, temperature is dropped to often in 20min
Temperature；
3) it uses molecular cut off to carry out ultra-filtration and separation for degradation solution of the ultrafiltration membrane of 5kDa to step 2), collects divide respectively
Ultrafiltrate of the son amount less than 5kDa and molecular weight are greater than 5kDa ultra-filter retentate.Molecular weight is taken to be less than the ultrafiltrate of 5kDa,
Vacuum concentration, obtains fucoidan oligosaccharide dry product after freeze-drying；
4) by after the ultra-filter retentate vacuum concentration in step 3), adjusting pH value using acetum is 3.0-4.0, then at
Degrade 90-120min in 120-130 DEG C of high-pressure steam；Degradation solution vacuum concentration, it is low to obtain chondroitin sulfate after freeze-drying
Glycan dry product；
It degrades in the high-pressure steam, is the heating rate 10min in high-pressure sterilizing pot or reaction kettle carry out
It is interior to be warming up to 120-130 DEG C by room temperature.Then 120-130 DEG C of degradation 90-120min is kept.It is warm in 20min after degradation
Degree drops to room temperature.
Acetum used in above-mentioned steps is the consumption level of acetic acid solution that concentration is 0.5-4mol/L.
The method of the present invention saves the ion chromatographic separation of fucoidan and chondroitin sulfate in sea cucumber, gel color
Spectrum purifying and desalination enormously simplify the separation and degradation work of fucoidan and chondroitin sulfate in sea cucumber
Skill.This method does not add any chemical reagents, with low in cost, easy to operate, the rate of recovery is high.Thermal degradation temperature is relatively low,
Heating time is short, therefore oligosaccharide and raw sugar structure having the same, and sulfate radical and monosaccharide composition are held essentially constant, molecular weight
Compare concentration, absorptivity greatly improves.This method is a kind of environmentally protective preparation and purification sea cucumber sulfate oligosaccharide sterling
Method.
Detailed description of the invention
Fig. 1: the high performance size exclusion chromatography analysis chart of sea cucumber fucoidan oligosaccharide,
Fig. 2: the high performance size exclusion chromatography analysis chart of sea cucumber chondroitin sulfate oligosaccharide；
Fig. 3: the high-efficient liquid phase chromatogram of the small monosaccharide composition analysis for having stichopus japonicus fucoidan oligosaccharide；
Fig. 4: the high-efficient liquid phase chromatogram of the small monosaccharide composition analysis for having stichopus japonicus fucosido chondroitin sulfate oligosaccharide；
Fig. 5: the total ion current figure of the small liquid chromatography-mass spectrometry for having stichopus japonicus fucoidan oligosaccharide；
Fig. 6: the small mass spectrogram for having stichopus japonicus fucoidan oligosaccharide on Q-E mass spectrum.
Fig. 7: the total ion current figure of the small liquid chromatography-mass spectrometry for having stichopus japonicus chondroitin sulfate oligosaccharide；
Fig. 8: the small mass spectrogram for having stichopus japonicus chondroitin sulfate oligosaccharide on Q-E mass spectrum.
Specific embodiment
The present invention provides it is a kind of using gradient thermal degradation method prepare and separate sea cucumber fucoidan oligosaccharide and
The method of fucosido chondroitin sulfate oligosaccharide.By control solution in the slightly acidic environment of pH3.0-5.0, make sea cucumber rock algae
Glycan sulfuric ester and chondroitin sulfate generate sulfate oligosaccharide with regard to fast degradation at 110-120 DEG C.And sea cucumber fucosan sulphur
1 → 4 key glycosidic bond thermal stability of α 1 → 3 and α of acid esters is poor, than β-Isosorbide-5-Nitrae-in chondroitin sulfate and β -1, the more appearance of 3 glycosidic bonds
It is degradable.
Embodiment 1
1) drying small to there is stichopus japonicus dried sea-cucumber to be pulverized into powder state.10g powder is weighed, 15mmol/L EDTA- is added
Na2,5mmol/L cysteine, the phosphate buffer solution of 300mL 0.1mol/L pH 8.0,500mg alkali protease and
150mg trypsase, 50 DEG C of stirring in water bath enzymatic hydrolysis are for 24 hours.It is later that reactant boiling water bath 5min enzyme deactivation is living, it is cooled to room temperature.From
The heart 5000 × g, 10min, take supernatant.
It is 10% cetylpyridinium chloride water that 16mL mass concentration will be added in gained supernatant (i.e. sea cucumber hydrolysate)
Solution is stored at room temperature 5h, is centrifuged 2000 × g, 15min, 20 DEG C, abandons supernatant, must precipitate.
By dissolution in the resulting ethanol solution (3mol/L sodium chloride, 86% ethyl alcohol) for being precipitated and dissolved in 150mL NaCl.
Then 95% ethyl alcohol of 300mL is added, 5h is placed at 4 DEG C, is centrifuged (4000 × g, 15min, 20 DEG C), takes precipitating.Precipitating is used
95% ethanol washing of 100mL 2 times, obtains sea cucumber sulfated polysaccharide.
2) the small aqueous solution for having stichopus japonicus sulfated polysaccharide to be made 1% adjusts the pH value of polysaccharide solution using 2mol/L acetic acid
It is 5.0.
3) 10mL polysaccharide solution is taken, is put into high-pressure sterilizing pot, degrade 60min in 110 DEG C of high-pressure steams, stops
Heating, degradation solution are cooled to room temperature.
4) it uses molecular cut off to carry out ultra-filtration and separation to degradation solution for the ultrafiltration membrane of 5kDa, collects molecular weight respectively and be less than
The ultrafiltrate of 5kDa, vacuum concentration, obtains fucoidan oligosaccharide dry product after freeze-drying.
5) ultra-filter retentate greater than 5kDa is taken, is concentrated into 1%, is 3.0 with 2mol/L vinegar acid for adjusting pH value.Take 10mL molten
Liquid is put into high-pressure sterilizing pot, and degrade 90min in 120 DEG C of high-pressure steams.Degradation solution vacuum concentration, freeze-drying obtain
Fucosido chondroitin sulfate oligosaccharide dry product.
The oligosaccharide dry product of above-mentioned fucoidan dry product and fucosido chondroitin sulfate is weighed, rock algae is calculated
The yield of glycan sulfuric ester oligosaccharide is 28.5%, the yield 50.2% of fucosido chondroitin sulfate oligosaccharide.
It is soft that fucoidan and fucosido sulfuric acid manufactured in the present embodiment are detected using high performance size exclusion chromatography
The molecular weight of ossein.The weight average molecular weight 4262.25Da of fucoidan oligosaccharide, number-average molecular weight 2547.67Da, point
Dissipating coefficient is 1.67 (Fig. 1).The equal 4357.43Da of weight of fucosido chondroitin sulfate oligosaccharide, number-average molecular weight 2485.41,
The coefficient of dispersion 1.75 (Fig. 2).
Using the sulfate radical content of the oligosaccharide of sulfate by ion chromatography preparation, ion-exchange chromatography is used in the prior art
The sea cucumber fucoidan and sodium sulphate chondroitin that method isolates and purifies, sulfate radical content are 28.87% He respectively
32.46%.The fucoidan oligosaccharide of this method preparation and the sulfate radical content difference of sodium sulphate chondroitin oligosaccharide
For 29.41% and 33.34%, illustrate the loss for not having sulfate radical in preparation process.
Fig. 3, Fig. 4 and table 1 are the monosaccharide composition that oligosaccharide is detected using PMP monosaccharide derivative-high performance liquid chromatography.Preparation
Fucoidan oligosaccharide contain 77.28% fucose and 10.79% galactolipin, contain a small amount of amido glucose, ammonia
Base galactolipin, glucuronic acid and glucose, monosaccharide composition are not significantly different with original fucoidan.Explanation
That obtain is sulfuric acid fucoidan oligosaccharide (Fig. 3)；
The monosaccharide of the chondroitin sulfate oligosaccharide of preparation mainly contains fucose 35.35%, amine-galactose 23.6% and Portugal
Grape uronic acid 18.79% forms (Fig. 4) close with original chondroitin sulfate.
Table 1: the comparison of the monosaccharide of sea cucumber fucoidan and fucosido chondroitin sulfate and its oligosaccharide composition
Table
Total monosaccharide content is 100%.Man: mannose；GlcNAc: Glucosamine；GlcA: glucuronic acid；
GalNAc: amine-galactose；Glc: glucose；Gal: galactolipin；Fuc: fucose
Embodiment 2
For specific verification step 3) and 5) in the fucoidan and rock algae of sample volume and degradation time to preparation
The influence of glycosyl chondroitin sulfate oligosaccharide, change step 3) and 5) in sample volume and degradation time, remaining is the same as embodiment 1.
The yield of specific sample volume, degradation time and corresponding fucoidan and fucosido chondroitin sulfate oligosaccharide,
Molecular weight and sulfate radical content are as shown in table 2.
2.1: sea cucumber sulfated polysaccharide liquor capacity being increased into 100mL and carries out thermal degradation, the fucoidan received
The yield of oligosaccharide is 21.2%, the yield 43.2% of fucosido chondroitin sulfate oligosaccharide.Fucoidan is oligomeric
The weight average molecular weight 4563.64Da of sugar, number-average molecular weight 3213.83Da, the coefficient of dispersion 1.42.Fucosido chondroitin sulfate
The weight of oligosaccharide equal 4243.57Da, number-average molecular weight 3082.94Da, the coefficient of dispersion 1.37 illustrate the volume for increasing degradation solution,
So that the degradation speed of sea cucumber sulfated polysaccharide declines, the molecular weight of the oligosaccharide of acquisition increases for meeting.Sulfate radical content detection proof,
The sulfate radical content of fucoidan oligosaccharide and sodium sulphate chondroitin oligosaccharide is respectively 28.34% and 32.05%, with
Raw sugar sulfate radical content (fucoidan 28.87% and sodium sulphate chondroitin 32.46%) is not significantly different.
2.2: taking 10mL sea cucumber sulfated polysaccharide aqueous solution that degradation time is extended to 90min and 120min respectively.It can by table 2
Know, the yield of the fucoidan oligosaccharide of acquisition is 26.7%, again respectively 3726.46Da, number-average molecular weight
2240.26Da, the coefficient of dispersion 1.66.The yield 47.6% of chondroitin oligosaccharide, weight average molecular weight 3849.33Da, the equal molecule of number
Measure 2641Da, the coefficient of dispersion 1.45.Sulfate radical content illustrates the time for extending thermal degradation, can make the drop of sea cucumber sulfated polysaccharide
It solves speed to accelerate, the molecular weight of the oligosaccharide of acquisition reduces.Detection proof, fucoidan oligosaccharide and sodium sulphate cartilage
The sulfate radical content of plain oligosaccharide is respectively 29.25% and 31.07%, is not significantly different with the sulfate radical content of raw sugar.
Table 2: the influence of different thermal degradation volumes and time to fucoidan and chondroitin sulfate oligosaccharide
Embodiment 3
In order to verify the purity of prepared fucoidan oligosaccharide and fucosido chondroitin sulfate oligosaccharide,
Using liquid chromatography-mass spectrometry, fucoidan oligosaccharide and fucosido chondroitin sulfate oligosaccharide are analyzed
Composition.
1) LC-MS analysis mode is as follows: 10mg/mL sulfated polysaccharide catabolite is mixed in equal volume with acetonitrile
It closes, is then centrifuged 10min at 10 000r/min.Supernatant is taken, is analyzed and is produced using high performance liquid chromatography-mass spectrometry technology
The Nomenclature Composition and Structure of Complexes of oligosaccharides in object.Chromatograph: 3000 type high performance liquid chromatograph of UltiMate, liquid-phase condition: chromatographic column is
3 μ HILIC of Luna (2.0mm × 150mm,), mobile phase A is that 5mmol/L ammonium acetate aqueous solution 10mL sea cucumber sulfuric acid is more
Sugared pH5 thermal degradation 90min, Mobile phase B are 98% acetonitrile of volume fraction (dissolution of 5mmol/L ammonium acetate solution).Time gradient: 0-
15-40min, concentration gradient: the wash-out concentration 2%-15%-40% of mobile phase A.Mass Spectrometry Conditions: mass spectrometer is selected
Thermo Scientific Q-ExactiveTMMass spectrum, using negative ion mode, electrospray ionization source, mass analyzer is
Orbitrap- Fourier transform, negative ion electrospray force mode, capillary voltage are -40V, t sleeve lens offset voltage: -50V, hair
Capillary temperature is set as 320, and laminar flow amount is 35.The flow for assisting gas is 10.Scanning charge-mass ratio 1-5min is 100-800u, 5-
40min is 300-1500u, resolution ratio 70 000, handles data in Xcalibur mass spectrum software.
2) Fig. 5 is total ion of the liquid chromatography-mass spectrometry of the small fucoidan oligosaccharide for having stichopus japonicus
Flow graph.According to the charge-mass ratio and MASS SPECTRAL DATA ANALYSIS of oligosaccharide, the extraction ion chromatography of 33 sulfuric acid rock algae oligosaccharide is parsed
Figure, calculates the total ionic strength of each oligosaccharides ion.Table 3 is sugar chain composition, the parent ion, electricity of 33 sulfuric acid rock algae oligosaccharide
Lotus number and ionic strength.Fig. 7 is the small liquid chromatography-mass spectrography for having representative sulfuric acid rock algae monosaccharide, disaccharides and tetrose in stichopus japonicus
Analysis chart and mass spectrogram.Liquid chromatography-mass spectrometry result illustrates, mainly contains 1- in fucoidan oligosaccharide
The rock algae monosaccharide (Fuc) of 2 sulfate radicals1(SO4)1-2, the rock algae disaccharides (Fuc) of 1-3 sulfate radical2(SO4)1-3, 1-3 sulfuric acid
The rock algae trisaccharide (Fuc) of root3(SO4)1-3, the rock algae tetrose (Fuc) of 1-4 sulfate radical4(SO4)1-4, 1-6 sulfate radical rock algae
Pentasaccharides (Fuc)5(SO4)1-6, the rock algae six of 1-7 sulfate radical it is sugared (Fuc)6(SO4)1-7, 2-7 sulfate radical seven sugar of rock algae
(Fuc)7(SO4)2-7.As a result illustrate, the mixture for the sulfuric acid rock algae oligosaccharide that catabolite difference sulfate radical replaces is in thermal degradation
In the process, the sulfate radical of fucoidan does not fall off.
The small charge-mass ratio for having stichopus japonicus fucoidan oligosaccharide of table 3:Thermo Q-E mass spectral analysis, charge number and total
Ionic strength.
Note: F: fucose；S: sulfate radical.
3) in order to determine that the fucosido chondroitin sulfate oligosaccharide of preparation derives from sea cucumber fucosido chondroitin sulfate,
Utilize the composition of liquid chromatography-mass spectrometry fucosido chondroitin sulfate oligosaccharide.Table 4 has stichopus japonicus sulfuric acid soft to be small
Charge-mass ratio, charge number, the ionic strength of ossein oligosaccharide.Fig. 8 is the liquid chromatograph mass spectrography point of anax on vascular endothelial sulfated oligosaccharide
The total ion current figure of analysis.Fig. 8 is the small LC-MS analysis figure for having representative oligosaccharides in stichopus japonicus chondroitin sulfate oligosaccharide
And mass spectrogram.Liquid chromatography-mass spectrometry result explanation, disaccharides (GalNAc) in catabolite1(Fuc)1(SO4)3Content
It is very high, illustrate that fucose is connected on acetylamino galactosamine, illustrates this disaccharides from fucosido chondroitin sulfate.1-
The disaccharides (GalNAc) of 3 sulfuric acid1(GlcA)1(SO4)1-3, 1-4 sulfate radical trisaccharide (GalNAc)1(GlcA)1(Fuc)1
(SO4)1-4(GalNAc)1(GlcA)2(Fuc)1(SO4)2-4With tetrose (GalNAc)2(GlcA)1(Fuc)1(SO4)2-6Presence,
Product oligomeric sugar still maintains the structure of fucosido chondroitin sulfate, and sulfate radical does not fall off.Oligosaccharide composition illustrates to produce
Object is degraded from sea cucumber fucosido chondroitin sulfate high-temperature pressurizing.
The small charge-mass ratio for having stichopus japonicus chondroitin sulfate oligosaccharide of table 4:Thermo Q-E mass spectral analysis, charge number and total ion
Intensity.
Note: N: amido galactolipin；A: glucuronic acid；F: fucose；S: sulfate radical.
Claims (5)
1. a kind of preparation method of sea cucumber fucoidan and chondroitin sulfate oligosaccharide, which is characterized in that the side
Method comprises the following steps that
1) sea cucumber sulfated polysaccharide mixture is configured to aqueous solution, stirred evenly；Then sea cucumber polysaccharide is adjusted using acetum
The pH value of aqueous solution is 5.0-6.0；
2) by the sea cucumber polysaccharide aqueous solution of step 1) preparation in 110-115 DEG C of compressed steam degradation 60-90min, cooling solution
To room temperature；
3) it uses molecular cut off to carry out ultra-filtration and separation for degradation solution of the ultrafiltration membrane of 5kDa to step 2), collects molecular weight respectively
Ultrafiltrate and molecular weight less than 5kDa are greater than 5kDa ultra-filter retentate；Molecular weight is taken to be less than the ultrafiltrate of 5kDa, vacuum
Concentration, obtains fucoidan oligosaccharide dry product after freeze-drying；
4) by after the ultra-filter retentate vacuum concentration in step 3), adjusting pH value using acetum is 3.0-4.0, then at 120-
Degrade 90-120min in 130 DEG C of high-pressure steams；Degradation solution vacuum concentration, obtains chondroitin sulfate oligosaccharide after freeze-drying
Dry product.
2. the method as described in claim 1, which is characterized in that sea cucumber sulfated polysaccharide mixture aqueous solution in the step 1)
Concentration be 0.05-2%.
3. the method as described in claim 1, which is characterized in that the step 2) is that sea cucumber polysaccharide aqueous solution goes out in high pressure
In bacterium pot or reaction kettle, heating rate is to be warming up to 110-115 DEG C by room temperature in 10min；Then 110-115 DEG C of degradation is kept
60-90min；After degradation, room temperature is dropped in 20min.
4. the method as described in claim 1, which is characterized in that it degrades in high-pressure steam described in the step 4),
It is in high-pressure sterilizing pot or reaction kettle carry out, heating rate is to be warming up to 120-130 DEG C by room temperature in 10min, is then protected
Hold 120-130 DEG C of degradation 90-120min；After degradation, temperature drops to room temperature in 20min.
5. the method as described in claim 1, which is characterized in that the acetum is that concentration is the edible of 0.5-4mol/L
Grade acetum.
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| CN110974849A (en) * | 2019-12-16 | 2020-04-10 | 华南理工大学 | Compound sea cucumber extract with hypoglycemic activity and preparation method and application thereof |
| CN111423525A (en) * | 2020-03-23 | 2020-07-17 | 中国海洋大学 | Method for extracting chondroitin sulfate, chondroitin sulfate and application thereof |
| CN111658664A (en) * | 2020-06-18 | 2020-09-15 | 大连工业大学 | Application of sea cucumber polysaccharide in resisting novel coronavirus |
| CN112175107A (en) * | 2020-10-16 | 2021-01-05 | 中国海洋大学 | Method for extracting fucosan sulfate and chondroitin sulfate from sea cucumber |
| CN112458126A (en) * | 2020-11-26 | 2021-03-09 | 中国海洋大学 | Preparation method and application of galactose sulfate compound with anti-inflammatory activity |
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| CN112175107B (en) * | 2020-10-16 | 2021-08-03 | 中国海洋大学 | Method for extracting fucosan sulfate and chondroitin sulfate from sea cucumber |
| CN112458126A (en) * | 2020-11-26 | 2021-03-09 | 中国海洋大学 | Preparation method and application of galactose sulfate compound with anti-inflammatory activity |
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