CN108148145B - A kind of high sulphation keratan sulfate and its preparation method and application - Google Patents
A kind of high sulphation keratan sulfate and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of high sulphation keratan sulfates and its preparation method and application.The present invention provides a kind of high sulphation keratan sulfates (KS) in Shark cartilage source, its molecular weight is 20 ~ 100 kDa, by 6 sulphation galactolipins and 6 sulphation acetylglucosamines with β -1,3 and β -1,4 glycosidic bonds are alternately formed by connecting, secondly two sugared content of sulphation is 80 ~ 95%, there are three sialylated sulfates to replace KS tetrose structure for non-reducing end, it is α -2,3 connections between sialic acid and galactolipin.The present invention further simultaneously discloses the preparation method of the high sulphation keratan sulfate.The polysaccharide, which has, adjusts enteric microorganism activity and immunoregulatory activity.Compound preparation process provided by the invention is at low cost, easy to operate, is easy industrialization production.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of high sulphation keratan sulfate and preparation method thereof
And application.
Background technique
Keratan sulfate (KS) is the mucopolysaccharide that nature is uniquely free of uronic acid, be mainly distributed on various animal corneals,
In the tissue such as cartilage, brain, repeating disaccharide unit skeleton is [→ 3Gal β l → 4GlcNAc β l →].The sulphation modification master of KS
It will be in the position C-6 of galactolipin (Gal) and N-acetylglucosamine (GlcNAc) two saccharide residues and degree is because of animal
Source is different and different, 10 ~ 50KDa of molecular weight ranges.According to the difference of connection type between sugar and protein, KS points are KS
I, KS II and KS III three classes.KS I is originally found in cornea, it is connected to the asparagine of core protein by GlcNAc
On, belong toNConnect glycan；KS II is primarily present in bone, is connected to core by N- acetylamino galactosamine (GalNAc)
The serine or threonine of albumen, belong toOConnect glycan；KS III then mainly has found in brain, is logical by mannose (Man)
It crossesO-It is connected to the serine of core protein.With both at home and abroad to the distribution of KS, chemical structure and physicochemical property etc. research
Deepen continuously, discovery KS has resisting rheumatoid arthritis, inhibits neural cell adhesion and neurite outgrowth, keeps cornea logical
The bioactivity such as permeability have potential medical value.As Chinese patent (publication number: CN102552312A) is disclosed including sulfuric acid
Glycosaminoglycan compound formulation including keratan treats osteoarthritis；Chinese patent (publication number: CN1682755) discloses sulfuric acid
Chondroitin or keratan sulfate and polyvinylpyrrolidone are as active treatments ulcer；Chinese patent (publication No.:
CN101370507 the traumatic nerve as caused by spinal cord injury can be improved by) disclosing keratan sulfate oligosaccharides or derivatives thereof
Disease and/or dyskinesia；Chinese patent (publication No.: CN1174557) prepares keratan sulfate using keratanase II
Oligosaccharides, and disclose its anti-inflammatory, antiallergy, immunological regulation, cell differentiation induction and cell it is apoptosis-induced etc. in effect；
United States Patent (USP) (publication No.: US8557536B2) discloses a kind of concentration evaluation joint by keratan sulfate in blood sample
Cartilage degradation or damage method；Chinese patent (publication No.: CN105732838A) discloses one kind and extracts sulfuric acid angle from egg white
The method of quality.Cornea, articular cartilage are concentrated mainly on using upper to the research of keratan sulfate at present, structural research is less.
Although Chinese patent (publication No.: 103755823 A of CN) discloses the purifying and inspection of keratan sulfate in a kind of chondroitin sulfate
Survey method and Chinese patent (publication No.: CN105777938A) disclose a kind of thick from chondroitin sulfate using chemical method
The method that keratan sulfate is removed in extract, but there is no KS structural information.Two sulphations two in KS structure reported at present
The ratio of sugar is 40 ~ 70%(Amanda Weyers, FEBS Journal, 2013；Li Fu et al.,Glycobiology, 2016), and in KS application aspect, it has no document or patent report KS and is adjusting enteric microorganism and tune
Save the effect of immunology.
Summary of the invention
The present invention provides a kind of high sulphation keratan sulfate and its preparation method and application.The present invention is from Shark cartilage
It is extracted a kind of high sulphation KS of structure novel, its structure is expounded, and further proves the high sulphation KS of gained
To enteric microorganism and it is immunized with good adjustment effect.
For achieving the above object, the present invention is achieved by the following scheme:
The present invention provides a kind of high sulphation keratan sulfate, the high sulphation keratan sulfate is soft from shark
Bone, molecular weight are 20 ~ 100 kDa, and the high sulphation keratan sulfate is by 6 sulphation galactolipins and 6 sulphation acetyl
Glucosamine is alternately formed by connecting with β -1,3 and β-Isosorbide-5-Nitrae glycosidic bond, secondly two sugared content of sulphation is 80 ~ 95%, it is non-reduced
End is three sialylated sulfate substituted sulfuric acid keratan tetroses, is α -2,3 connections, structure between sialic acid and galactolipin
Formula is as follows:
。
Further: the three sialylated sulfates replace KS tetrose, and structure is as follows:
Wherein two substituent Rs in the structural formula1And R2One of them is SO3H, another substituent group are then H.
Further: the Shark cartilage is soft for the joint of shark, rib cage, skull, vertebra, interverbebral disc or tracheae position
Bone.
The present invention also provides the preparation method of the high sulphation keratan sulfate, it the following steps are included:
(1) proteasome degradation: being scattered in enzymatic hydrolysis buffer after Shark cartilage is crushed, papain be then added,
The reaction of 50 ~ 70 DEG C of shaking tables, after boil and make enzyme-deactivating；Add trypsase after cooling, 30 ~ 55 DEG C of shaking tables reactions, after boil
Make enzyme-deactivating, removal insoluble impurities is centrifuged after cooling；
(2) ethanol precipitation: ethyl alcohol is added in the solution obtained to step (1), low temperature is placed 12 ~ 24 hours, after centrifugation
Dialyse after taking precipitating, precipitating to be redissolved with distilled water, be concentrated under reduced pressure, be lyophilized after obtain Shark cartilage mucopolysaccharide component；
(3) Anion exchange resin separation: Shark cartilage mucopolysaccharide component obtained by step (2) is dissolved with distilled water, with
Sodium-chloride water solution is mobile phase linear elution, is purified through Anion exchange resin separation, Phenol-sulphate acid method detection, and distilled water is saturating
It is concentrated under reduced pressure, is lyophilized after analysis, obtain high-purity sulfuric acid keratan；
Or chondroitin enzymatic hydrolysis separation: Shark cartilage mucopolysaccharide component buffer solution obtained by step (2) is added soft
Ossein ABC enzyme, 25 ~ 40 DEG C of shaking tables, which react to boil after 4 ~ 6h, makes enzyme-deactivating, ethyl alcohol is added after centrifugation into supernatant, low temperature is put
It sets 12 ~ 24 hours, precipitating is collected by centrifugation, redissolve Yu Shuihou dialysis desalting, freeze-drying, obtain high-purity sulfuric acid keratan.
Further: the dosage of papain is 10 ~ 100 Ug in the step (1)-1, pH 7.0；The pancreas egg
The dosage of white enzyme is 0.1 ~ 10%, pH 7.5.
It is further: papain being added in the step (1) and the reaction time of trypsase is 8 ~ 12 h.
Further: the filler of anion exchange resin described in the step (3) is Q-Sepharose FF, DEAE-
Sepharose FF, Capto-Q Sepharose FF, DEAE-Cellulose or Capto-DEAE Sepharose FF.
Further: the dosage of Chondroitin A BC enzyme is 300 ~ 600 mUg in the step (3)-1。
The present invention also provides the high sulphation keratan sulfates in the preparation that preparation adjusts enteric microorganism
Using.
The present invention also provides application of the high sulphation keratan sulfate in the preparation of preparation strengthen immunity.
Compared with prior art, the advantages and positive effects of the present invention are:
(1) keratan sulfate preparation process provided by the invention is at low cost, easy to operate, is easy industrialization production.
(2) keratan sulfate product purity prepared by the present invention is high, and molecular weight is 20 ~ 100 kDa, the polysaccharide mainly by
With β -1,3 and β-Isosorbide-5-Nitrae glycosidic bond is alternately formed by connecting for 6 sulphation galactolipins and 6 sulphation acetylglucosamines, secondly
Two sugared content of sulphation is 80 ~ 95%, and there are three sialylated sulfates to replace KS tetrose, sialic acid and gala for non-reducing end
It is α -2,3 connection between sugar.
(3) high sulphation keratan sulfate provided by the invention has good adjustment effect to enteric microorganism.It can change
Become the structure of enteric microorganism in female mice and male mouse body, hence it is evident that increase the abundance of Bacillus acidi lactici in male and female mouse body, and this
Change closely related with gender.
(4) high sulphation keratan sulfate provided by the invention has preferable enhancing immunocompetence.
After a specific embodiment of the invention is read in conjunction with the figure, further advantage of the invention and feature will become more clear
It is clear.
Detailed description of the invention
Fig. 1 is the High Performance Gel Permeation chromatogram of high sulphation keratan sulfate of the present invention.
Fig. 2 is the one-dimensional nuclear magnetic resonance hydrogen spectrogram of high sulphation keratan sulfate of the present invention.
Fig. 3 is the one-dimensional nuclear magnetic resonance carbon spectrogram of high sulphation keratan sulfate of the present invention.
Fig. 4 is high sulphation keratan sulfate enzymolysis product HILIC-UPLC-FT-MS analysis chart of the present invention.
Fig. 5 is the two dimension that three sialylated sulfates replace KS tetrose1H-13C HMBC (A) and1H-1H COSY (B)
Spectrogram.
Fig. 6 is that high sulphation keratan sulfate of the present invention changes enteric microorganism structure thermal map in Mice Body.
Fig. 7 is that high sulphation keratan sulfate of the present invention adjusts Bacillus acidi lactici figure in Mice Body.
Fig. 8 is that high sulphation keratan sulfate of the present invention promotees cell phagocytosis dimethyl diaminophenazine chloride figure.
Specific embodiment
Technical solution of the present invention is further described in detail with reference to the accompanying drawings and detailed description, but the present invention
Claimed range is not limited to the range of example statement.
Embodiment 1: the preparation of high sulphation keratan sulfate
The preparation method of high sulphation keratan sulfate of the present invention the following steps are included:
(1) proteasome degradation: PBS(phosphate-buffered is scattered in after Shark cartilage (buying from commercially available company) is crushed
Liquid) in, papain is then added, the Papain enzyme dosage (E/S) is 10 ~ 100 Ug-1, pH 7.0,50 ~ 70
DEG C shaking table, which reacts to boil after 8 ~ 12 h, makes enzyme-deactivating；Trypsase is added after cooling, the trypsase dosage (E/S) is
0.1 ~ 10%, pH 7.5.Boiling after 30 ~ 55 DEG C of 8 ~ 12 h of shaking tables reaction makes enzyme-deactivating, and it is insoluble miscellaneous to be centrifuged removal after cooling
Matter.
(2) ethyl alcohol of 2 ~ 10 times of volumetric concentration >=95%, 4 DEG C of placements ethanol precipitation: are added into step (1) solution
It 12 ~ 24 hours, dialyses after taking precipitating, precipitating to be redissolved after centrifugation with distilled water, to obtain Shark cartilage viscous after being concentrated under reduced pressure, being lyophilized
Polysaccharide component.
(3) Anion exchange resin separation: 1 g of Shark cartilage mucopolysaccharide component obtained by step (2) is distilled with 4 mL
Water dissolution, with 0 ~ 3.0 molL-1Sodium-chloride water solution is mobile phase linear elution, is purified through Anion exchange resin separation,
It collects after component distilation water is dialysed and is concentrated under reduced pressure, is lyophilized, obtain high-purity sulfuric acid keratan.
Chondroitin enzymatic hydrolysis separation: 1 g of Shark cartilage mucopolysaccharide component enzymatic hydrolysis buffer solution obtained by step (2) adds
Enter Chondroitin A BC enzyme (300 ~ 600 mUg-1), 25 ~ 40 DEG C of shaking tables, which react to boil after 4 ~ 6h, makes enzyme-deactivating, Xiang Shangqing after centrifugation
The middle ethyl alcohol that 2 ~ 10 times of volumetric concentration >=95% are added, 4 DEG C are placed 12 ~ 24 hours, and precipitating is collected by centrifugation, and redissolve Yu Shuihou
Dialysis desalting, freeze-drying obtain high-purity sulfuric acid keratan.
Embodiment 2: the structural characterization of high sulphation keratan sulfate
(1) it absolute molecular weight measurement and purity analysis: is dissipated using High Performance Gel Permeation chromatography (HPGPC)-multi-angle laser
Penetrate absolute molecular quality and purity that method (MALLS) combination method measures the high sulphation keratan sulfate.
By 0.1 molL of the high sulphation keratan sulfate sample obtained-1 Na2SO4Dissolution is configured to 5 mg
mL-1Aqueous solution.Chromatographiccondition: chromatographic column is Shodex OHpak SB-804 HQ and Shodex OHpak SB-802.5
HQ is used in series, and mobile phase is 0.1 molL-1 Na2SO4Aqueous solution, sample volume are 100 μ L, and column temperature is 35 DEG C, and flow velocity is
0.6 mL·min-1, acquisition time 45min, detector is that Composition distribution and multiple angle laser light scattering instrument are combined.Experiment knot
Fruit is as shown in Figure 1, single symmetrical peak is presented in sample, and molecular weight is between 20 ~ 100kDa.
(2) spectral analysis of the nuclear magnetic resonance: nuclear magnetic tube is gone to after taking 30 mg high sulphation keratan sulfates to be exchanged with heavy water
In, using deuterated acetone as internal standard, analyzed using 600MHz nuclear magnetic resonance chemical analyser.From such as Fig. 2,3 it is found that high sulphation sulfuric acid angle
Quality purity is very high, without albumen and nucleic acid and other impurities, and can find that there are sialic acid (1.74ppm) signals in molecule
Peak.At it1In H-NMR, 2.05 ppm are the absorption peaks of methyl hydrogen in GlcNAc acetyl group, are saccharide rings between 3.5 ~ 4.5 ppm
Upper each hydrogen signal；4.66 and 4.48 ppm are respectively the anomeric proton absorption peak of GlcNAc and Gal.?13In C-NMR, 22.07
The peak ppm is the absorption peak of methyl in acetyl group on the position GlcNAc C-2, and 102.83 ppm or so are the different heads of Gal and GlcNAc
Carbon absorption peak, 82.06ppm and 78.72 ppm are the absorption peak of glucosides key position respectively；67.66ppm and 66.43 ppm difference
It is the C6 absorption peak of Gal6S and GlcNAc6S.In addition, in high field area, it was found that 1.74ppm and 2.68ppm sialic acid C3
Two hydrogen signals on position.
(3) HILIC-UPLC-FTMS is analyzed: the high sulphation keratan sulfate carries out HILIC- after thoroughly degrading
UPLC-FTMS analysis.Chromatographic condition: chromatographic column: Phenomenex Luna HILIC 200 (150 × 2.00 mm, 3 μm), stream
Dynamic phase: A: acetonitrile (contains 5 mM ammonium acetates), B:5 mM ammonium acetate aqueous solution, and 25 DEG C of column temperature, 0.15 mLmin of flow velocity-1, wash
De- gradient: 0 ~ 58 min, 92 ~ 60% A; 58~60 min, 60~30% A; 60~70 min, 30% A; 70~71 min,
30~92% A; 71~90 min, 92% A.As a result as shown in figure 4, main disaccharide unit is the disaccharides of two sulphations in KS,
Content is 80 ~ 95%, it has been found that the presence of sialylated disaccharides and tetrose further demonstrates NMR spectrum result.
Embodiment 3: the preparation of three sialylated sulfate substituted sulfuric acid keratan tetroses and structural characterization
(1) 200 mg high sulphation keratan sulfate samples is taken to be dissolved in 20 mL buffers (0.05 mol/L ammonium acetate
With 2 mmol/L calcium chloride) in, 100 mU Keratanase II are added, 37 DEG C hydrolyze in 10 Kd ultra-filtration centrifuge tubes.Every
5 h or so are centrifugated oligosaccharides, and constantly add sample and enzyme.Then isolated oligosaccharides Bio-Gel P6 is splined on to coagulate
Glue penetration chromatographic column, with 0.2 mol/L NH4HCO3Elution, flow velocity are 0.2 mL/min.Phenol-sulphate acid method detection, is obtained five
Kind different polymerization degree oligosaccharides.It is detected by first mass spectrometric, determines that each oligosaccharides degree of polymerization is 2 ~ 8, predominantly high sulfated oligosaccharide,
The oligosaccharides of the middle degree of polymerization 5 is the three sialylated sulfate substituted sulfuric acid keratan tetroses positioned at non-reducing end.
Above-mentioned three sialylated sulfates are taken to replace 5 mg of KS tetrose, it is total to carry out one-dimensional and two-dimentional nuclear-magnetism after heavy water exchange
Vibration wave spectrum analysis.As a result pass through α -2 between the galactolipin in sialic acid and tetrose as shown in figure 5, can determine by analysis, 3
Connection.
(2) multi-stage ms are analyzed: in order to determine sulfate radical the position of substitution, replacing KS to the three sialylated sulfates
Tetrose carries out multi-stage ms detection.Determine that it at least has two kinds of structures, i.e., the position that sulfate radical substitution does not occur is located at reduction
On the acetylglucosamine or galactolipin at end.It is possible thereby to determine that the high sulphation keratan sulfate structure is as follows: its
Described in two substituent Rs in structural formula1And R2One of them is SO3H, another substituent group are then H, that is, work as R1=SO3When H, R2=
H；Or R1When=H, R2= SO3H。
。
Embodiment 4: adjustment effect of the high sulphation keratan sulfate to enteric microorganism
(1) 6 groups are selected with a batch of kunming mice, each three groups of male and female, be control, remaining two component with diet group
It Yong not low dosage (8 mgKg-1) and high dose (40 mgKg-1) KS to its stomach-filling raise, put to death after six weeks, take caecum
DNA is extracted, its intestinal flora is analyzed by high-flux sequence.
(2) such as the thermal map that Fig. 6 is high sulphation keratan sulfate adjusting enteric microorganism, N, L, H respectively indicate normal right
According to group, low dosage administration group, high dose administration group, firmicutes (Firmicutes), bacteroid (Bacteroidetes) and deformation
Bacterium (Proteobacteria) is the higher bacterium of abundance.As a result, it has been found that in male mouse body bacteroid abundance reduce and firmicutes are rich
Degree increases, and in female mice body, bacteroid abundance reduces and mycetozoan abundance increases, and illustrates Flora dynamics and the close phase of gender
It closes.Moreover, it has been found that high sulphation keratan sulfate increases the abundance (such as Fig. 7) of Bacillus acidi lactici in male and female mouse body simultaneously, and
And this change becomes apparent in female mice body.Due to the adjustable fat, diabetes of Bacillus acidi lactici, a variety of enteron aisle diseases such as diarrhea
Disease, this treats related disease for high sulphation keratan sulfate and provides theoretical foundation.
Embodiment 5: high sulphation keratan sulfate is to immune adjustment effect
50000/hole of logarithmic growth phase RAW264.7 cell is inoculated in 96 orifice plates, 90 holes μ L/；It is added afterwards for 24 hours to be measured
High sulphation keratan sulfate sample (10 hole μ L/).Drug effect terminates afterwards be incubated for for 24 hours, gently siphons away culture medium, PBS is added to wash
Once, 0.075% dimethyl diaminophenazine chloride (100 hole μ L/) is added, incubator is incubated for 30 min, siphons away dimethyl diaminophenazine chloride, adds PBS to wash three times, add and split
Liquid (dehydrated alcohol: glacial acetic acid=1:1 volume ratio) 200 holes μ L/ are solved, survey OD540 after cracking.As shown in figure 8, the height of various concentration
Sulphation keratan sulfate has the function of enhancing macrophage phagocytic function, and in 100 μ g/mL, effect is best, illustrates it
Have effects that enhancing is immune.
To sum up, the present invention prepares high sulphation keratan sulfate by feed purification of Shark cartilage, it is found that its molecular weight is
20~100 kDa.Structural analysis, the high sulfuric acid of gained are carried out to high sulphation keratan sulfate based on HILIC-UPLC-FTMS technology
Change keratan sulfate to be dissolved in enzymatic hydrolysis buffer, the Ug of Keratanase II(100 ~ 800 is added-1), 37 DEG C of shaking table reactions 24
~ 48 h digest it completely, and enzymolysis product carries out HILIC-UPLC-FTMS analysis, find that this is more by bioinformatic analysis
Sugar is mainly alternately connected by 6 sulphation galactolipins and 6 sulphation acetylglucosamines with β -1,3 and β -1,4 glycosidic bond
It forms, secondly two sugared content of sulphation is 80 ~ 95%, there are three sialylated sulfates to replace KS tetrose, saliva for non-reducing end
It is α -2,3 connection between acid and galactolipin.High sulphation keratan sulfate prepared by the present invention, which has, to be adjusted in enteric microorganism
Effect, the structure of enteric microorganism in female mice and male mouse body can be changed, hence it is evident that increase the rich of Bacillus acidi lactici in male and female mouse body
Degree, and this change is closely related with gender.Meanwhile the present invention prepared by high sulphation keratan sulfate also have it is good
Immunoregulation effect.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (7)
1. a kind of high sulphation keratan sulfate, it is characterised in that: the high sulphation keratan sulfate derives from Shark cartilage,
Molecular weight is 20~100kDa, and the high sulphation keratan sulfate is by 6 sulphation galactolipins and 6 sulphation acetylaminos
Glucose is alternately formed by connecting with β -1,3 and β-Isosorbide-5-Nitrae glycosidic bond, secondly two sugared content of sulphation is 80~95%, non-reducing end
It is α -2,3 connections, structural formula between sialic acid and galactolipin for three sialylated sulfate substituted sulfuric acid keratan tetroses
It is as follows:
Wherein R in the structural formula1=SO3 -, R2=H or R1=H, R2=SO3 -；
The three sialylated sulfates replace KS tetrose, and structure is as follows:
Wherein two substituent Rs in the structural formula1And R2One of them is SO3 -, another substituent group is then H.
2. high sulphation keratan sulfate according to claim 1, it is characterised in that: the Shark cartilage is the pass of shark
Section, rib cage, skull, vertebra, interverbebral disc or the cartilage at tracheae position.
3. the preparation method of high sulphation keratan sulfate described in claim 1, it is characterised in that it the following steps are included:
(1) proteasome degradation: being scattered in enzymatic hydrolysis buffer after Shark cartilage is crushed, be then added papain, 50~
The reaction of 70 DEG C of shaking tables, after boil and make enzyme-deactivating；Add trypsase after cooling, 30~55 DEG C of shaking tables reactions, after boil and make enzyme
Inactivation is centrifuged removal insoluble impurities after cooling；
(2) ethanol precipitation: being added ethyl alcohol in the solution obtained to step (1), low temperature is placed 12~24 hours, and it is heavy to take after centrifugation
Form sediment, precipitating dialyses after being redissolved with distilled water, be concentrated under reduced pressure, be lyophilized after obtain Shark cartilage mucopolysaccharide component；
(3) Anion exchange resin separation: Shark cartilage mucopolysaccharide component obtained by step (2) is dissolved with distilled water, with chlorination
Sodium water solution is mobile phase linear elution, is purified through Anion exchange resin separation, Phenol-sulphate acid method detection, after distilled water dialysis
It is concentrated under reduced pressure, freeze-drying, obtains high-purity sulfuric acid keratan；
Or chondroitin enzymatic hydrolysis separation: by Shark cartilage mucopolysaccharide component buffer solution obtained by step (2), chondroitin is added
ABC enzyme, 25~40 DEG C of shaking tables, which react to boil after 4~6h, makes enzyme-deactivating, ethyl alcohol is added after centrifugation into supernatant, low temperature places 12
~24 hours, precipitating is collected by centrifugation, redissolves Yu Shuihou dialysis desalting, freeze-drying, obtains high-purity sulfuric acid keratan；
The filler of anion exchange resin described in the step (3) be Q-Sepharose FF, DEAE-Sepharose FF,
Capto-Q Sepharose FF, DEAE-Cellulose or Capto-DEAE Sepharose FF；
The dosage of Chondroitin A BC enzyme is 300~600mUg in the step (3)-1。
4. the preparation method of high sulphation keratan sulfate according to claim 3, it is characterised in that: the step (1)
The dosage of middle papain is 10~100Ug-1, pH 7.0；The dosage of the trypsase is that 0.1~10%, pH is
7.5。
5. the preparation method of high sulphation keratan sulfate according to claim 3, it is characterised in that: the step (1)
The middle papain and the reaction time of trypsase of being added is 8~12h.
6. application of the high sulphation keratan sulfate of any of claims 1 or 2 in the preparation that preparation adjusts enteric microorganism.
7. application of the high sulphation keratan sulfate of any of claims 1 or 2 in the preparation of preparation strengthen immunity.
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| Title |
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| 鲨鱼软骨多糖中硫酸角质素分离方法研究;李燕妮 等;《食品研究与开发》;20160930;第37卷(第18期);第1.2.1、1.2.2、3节 |
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