CN106916863A - A kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying - Google Patents
A kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying Download PDFInfo
- Publication number
- CN106916863A CN106916863A CN201710291565.3A CN201710291565A CN106916863A CN 106916863 A CN106916863 A CN 106916863A CN 201710291565 A CN201710291565 A CN 201710291565A CN 106916863 A CN106916863 A CN 106916863A
- Authority
- CN
- China
- Prior art keywords
- kappa
- purifying
- carrageenin
- monomer
- carrageenin oligose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying, belong to Tang oligosaccharide extraction and separation technology field.Comprise the following steps that：1）It is prepared by the enzymolysis of κ carrageenan oligosaccharides：Using biologic enzymolysis method degraded κ carragheen substrate solutions, reaction solution water bath with thermostatic control digests 48 h, heating termination reaction, the insoluble fragment that removal is not digested is centrifuged after being cooled to room temperature, supernatant uses tangential ultrafiltration, collects the component less than 3kDa, and freeze-drying obtains oligosaccharide mixture dry powder；2）Using flash mesolow preparative chromatography fast separating and purifying oligosaccharide monomer；3）Detected according to analysis, obtain four kinds of κ carrageenan oligosaccharide monomers, a circulation takes only 1.5 h, and oligosaccharides total amount yield is 5%.The present invention is easy to operate, while preparation amount is big, sample concentration is up to gram rank, and purity is high, and separation is time-consuming short, and stable technical process reappearance is high, and prepared post used and instrument are reusable, is capable of achieving large-scale production.
Description
Technical field
The invention belongs to Tang oligosaccharide extraction and separation technology field, it is related to a kind of kappa-carrageenin oligose monomer quick separating
The method of purifying.
Background technology
Carrageenan oligosaccharide is with other sulfate oligosaccharides and its derivative species seemingly, active with various biological, such as antitumor,
Antiviral, anti-oxidant and regulation is immune etc..The degree of polymerization of its functional activity and carrageenan oligosaccharide, the substitution quantity of sulfate group and
The position of substitution is relevant, has caused the concern of researcher, the focus as food, medicine and other fields R and D.Try to gain
Hai Jin etc. obtains the new kappa-carrageenan oligosaccharide of different polymerizations using enzymatic isolation method, and obtains its derivative using sulfonation modification,
Sample can substantially suppress mouse S 180 sarcoma activity, as a result show that its antitumous effect is reduced with increasing for sulfate group,
Therefore the antitumous effect of carraoligose has close ties with the content of sulfate group.The research discovery such as Wei Wang,
The carraoligose and its sulfated derivative of low-molecular-weight can effectively suppress influenza A virus in mdck cell（IVA）
Growth, while antiviral activity it is optimal for molecular weight in 1 ~ 3kDa, sulfuric acid content between 0.8 ~ 1.0mole/mole
Oligomeric bglii fragment, the antiviral activity and its molecular weight and sulfate group content for further illustrating carraoligose has closely
Relation.In addition, Wei Wang etc. further demonstrate the activity and its molecular weight of the anti-H1NI viruses of kappa-carrageenin oligose
There are substantial connection, i.e. 2kDa ＞ 3kDa ＞ 5kDa.Ling Xu etc., Zi-ang Yao etc. have studied kappa-carrageenin oligose and LPS lured
The immunoregulatory activity of the microglia led, as a result shows that kappa-carrageenin oligose and desulfurization kappa-carrageenin oligose can suppress LPS
The activity of the small colloid inflammatory cell of induction, and in dose-dependence, meanwhile, the antiphlogistic effects of desulfurization kappa-carrageenin oligose
It is weaker than kappa-carrageenin oligose.
At present, oligosaccharides separation method is a lot, and traditional has step-by-step precipitation method, salting out method, ultrafiltration, gel filtration chromatography, ion
Exchange etc., what is grown up recently has high performance liquid chromatography, polyacrylamide gel electrophoresis, Capillary Electrophoresis, electroblotting, ion
The new methods such as chromatogram.In recent years, researcher has employed ethanol step-by-step precipitation method, gel-filtration chromatography（a Bio-Gel
P-2 column、a Bio-gel P6 column、Q-Sepharose Fast Flow）, High Performance Gel Permeation Chromatography（PL
aquagel-OH column）, size exclusion chromatography（SEC）, efficient liquid phase system（Superdex peptide 10/300
GL column）, Ion-pair Liquid Chromatography method（Spherisorb ODS1 column）, AKTA fast protein liquid chromatographies
（Superdex 30 colunm）Isolated and purified Deng to carrageenan oligosaccharide.But all because its efficiency is low, preparation amount is small, limits
Its industrial applications.Wherein the colunm of gel column Superdex 30 and Bio-gel are two kinds of the most frequently used fillers, because its is preferable
Sensitivity and resolution ratio, for low-molecular-weight oligosaccharides, with preferable separating effect.But to prepare post how efficient due to these
The chromatographic systems such as liquid phase systems are combined, although can monitor on-line, but its preparation amount and operating efficiency, leverage its scale
Using.
Flash preparative chromatography technology, according to pressure applied, can be divided into flash chromatography (0.1-5/10 bars) or in
Pressure liquid chromatography MPLC (5/10-50 bars).Flash chromatography system is used primarily for the application that fast purifying synthesizes compound, very
It is rare to be applied in the separation of mixture obtained by complicated natural product extraction to by this system.Compared to high performance liquid chromatography and
High-speed countercurrent chromatography, its preparation amount is big, and sample applied sample amount can be from milligram level to hectogram, and test period is shorter, the same to time-division
Can independently be filled from column packing, increased separation selectivity, effectively save experiment production cost, in dividing for natural products
Played an important role in purifying research work.Meanwhile, the preparative chromatography of general configuration only has UV-detector, and this is simultaneously uncomfortable
For the compound test without ultraviolet absorption peak, such as carbohydrate；And the offline inspections such as phend-sulphuric acid are used, often
Need to be more long using harmful chemical reagent, and poor specificity, analysis time, sample waste is also more serious.Lu Xu et al., has adopted
Lotus seeds oligosaccharide is preferably isolated and purified with mesolow preparing chromatography system, and applied sample amount is larger, and yield is higher, effect
Fruit is significantly.
The content of the invention
The purpose of the present invention is to solve the shortcomings of the prior art, there is provided a kind of kappa-carrageenin oligose monomer quick separating is pure
The preparation method of change.With Rs resolution ratio as evaluation index, by optimizing sample concentration, loading volume, elution flow rate etc., it is determined that most
Good separation purifying technique, to be laid the foundation for kappa-carrageenin oligose monomer prepare with scale；The method is easy to operate, while system
Standby amount is big, and sample concentration is up to gram rank, and purity is high, separates time-consuming short, and stable technical process reappearance is high, prepared post used and
Instrument is reusable, is capable of achieving large-scale production.
For achieving the above object, technical program of the present invention lies in：
A kind of method of kappa-carrageenin oligose monomer fast separating and purifying, comprises the following steps that：
1）It is prepared by the enzymolysis of kappa-carrageenin oligose：The kappa-carrageenin substrate that mass concentration is for 0.2% ~ 0.5% is prepared with deionized water
Solution, the kappa-carrageenan enzyme liquid that consumption is react cumulative volume 5% ~ 10% is added toward solution, in 45 DEG C of water bath with thermostatic control reactions 48
5 min terminating reactions are boiled in h, heating, are cooled to room temperature, and hydrolyzate is centrifuged 10 min to remove what is do not digested in 10000r/min
Insoluble fragment；
2）Centrifuged supernatant is collected, 10kDa, 3kDa tangential ultrafiltration film are crossed successively, collect enzymolysis liquid of the molecular weight less than 3kDa, adopted
Albumen, the lipid in supernatant are removed with Sevag methods, centrifuging and taking supernatant concentrated by rotary evaporation, freeze-drying again obtains κ-OK a karaoke club
Glue oligosaccharides solid mixture；
3）Kappa-carrageenin oligose solid mixture is configured to kappa-carrageenin oligose solution, using mesolow preparing chromatography system
（Medium Pressure Preparative Liquid Chromatography System, abbreviation MPLC, including MPLC colors
Spectrum pump）Oligosaccharide monomer is isolated and purified, purification condition is：Isocratic elution, column temperature：25 DEG C, sample concentration is 0.3 ~ 1.2 g/
ML, loading volume is 0.5 ~ 3 mL, eluent 0.1M NH4HCO3, elution flow rate is 1 ~ 4 mL/min；
4）Merge the consistent solution of component, concentrated by rotary evaporation, freeze-drying is detected using TLC and ESI-MS, obtains the degree of polymerization different
Kappa-carrageenin oligose monomer.
The kappa-carrageenan enzyme liquid is that the autonomous bacterium fermentation in laboratory is obtained.Fermentation process is as follows：κ-the OK a karaoke club
The preparation method of glue enzyme liquid is as follows：By sea rotation bacterium（Thalassospira sp）Fjfst-332 bacterial strains are activated in slant medium
After 24 h, liquid seed culture medium is seeded to, inoculum concentration is 3ml/25ml, in 25 DEG C, the h of 125 r/min concussion and cultivates 12
Afterwards, seed culture fluid is obtained；Nutrient solution is seeded in fermentation medium, inoculum concentration is 3ml/25ml, in 25 DEG C, 125 r/
The h of min concussion and cultivates 36, by zymotic fluid with 8000 r/min rotating speeds be centrifuged 20min, remove culture medium in graininess impurity and
Somatic cells, collect supernatant, and the milipore filter of 50kD and 10kD is crossed respectively, collect concentration enzyme liquid and are kappa-carrageenan enzyme liquid.
Preferably, step 3）In, the condition that mesolow preparing chromatography system isolates and purifies oligosaccharide monomer is：Sample concentration is
1.2 g/mL, loading volume is 1 mL, and elution flow rate is 4 mL/min.
Step 3）In, each sample introduction flows the chromatographic column that balances each other using 3 Initial Gradients of column volume, isolates and purifies every time
After the completion of, mobile phase retracts one column volume of former gradient washes, and next sample introduction is then prepared again.
Step 3）Described mesolow preparing chromatography system is evaporated using the ultraviolet full wavelength scanner devices of DAD with FLASH ELSD
Light scattering detector is used in combination, and the Control ＆ data acquisition of system is operated using the softwares of Interchim Software 5.0；
Ultraviolet full wavelength scanner device wave-length coverage：190-840 nm, EISD parameter setting：Purging nitrogen pressure is
3.4 bar, 100 DEG C of drift tube temperature.
Beneficial benefit of the invention is：
1st, invention prepares carrageenan oligosaccharide using the active bacterial strain enzymolysis of autonomous screening, the wild strain（Sea rotation bacterium
（Thalassospira sp）fjfst-332）Fermentation enzymolysis efficiency high, and product species are single-minded, impurity is few, and the product degree of polymerization is simultaneously
It is not broken degraded further with the extension of enzymolysis time；
2nd, the present invention using mesolow preparative chromatography joint evaporative light-scattering system and combines the colunm of Superdex 30 first
Preparation post, isolates and purifies kappa-carrageenin oligose, easy to operate, and applied sample amount is big, time-consuming short, and gained oligosaccharide monomer purity is high, is sea
The prepare with scale of algae oligosaccharides is laid a good foundation.
Brief description of the drawings
Enzymolysis product TLC analysis charts under Fig. 1 difference enzymolysis times；
Fig. 2A, Fig. 2 B, Fig. 2 C, Fig. 2 D are kappa-carrageenin oligose chromatograms under different elution flow rates（The g/ml of sample concentration 0.6,
The ml of loading volume 1；Mobile phase 0.1M NH4HCO3；A：1 ml/min；B：2 ml/min；C：3 ml/min；D：4 ml/min）；
Fig. 3 A, Fig. 3 B, Fig. 3 C, Fig. 3 D are kappa-carrageenin oligose chromatograms under different sample concentrations（The ml of loading volume 1；Wash-out stream
4 ml/min of speed；Mobile phase 0.1M NH4HCO3；A：0.3 g/ml；B：0.6 g/ml；C：1 g/ml；D：1.2 g/ml）；
Fig. 4 A, Fig. 4 B, Fig. 4 C, Fig. 4 D are kappa-carrageenin oligose chromatograms under different loading volumes（The g/ ml of sample concentration 1.2；
The ml/min of elution flow rate 4；Mobile phase 0.1M NH4HCO3；A：0.5 ml；B：1 ml；C：2 ml；D：3 ml）；
Fig. 5 kappa-carrageenin oligose monomer TLC analysis charts；
Fig. 6 A, Fig. 6 B, Fig. 6 C, Fig. 6 D are the ESI-MS collection of illustrative plates of kappa-carrageenin oligose monomer（A：Kappa-carrageenan disaccharides；B：κ-OK a karaoke club
Glue tetrose；C：The sugar of kappa-carrageenan six；D：The sugar of κ/ι-carragheen six）.
Specific embodiment
The following is explanation specific embodiment of the invention, but the present invention is not limited thereto.
First, influence of the different enzymolysis times to enzymolysis product
It is prepared by the enzymolysis of kappa-carrageenin oligose：The kappa-carrageenin substrate solution of mass concentration 0.4% is prepared with deionized water, by anti-
Answer cumulative volume to add 7.5% kappa-carrageenan enzyme liquid, 1 h, 4 h, 8 h, 12 h, 24 h, 30 are reacted in 45 DEG C of difference waters bath with thermostatic control
5 min terminating reactions are boiled in h, 36 h, 48 h, 72 h, heating, are cooled to room temperature, and hydrolyzate is centrifuged 10 in 10000r/min
Min is removing the insoluble fragment not digested.
The degraded sample under different enzymolysis time points, point plate observation product situation, as shown in Figure 1, with enzymolysis are taken respectively
The extension of time, the species of enzymolysis product is not changed, and yield extension over time and accumulate, therefore consider enzyme
Solution selection of time is 48h.
2nd, kappa-carrageenin oligose separates situation under different elution flow rates
1）It is prepared by the enzymolysis of kappa-carrageenin oligose：The kappa-carrageenin substrate solution of mass concentration 0.4% is prepared with deionized water, it is past
The kappa-carrageenan enzyme liquid that consumption is react cumulative volume 7.5% is added in solution, 48 h are reacted in 45 DEG C of difference waters bath with thermostatic control, plus
Heat boils 5 min terminating reactions, is cooled to room temperature, and hydrolyzate is centrifuged 10 min to remove do not digest insoluble in 10000r/min
Property fragment；
2）Centrifuged supernatant is collected, 10kDa, 3kDa tangential ultrafiltration film are crossed successively, collect enzymolysis liquid of the molecular weight less than 3kDa, adopted
Albumen, the lipid in supernatant are removed with Sevag methods, centrifuging and taking supernatant concentrated by rotary evaporation, freeze-drying again obtains κ-OK a karaoke club
Glue oligosaccharides solid mixture；
3）Kappa-carrageenin oligose solid mixture is configured to kappa-carrageenin oligose solution, using mesolow preparing chromatography system
（Medium Pressure Preparative Liquid Chromatography System, abbreviation MPLC, including MPLC colors
Spectrum pump）Oligosaccharide monomer is isolated and purified, Parameter Conditions are：Isocratic elution, column temperature is：25 DEG C, the g/mL of sample concentration 0.6, loading body
Product 1 mL, eluent 0.1M NH4HCO3, elution flow rate is respectively：1、2、3、4 mL/min；
4）Merge the consistent solution of component, concentrated by rotary evaporation, freeze-drying.
From Fig. 2 and the result of Biao 1, comprehensive selection elution flow rate is 4 mL/min.
3rd, kappa-carrageenin oligose separates situation under different sample concentrations
1）It is prepared by the enzymolysis of kappa-carrageenin oligose：The kappa-carrageenin substrate solution of mass concentration 0.4% is prepared with deionized water, it is past
The kappa-carrageenan enzyme liquid that consumption is react cumulative volume 7.5% is added in solution, 48 h are reacted in 45 DEG C of waters bath with thermostatic control, heating is boiled
5 min terminating reactions are boiled, room temperature is cooled to, hydrolyzate is centrifuged 10 min to remove insoluble not digested in 10000r/min
Section；
2）Centrifuged supernatant is collected, 10kDa, 3kDa tangential ultrafiltration film are crossed successively, collect enzymolysis liquid of the molecular weight less than 3kDa, adopted
Albumen, the lipid in supernatant are removed with Sevag methods, centrifuging and taking supernatant concentrated by rotary evaporation, freeze-drying again obtains κ-OK a karaoke club
Glue oligosaccharides solid mixture.
3）Configuration kappa-carrageenin oligose solution, using mesolow preparing chromatography system（Medium Pressure
Preparative Liquid Chromatography System, abbreviation MPLC, including MPLC chromatogram pumps）Isolate and purify oligosaccharides
Monomer, Parameter Conditions are：The g/mL of sample concentration 0.3,0.6,1.0,1.2, loading volume 1 mL, eluent 0.1M NH4HCO3,
Elution speed：4 mL/min；
4）Merge the consistent solution of component, concentrated by rotary evaporation, freeze-drying.
From Fig. 3 and the result of Biao 2, comprehensive selection sample concentration is 1.2 g/mL.
3rd, kappa-carrageenin oligose separates situation under different loading volumes
1）It is prepared by the enzymolysis of kappa-carrageenin oligose：The kappa-carrageenin substrate solution of mass concentration 0.4% is prepared with deionized water, it is past
The kappa-carrageenan enzyme liquid that consumption is react cumulative volume 7.5% is added in solution, 48 h are reacted in 45 DEG C of waters bath with thermostatic control, heating is boiled
5 min terminating reactions are boiled, room temperature is cooled to, hydrolyzate is centrifuged 10 min to remove insoluble not digested in 10000r/min
Section；
2）Centrifuged supernatant is collected, 10kDa, 3kDa tangential ultrafiltration film are crossed successively, collect enzymolysis liquid of the molecular weight less than 3kDa, adopted
Albumen, the lipid in supernatant are removed with Sevag methods, centrifuging and taking supernatant concentrated by rotary evaporation, freeze-drying again obtains κ-OK a karaoke club
Glue oligosaccharides solid mixture.
3）Configuration kappa-carrageenin oligose solution, using mesolow preparing chromatography system（Medium Pressure
Preparative Liquid Chromatography System, abbreviation MPLC, including MPLC chromatogram pumps）Isolate and purify oligosaccharides
Monomer, Parameter Conditions are：The g/mL of sample concentration 1.2, loading volume 0.5,1.0,2.0,3.0 mL, eluent 0.1M
NH4HCO3, elution speed：4 mL/min；
4）Merge the consistent solution of component, concentrated by rotary evaporation, freeze-drying.
From Fig. 4 and the result of Biao 3, comprehensive selection loading volume is 1.0 mL.
Gel chromatography chromatogram be biased sample fixed equipped with gel as mobile phase is flowed through phase chromatographic column when, sample
According to the difference of molecular size range, separated successively according to sequencing.Carried out simultaneously when each Middle Molecular Substance passes through filler
Vertical and diverging flow.The big material of molecular weight is larger due to particle diameter, is not easily accessible gel pore, can only be distributed in gel
In particulate interspaces, as mobile phase is quickly moved down.The small material of molecular weight is not only spread in gel particle gap, may be used also
Into in granule pores, constantly distributed between gap in gel particle, so that the material of macromolecule is faster than small-molecular-weight thing
Mass flow goes out outside post, and therefore sample mixture is isolated and purified.Resolution factor（Rs）Resolution ratio is used to evaluate solidifying
The separating effect of glue thin layer chromatography, depending on selectivity（selectivity）Imitated with post（efficiency）.Peak separation dimension
Height, its selectivity is good, and high selectivity can reach baseline separation；Post effect measured with peak width it is relevant, post imitate more peak it is narrower, Gao Zhu
Effect can make up selective deficiency.Post effect depends on flow rate of mobile phase, packing material size size, particle diameter distribution, the dress of chromatographic column
Fill out and sample volume and concentration.Research has been separately optimized elution flow rate, sample concentration and loading volume to isolating and purifying effect
Influence.Obtaining optimal purifying parameter is：Sample concentration 1.2 g/ ml, loading volume 1mL, the ml/min of elution flow rate 4.
Rs and oligosaccharides yield under the different elution flow rates of table 1
Rs and oligosaccharides yield under the different sample concentrations of table 2
Rs and oligosaccharides yield under the different loading volumes of table 3
Embodiment 1
A kind of method of kappa-carrageenin oligose monomer fast separating and purifying, comprises the following steps that：
1）It is prepared by the enzymolysis of kappa-carrageenin oligose：The kappa-carrageenin substrate solution of mass concentration 0.4% is prepared with deionized water, it is past
The kappa-carrageenan enzyme liquid that consumption is react cumulative volume 7.5% is added in solution, 48 h are reacted in 45 DEG C of waters bath with thermostatic control, heating is boiled
5 min terminating reactions are boiled, room temperature is cooled to, hydrolyzate is centrifuged 10 min to remove insoluble not digested in 10000r/min
Section；
2）Centrifuged supernatant is collected, 10kDa, 3kDa tangential ultrafiltration film are crossed successively, collect enzymolysis liquid of the molecular weight less than 3kDa, adopted
Albumen, the lipid in supernatant are removed with Sevag methods, centrifuging and taking supernatant concentrated by rotary evaporation, freeze-drying again obtains κ-OK a karaoke club
Glue oligosaccharides solid mixture.
3）Configuration kappa-carrageenin oligose solution, using mesolow preparing chromatography system（Medium Pressure
Preparative Liquid Chromatography System, abbreviation MPLC, including MPLC chromatogram pumps）Isolate and purify oligosaccharides
Monomer, Parameter Conditions are：The g/mL of sample concentration 1.2, loading volume 1.0 mL, eluent 0.1M NH4HCO3, elution speed：4
mL/min；
4）Merge the consistent solution of component, concentrated by rotary evaporation, freeze-drying.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modification, should all belong to covering scope of the invention.
Claims (6)
1. a kind of method of kappa-carrageenin oligose monomer fast separating and purifying, it is characterised in that comprise the following steps that：
1）It is prepared by the enzymolysis of kappa-carrageenin oligose：The kappa-carrageenin substrate that mass concentration is for 0.2% ~ 0.5% is prepared with deionized water
Solution, the kappa-carrageenan enzyme liquid that consumption is react cumulative volume 5% ~ 10% is added toward solution, in 45 DEG C of water bath with thermostatic control reactions 48
5 min terminating reactions are boiled in h, heating, are cooled to room temperature, and hydrolyzate is centrifuged 10 min to remove what is do not digested in 10000r/min
Insoluble fragment；
2）Centrifuged supernatant is collected, 10kDa, 3kDa tangential ultrafiltration film are crossed successively, collect enzymolysis liquid of the molecular weight less than 3kDa, adopted
The albumen and lipid in supernatant are removed with Sevag methods, centrifuging and taking supernatant concentrated by rotary evaporation, freeze-drying again obtains κ-card
Draw glue oligosaccharides solid mixture；
3）Kappa-carrageenin oligose solid mixture is configured to kappa-carrageenin oligose solution, using mesolow preparing chromatography system point
From purifying oligosaccharide monomer, purification condition is：Isocratic elution, column temperature：25 DEG C, the g/mL of sample concentration 0.3 ~ 1.2, loading body
Product is 0.5 ~ 3 mL, and eluent is 0.1M NH4HCO3, elution flow rate is 1 ~ 4 mL/min；
4）Merge the consistent solution of component, concentrated by rotary evaporation, freeze-drying is detected using TLC and ESI-MS, obtains the degree of polymerization different
Kappa-carrageenin oligose monomer.
2. the method for kappa-carrageenin oligose monomer fast separating and purifying according to claim 1, it is characterised in that：The κ-
The preparation method of carragheen enzyme liquid is as follows：By sea rotation bacterium（Thalassospira sp）Fjfst-332 bacterial strains are in slant medium
After activating 24 h, liquid seed culture medium is seeded to, inoculum concentration is 3ml/25ml, in 25 DEG C, 125 r/min concussion and cultivates
After 12 h, seed culture fluid is obtained；Nutrient solution is seeded in fermentation medium, inoculum concentration is 3ml/25ml, 25 DEG C, 125
The h of r/min concussion and cultivates 36,20min is centrifuged by zymotic fluid with 8000 r/min rotating speeds, removes the graininess impurity in culture medium
And somatic cells, supernatant is collected, the milipore filter of 50kD and 10kD is crossed respectively, collect concentration enzyme liquid and be kappa-carrageenan enzyme liquid.
3. the method for kappa-carrageenin oligose monomer fast separating and purifying according to claim 1, it is characterised in that：Step 3）
In, the condition that mesolow preparing chromatography system isolates and purifies oligosaccharide monomer is：Sample concentration is 1.2 g/mL, and loading volume is 1
ML, elution flow rate is 4 mL/min.
4. the method for kappa-carrageenin oligose monomer fast separating and purifying according to claim 1, it is characterised in that：Step 3）
In, each sample introduction flows balance each other chromatographic column, after the completion of isolating and purifying every time, mobile phase using 3 Initial Gradients of column volume
One column volume of former gradient washes is retracted, next sample introduction is then prepared again.
5. the method for kappa-carrageenin oligose monomer fast separating and purifying according to claim 1, it is characterised in that：Step 3）
Described mesolow preparing chromatography system is joined using the ultraviolet full wavelength scanner devices of DAD with FLASH ELSD EISDs
Conjunction is used, and the Control ＆ data acquisition of system is operated using the softwares of Interchim Software 5.0.
6. the method for kappa-carrageenin oligose monomer fast separating and purifying according to claim 5, it is characterised in that：It is ultraviolet complete
Length scanning device wave-length coverage：190-840 nm, EISD parameter setting：Purging nitrogen pressure is 3.4 bar,
100 DEG C of drift tube temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710291565.3A CN106916863A (en) | 2017-04-28 | 2017-04-28 | A kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710291565.3A CN106916863A (en) | 2017-04-28 | 2017-04-28 | A kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106916863A true CN106916863A (en) | 2017-07-04 |
Family
ID=59567788
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710291565.3A Pending CN106916863A (en) | 2017-04-28 | 2017-04-28 | A kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106916863A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536548A (en) * | 2018-11-20 | 2019-03-29 | 青岛博智汇力生物科技有限公司 | A kind of preparation method of new kappa- carrageenan oligosaccharide monomer |
| CN115181194A (en) * | 2022-06-21 | 2022-10-14 | 浙江大学 | Rapid purification degradation and structure analysis method of pectin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037448A (en) * | 2015-09-10 | 2015-11-11 | 福建农林大学 | Method for quick and large-scale extraction and separation of coix seed oligosaccharide monomers |
| CN105153321A (en) * | 2015-09-10 | 2015-12-16 | 福建农林大学 | Quick separation method of lotus seed oligosaccharide monomers with prebiotic effect |
| CN106047855A (en) * | 2016-08-22 | 2016-10-26 | 福建农林大学 | Immobilized bacteria and method for preparing kappa-carrageenan oligosaccharide by using immobilized bacteria |
-
2017
- 2017-04-28 CN CN201710291565.3A patent/CN106916863A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037448A (en) * | 2015-09-10 | 2015-11-11 | 福建农林大学 | Method for quick and large-scale extraction and separation of coix seed oligosaccharide monomers |
| CN105153321A (en) * | 2015-09-10 | 2015-12-16 | 福建农林大学 | Quick separation method of lotus seed oligosaccharide monomers with prebiotic effect |
| CN106047855A (en) * | 2016-08-22 | 2016-10-26 | 福建农林大学 | Immobilized bacteria and method for preparing kappa-carrageenan oligosaccharide by using immobilized bacteria |
Non-Patent Citations (1)
| Title |
|---|
| 卢旭等: "莲子低聚糖快速制备工艺研究", 《中国食品学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536548A (en) * | 2018-11-20 | 2019-03-29 | 青岛博智汇力生物科技有限公司 | A kind of preparation method of new kappa- carrageenan oligosaccharide monomer |
| CN115181194A (en) * | 2022-06-21 | 2022-10-14 | 浙江大学 | Rapid purification degradation and structure analysis method of pectin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104817604B (en) | A kind of purification process of β nicotinamide mononucleotides | |
| CN108026132A (en) | A kind of purification process of nicotinamide mononucleotide | |
| US20200369789A1 (en) | Cordyceps militaris medium polysaccharide, method for separating and purifying same, and use thereof | |
| CN104774182B (en) | The extraction of erythrothioneine and purification process | |
| CN101508715A (en) | Extraction and purification process for cordycepin in cordyceps militaris link | |
| CN101418327A (en) | The new process of production of high purity 5 ' Nucleotide | |
| CN106916863A (en) | A kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying | |
| CN100509760C (en) | Method for separating and purifying glutamine from fermentation liquor by four-area simulation moving bed | |
| Shimokawa et al. | Preparation of two series of oligo-guluronic acids from sodium alginate by acid hydrolysis and enzymatic degradation | |
| CN102617746A (en) | Method for preparing multiple oligosaccharides by separating and purifying Chinese dates | |
| CN107011433A (en) | A kind of phycocyanin of the extraction of nostoc phycobniliprotein, purification process and purifying | |
| CN108070627A (en) | A kind of preparation method of chondroitin sulfate D tetroses | |
| Bernardi et al. | Separation and quantitative determination of carbohydrates in microbial submerged cultures using different planar chromatography techniques (HPTLC, AMD, OPLC) | |
| CN106008729B (en) | A kind of sunset abelmoschus stem or bark leaf polyose and preparation method and application | |
| CN102838684B (en) | Separating and purifying process of isochrysis galbana exopolysaccharide | |
| CN102018955A (en) | Purification method for producing viral vaccine in large scale | |
| CN109942723A (en) | A kind of separation method of polysaccharides | |
| CN101456898B (en) | Method for separating and purifying polypeptide by using hydrogen bond adsorption chromatogram | |
| CN106262638A (en) | A kind of flavouring agent | |
| CN106831943B (en) | Method for purifying transdermal peptide at low cost | |
| CN105254746A (en) | Method for desalinating thymopeptide alpha 1 | |
| CN110194758A (en) | A method of the fast separating and purifying Aristolochic Acid compound from caulis aristologhiae manshuriensis | |
| CN111135810B (en) | Preparation method of special chromatographic separation medium for cannabidiol separation | |
| CN110028531B (en) | Method for extracting and separating flavonoid substances from soil | |
| CN102827244A (en) | Method for refining glutathione fermentation broth |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170704 |