CN115181194A - Rapid purification degradation and structure analysis method of pectin - Google Patents

Rapid purification degradation and structure analysis method of pectin Download PDF

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CN115181194A
CN115181194A CN202210708470.8A CN202210708470A CN115181194A CN 115181194 A CN115181194 A CN 115181194A CN 202210708470 A CN202210708470 A CN 202210708470A CN 115181194 A CN115181194 A CN 115181194A
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pectin
solution
oligosaccharide
polysaccharide
analysis
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CN115181194B (en
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陈士国
朱凯
叶兴乾
陈健乐
潘海波
侯志强
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Zhejiang University ZJU
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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
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8696—Details of Software

Abstract

The invention discloses a method for purifying, degrading and structurally analyzing pectin. The method adopts the quaternary ammonium strong anion exchange material to separate and purify pectin, and has the characteristics of less sample consumption, short time and simple operation; the pectin obtained by purification is degraded by taking hydrogen peroxide as a reactant and ascorbic acid as a catalyst, so that the free radical degradation (H) based on a Fenton system is optimized 2 O 2 ‑V C ) The method ensures that the molecular size of the prepared pectin oligosaccharide is more suitable for mass spectrum detection and reduces the structure loss; finally, HILIC-MS/MS and bioinformatics software GlycReSoft 2.0 are adopted for combined analysis,realizing automatic qualitative and quantitative analysis of mass spectrum data and analyzing fine structure of pectin.

Description

Rapid purification degradation and structure analysis method of pectin
Technical Field
The invention relates to the field of plant pectin polysaccharide oligosaccharides, in particular to a method for purifying, degrading and structurally analyzing pectin.
Background
Pectin (Pectin) is a type of galacturonic acid (GalA) -rich heteropolysaccharide that is found extensively in the intercellular space and primary wall of plant cells. The prior research roughly divides pectin molecules into 4 types of structural domains according to the difference of main chain and branched chain structures of the pectin molecules: homogalacturonans (HG), rhamnogalacturonans I (RGI), rhamnogalacturonans II (RGII), and xylogalacturonans (Xylogalactaronan, XG). Pectin is structurally complex (50-1000 kDa) and has a high viscosity, and is also diversified in its composition according to source, extraction conditions, plant development stage, plant species, cell wall location and environmental factors, and questions remain as to the relative positions of pectin domains and how these domains are linked together in pectin. Earlier researches find that the structure of pectin is closely related to the function of pectin, and pectin with different structures has obvious difference in aspects of gel composition, intestinal flora regulation, enteritis resistance and the like. Therefore, the analysis of the fine structure of pectin is a precondition for utilizing abundant pectin resources.
The structural analysis of the complete pectin polysaccharide is very difficult. The extracted pectin crude polysaccharide contains more impurities (protein, polyphenol, starch, small molecular substances and the like) to influence the subsequent analysis, and the pectin crude polysaccharide needs to be separated and purified. The traditional method combines the methods of multi-stage separation and purification, periodic acid-Smith degradation, methylation, nuclear magnetic resonance spectroscopy and the like to analyze the pectin polysaccharide, and has the problems of time consumption, complex operation and fine structure loss. Therefore, the development of a rapid, efficient and trace method for analyzing the fine structure of pectic polysaccharides is a trend of necessity.
Currently, more and more research is being conducted to degrade pectin into oligosaccharides using controlled methods and analysis using liquid mass spectrometry. Commonly used polysaccharide degradation methods include acid degradation, enzymatic degradationDegradation of free radicals, and the like. Wherein, the acid degradation reaction is violent and difficult to control, and the fine structure of the polysaccharide can be lost; enzymatic degradation has the advantages of strong specificity, mildness and controllability, but the related enzymes have few varieties and cannot completely degrade complex pectin polysaccharide. Based on the Fenton system (H) 2 O 2 ) The free radical degradation is different from the specificity of enzymolysis and the violent reaction of an acid method, the free radical degradation can be applied to all kinds of polysaccharide, the reaction can be controlled through relevant conditions, the branch breakage or the loss of sulfate groups is not caused, the reproducibility is good, and the method generally needs to use metal Cu 2+ And Fe 2+ Auxiliary ultrasound is carried out, but the two metal ions need to be removed by a certain method after the reaction, so that the reaction time is prolonged, and the analysis efficiency is reduced. Therefore, the improvement of the free radical degradation method is needed to establish a non-metallic controllable free radical degradation method.
Disclosure of Invention
Aiming at the defect of the existing pectin structure analysis method in the background art, the quaternary ammonium strong anion exchange chromatographic column is applied to the purification and separation of pectin for the first time, so that the purification efficiency can be obviously improved; a free radical degradation method after pectin purification is established, so that pectin degradation is thorough, structure loss is reduced, and the method is particularly suitable for LC-MS/MS detection and realizes the analysis of a pectin fine structure; and the quick processing (10 min) of mass spectrum data is realized by combining with biological information software, and the analysis efficiency is obviously improved.
The invention provides a method for rapid purification of pectin, nonmetallic Fenton degradation of pectin and HILIC-MS/MS analysis of pectin oligosaccharide obtained by degradation, which can be used for preparation of pectin oligosaccharide and fine structure analysis of pectin, and specifically comprises the following steps:
1. purification and degradation of pectin
(1) Loading the crude polysaccharide solution to a chromatographic column for elution and separation, wherein the filler of the chromatographic column is quaternary ammonium type anion exchange filler;
(2) Dialyzing the eluent obtained in the step (1), and freeze-drying to obtain pectin pure polysaccharide;
(3) Dissolving the pectin pure polysaccharide obtained in the step (2) in water to obtain a pectin polysaccharide water solution with the concentration of 1-2 mg/ml; adding ascorbic acid, uniformly mixing, adding a hydrogen peroxide solution, and reacting at a constant temperature of 80-100 ℃; wherein the concentration of the ascorbic acid is 10-12 mM, the volume fraction of the hydrogen peroxide solution is 1%, and the pectin oligosaccharide is obtained by freeze drying after the reaction is finished.
In step (1), the eluent can be efficiently collected by a collection method commonly used in the art. For example, 1 tube of eluent is collected every minute, the peak position of the eluent is analyzed to detect the components of the eluent, and the same components are screened and combined.
The analytical method may be a trace phenol-sulfuric acid method: after 20. Mu.L of 5% (v/v) phenol aqueous solution was added to 40. Mu.L of the sample, 200. Mu.L of concentrated sulfuric acid was added thereto and mixed, followed by standing for color development for 15min and detection at 490nm wavelength. Or a trace of m-hydroxybiphenyl method: adding 4 mu L of sulfamic acid reagent into 40 mu L of sample, mixing uniformly, adding 250 mu L of concentrated sulfuric acid, incubating in an oven at 90 ℃ for 20min, cooling to room temperature, adding 4 mu L of m-hydroxybiphenyl, mixing uniformly, and detecting at the wavelength of 525 nm.
Further, the concentration of the crude polysaccharide solution is 5 to 6mg/mL.
Further, the elution procedure of step (1) is: deionized water for 0-10 min, naCl solution for 10-50 min and 0-0.8 mol/L, naCl solution for 50-60min and deionized water for 60-70 min, and the flow rate of the eluent is 2.5mL/min.
Further, in the step (3), the isothermal reaction time is 6h.
Further, the quaternary ammonium anion exchange filler in the step (1) takes agarose gel as a matrix.
2. Performing structure identification on the obtained pectin oligosaccharide
The invention adopts a structure analysis method based on HILIC-MS/MS combined bioinformatics software, and the specific method comprises the following steps:
a. dissolving the pectin oligosaccharide obtained in the step (3) in acetonitrile aqueous solution with volume fraction of 50%, wherein the concentration is 1-2 mg/ml;
b. and (b) separating the oligosaccharide and detecting the mass spectrum of the solution obtained in the step a by using an ultra-high performance liquid phase system and combining a flight time Q-TOF mass spectrum.
Liquid phase detection conditions: ultra high performance liquid chromatograph Waters Acqtity UPLC class; column UPLC-Amide (2.1X 150mm,1.7 μm); mobile phase a-20% acetonitrile +10mM ammonium formate +0.2% formic acid (v/v), phase B-80% acetonitrile +10mM ammonium formate +0.2% formic acid (v/v); the flow rate is 0.25mL/min; the column temperature is 35 ℃; time gradient: 0 → 35 → 50 → 55 → 56; concentration gradient: 100% → 70% → 20% → 20% → 100%, b phase, sample volume: 5 μ L.
Mass spectrum conditions: triple-TOF 5600 + Time-of-flight LC-MS; a negative ion scanning mode; scanning range: m/z is 100-1500; atomizing: 55psi; atomizing: 55psi; air curtain gas (CUR): 35psi; ion source Temperature (TEM): 550 ℃; ion source voltage (IS): -4500V; primary scanning: declustering voltage (DP): 100V; focus voltage (CE): 10V; secondary scanning: and (3) acquiring mass spectrum data by using a TOF MS-Product Ion-IDA mode, wherein the CID energy is 40 +/-20 eV, and before sample injection, a CDS (compact disc) pump is used for mass axis correction to ensure that the mass axis error is less than 2ppm.
c. The method for automatically acquiring and quantifying the UPLC Q-TOF MS data by directly using the GlycReSoft 2.0 software comprises the following two steps: firstly, establishing a pectin oligosaccharide database; then, the oligosaccharide which accords with the database structure in the sample is searched, and the specific parameters are set as follows: minimum abundance: 1.0; minimum number of scans: 1; lower molecular weight bound: 200 of a carrier; upper limit of molecular weight: 2,000; mass transfer: CHOOH; matching error: 5.0ppm; grouping errors: 80ppm; and adduct tolerances: 5.0ppm.
The invention has the following beneficial effects:
(1) Compared with the prior art, the separation and purification method adopts novel strong anion column chromatography, can ensure high binding capacity and high flow rate of the filler and purify the viscous sample at high flow rate, has the advantages of trace quantity, high efficiency, high speed and simple operation, and can shorten the purification time from the original 1 day to 1 hour.
(2) The degradation process of the present invention employs modified H 2 O 2 -V C The technology has the advantages of easily controlled reaction conditions, no metal ions, high efficiency, high speed and good degradation effect, leads the fructose to be degraded thoroughly, reduces the structure loss and is beneficial to the post-treatmentAnd (4) performing LC-MS fine structure analysis.
(3) According to the invention, the HILIC-MS/MS and the bioinformatics software GlycReSoft 2.0 are combined to analyze the structure of the pectin oligosaccharide, so that the automatic qualitative and quantitative analysis of mass spectrum data is realized, the structure analysis rate is greatly improved, and the structure analysis time can be shortened from 2 days to 10 minutes.
Drawings
FIG. 1 is a schematic diagram of an embodiment of the present invention;
FIG. 2 shows the rapid separation and purification of citrus pectin;
FIG. 3 gel permeation chromatogram after purification of citrus pectin;
FIG. 4 shows Superdex Peptide 30 chromatogram after Fenton degradation of citrus pectin purified sample;
FIG. 5 shows HILIC-MS/MS of citrus pectin oligosaccharide;
FIG. 6 is a citrus pectin oligosaccharide quantification calculated from GlycReSoft;
FIG. 7 predicted structural formula of citrus pectin;
FIG. 8 shows the rapid separation and purification of apple pectin;
FIG. 9 gel permeation chromatogram after apple pectin purification;
FIG. 10 Superdex Peptide 30 chromatogram after Fenton degradation of apple pectin purified sample;
FIG. 11 is a HILIC-MS/MS diagram of apple pectin oligosaccharide;
FIG. 12 is the apple pectin oligosaccharide quantification results from GlycReSoft calculations;
FIG. 13 shows the conventional strong anion chromatography separation and purification of citrus pectin;
FIG. 8978 NMR spectrum of zxft 8978; a: 1 H NMR；b： 13 C NMR；c： 13 C NMR(160～185ppm)；d： 13 C NMR(160～185ppm)(D2O，25℃)。。
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, structures, features and effects according to the present invention will be made with reference to the accompanying drawings and preferred embodiments.
The invention is illustrated in FIG. 1 and is further described below by way of example.
Example one: preparation and structure identification of citrus pectin oligosaccharide
Separation and purification are carried out by using HiTrap Capto Q strong anion exchange column. The sample concentration is 10mg/mL, the sample is centrifuged at 12000rpm for 20min, the sample is loaded by 2.5mL, the highest concentration of the gradient elution solution is 1mol/L NaCl solution, the peristaltic pump controls the flow rate to be 2.5mL/min, and the gradient elution is carried out: 0-10min, 0mol/L NaCl; 10-50min, 0-0.8 mol/L NaCl; 50-60min, 1mol/L NaCl; 60-70min, 0mol/L NaCl. One tube was collected every 1min and total sugar and uronic acid content were determined and elution curves were plotted. Samples of the same peak on the combined elution curve are respectively collected and combined, the samples are lyophilized after dialysis of 10kDa, the elution condition is shown in figure 2, the samples are respectively named as SCPW, SCP1, SCP2, SCP3, SCP4 and SCP5 according to the peak appearance sequence, the recovery rates (mass fractions) are respectively 4.3%, 8.5%, 10.3%, 10.7%, 20.6% and 6.2%, the total recovery rate is as high as 60.6%, and the purification efficiency is remarkably improved. Gel chromatogram of purified citrus pectin (SCP 4) is shown in fig. 3, with a molecular weight of 76.18kda, mw/Mn =2.61, and purification significantly improved the homogeneity of the polysaccharide sample compared to pre-purification SCP (Mw/Mn = 4.65).
Taking 6mg of purified SCP4, completely dissolving in 6ml of newly configured 10mmol/L ascorbic acid aqueous solution, adding 60 mu L of 30% hydrogen peroxide solution, placing the reaction system at 100 ℃ for constant temperature reaction for 6h, and then freeze-drying to obtain citrus pectin (SCP 4) oligosaccharide, wherein a Superdex Peptide 30 chromatogram of the citrus pectin (SCP 4) oligosaccharide is shown in figure 4, and after Fenton degradation, SCP4 is almost completely degraded into oligosaccharide with the Degree of Polymerization (DP) of 2-10. The oligosaccharide is re-dissolved in 50% (v/v) acetonitrile water solution, samples are injected in a HILIC-MS/MS system after passing through a membrane, decon Tools are used for deconvolution, glycReSoft 2.0 is used for quantitative analysis, the analysis result is shown in figures 5 and 6, and the data analysis result shows that HG is mainly used in SCP4, a small amount of RG-I structural domain is contained, only a small amount of monosaccharide is detected, and further, the nonmetal Fenton degrades SCP4 completely and has little structural loss. Based on the analysis results, it is speculated that the SCP4 results may be as shown in fig. 7.
Example two: preparation and structure identification of apple pectin oligosaccharide
Separation and purification are carried out by using HiTrap Capto Q strong anion exchange column. The sample concentration is 10mg/mL, the sample is centrifuged at 12000rpm for 20min, the sample is loaded by 2.5mL, the solution with the highest concentration of gradient elution is 1mol/L NaCl solution, the peristaltic pump is controlled by the flow rate to be 2.5mL/min, and the gradient elution is carried out: 0-10min, 0mol/L NaCl; 10-50min, 0-0.8 mol/L NaCl; 50-60min, 1mol/L NaCl; 60-70min, 0mol/L NaCl. One tube was collected every 1min and total sugar and uronic acid content were determined and elution curves were plotted. Samples of the same peak on the combined elution curve are respectively collected and combined, the samples are lyophilized after dialysis of 10kDa, the elution conditions are shown in figure 8, the samples are respectively named as SAPW, SAP1, SAP2, SAP3, SAP4, SAP5 and SAP6 according to the peak appearance sequence, the recovery rates (mass fractions) are respectively 8.4%, 6.3%, 14.6%, 18.9%, 10.7% and 3.3%, the total recovery rate is as high as 62.2%, and the purification efficiency is remarkably improved. The gel chromatogram of apple pectin after purification (SAP 3) is shown in fig. 9, with a molecular weight of 45.82kda and Mw/Mn =1.78, and purification significantly improved the homogeneity of the polysaccharide sample compared to SAP before purification (Mw/Mn = 4.65).
And (3) completely dissolving 6mg of purified SAP3 into 6mL of newly-prepared 10mmol/L ascorbic acid aqueous solution, adding 60 mu L of 30% hydrogen peroxide solution, reacting the reaction system at a constant temperature of 100 ℃ for 6h, and then freeze-drying to obtain the apple pectin (SAP 3) oligosaccharide. Superdex Peptide 30 chromatogram of apple pectin (SAP 3) oligosaccharide is shown in FIG. 10, and it can be seen that SAP3 is almost completely degraded into DP 2-10 oligosaccharide after Fenton degradation. The oligosaccharide was redissolved in 50% acetonitrile water solution, sample was injected in HILIC-MS/MS system after passing through the membrane, and the results were shown in FIG. 11, where FIG. 11 shows the structure of SAP3 oligosaccharide, and it can be seen from the figure that pectin degradation was complete and the loss of structure was small. The data were deconvoluted using Decon Tools followed by quantitative analysis using GlycReSoft, the results of which are shown in FIG. 12, and it can be seen from the data analysis that SAP3 possesses more RG-I domains than SCP4, arabinoglycans and galactosans are linked to RG-I via O-4, and the ratio of HG to RG-I is 3:1.
Comparative example 1: preparation and structure analysis of citrus pectin oligosaccharide
300mL of QFF filler is filled into a 2.6X 30mL glass chromatographic column, and the citrus pectin is separated and purified. Because the chromatographic column is filled with different materials, experimental parameters need to be correspondingly adjusted. The sample concentration is 16mg/mL, the sample is centrifuged for 20min at 8000r/min, the sample is loaded for 50mL, the solution with the highest concentration of gradient elution is 0.6mol/L NaCl solution, the peristaltic pump is controlled to control the flow rate to be 3mL/min, and the gradient elution is carried out: distilled water is used for 0-120 min respectively; 120-240min, 0.2mol/L; 240-360min, 0.4mol/L; 360-480min, 0.6mol/LNaCl, collecting one tube every 5min, measuring the total sugar and uronic acid content, and drawing an elution curve. Respectively collecting samples of the same peak on the combined elution curve, dialyzing by 10kDa and then freeze-drying. The elution conditions are shown in fig. 13, which are respectively named SCP0M, SCP0.2M, SCP0.4M according to the order of their peaks, the recovery (mass fraction) is 4.5%, 19.75% and 21.24%, respectively, the total recovery is 44.99%, which is significantly lower than HiTrap Capto Q strong anion exchange column, and the purification takes 1 day.
Since the sample was not degraded, it took 3 days for the SCP0.4M structure analysis using nuclear magnetic technology, as shown in fig. 14, the structural information was partially lost compared to the mass spectrum, which revealed partial neutral sugars (1-4 linked gal,1-5linked Ara), galacturonic acid backbone information (1-4 linked GalA), and the data processing took 2 days, hindering the pectin structure analysis efficiency.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (6)

1. A method for the purification degradation of pectin, comprising:
(1) Loading the crude polysaccharide solution to a chromatographic column for elution and separation, wherein the filler of the chromatographic column is quaternary ammonium type anion exchange filler;
(2) Dialyzing the eluent obtained in the step (1), and freeze-drying to obtain pectin pure polysaccharide;
(3) Dissolving the pectin pure polysaccharide obtained in the step (2) in water to obtain a pectin polysaccharide water solution with the concentration of 1-2 mg/ml; adding ascorbic acid, mixing uniformly, adding a hydrogen peroxide solution, and reacting at a constant temperature of 80-100 ℃; wherein the concentration of the ascorbic acid is 10-12 mM, the volume fraction of the hydrogen peroxide solution is 1%, and the pectin oligosaccharide is obtained by freeze drying after the reaction is finished.
2. The method according to claim 1, wherein in step (1), the concentration of the crude polysaccharide solution is 5 to 6mg/mL.
3. The method according to claim 1, wherein the elution procedure of step (1) is: deionized water for 0-10 min, naCl solution for 10-50min 0-0.8 mol/L, naCl solution for 50-60min 1mol/L, deionized water for 60-70 min and eluent flow rate of 2.5mL/min.
4. The method according to claim 1, wherein in the step (3), the isothermal reaction time is 6 hours.
5. The method according to claim 1, wherein the quaternary ammonium type anion exchange packing in step (1) is agarose gel based.
6. A pectin fine structure rapid analysis method based on mass spectrum is characterized in that: the pectin oligosaccharide obtained in the claim 1 is subjected to combined analysis by HILIC-MS/MS and bioinformatics software GlycReSoft 2.0, so that the automatic qualitative and quantitative analysis of mass spectrum data is realized, and the fine structure of pectin is analyzed.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101891904A (en) * 2010-06-23 2010-11-24 中国农业科学院植物保护研究所 Kelp oligosaccharide and preparation method and application thereof
CN102241754A (en) * 2010-05-12 2011-11-16 曹敏杰 Method for separating and purifying phycoerythrin from bangia fusco-purpurea
CN106893001A (en) * 2017-02-22 2017-06-27 浙江大学 A kind of preparation method of the Ultra-low molecular weight pectin rich in RG I
CN106916863A (en) * 2017-04-28 2017-07-04 福建农林大学 A kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying
CN113667035A (en) * 2021-07-26 2021-11-19 厦门大学 Preparation method of apple pectin oligosaccharide
CN113912745A (en) * 2021-08-12 2022-01-11 浙江大学 Separation and purification method of glycosaminoglycan and preparation method of sulfated oligosaccharide

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102241754A (en) * 2010-05-12 2011-11-16 曹敏杰 Method for separating and purifying phycoerythrin from bangia fusco-purpurea
CN101891904A (en) * 2010-06-23 2010-11-24 中国农业科学院植物保护研究所 Kelp oligosaccharide and preparation method and application thereof
CN106893001A (en) * 2017-02-22 2017-06-27 浙江大学 A kind of preparation method of the Ultra-low molecular weight pectin rich in RG I
CN106916863A (en) * 2017-04-28 2017-07-04 福建农林大学 A kind of method of κ carrageenan oligosaccharides monomer fast separating and purifying
CN113667035A (en) * 2021-07-26 2021-11-19 厦门大学 Preparation method of apple pectin oligosaccharide
CN113912745A (en) * 2021-08-12 2022-01-11 浙江大学 Separation and purification method of glycosaminoglycan and preparation method of sulfated oligosaccharide

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