CN115181194B - Quick purification degradation and structure analysis method of pectin - Google Patents
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Abstract
The invention discloses a method for purifying and degrading pectin and analyzing structure. The pectin is separated and purified by adopting the quaternary ammonium strong anion exchange material, and the method has the characteristics of small sample consumption, short time and simple operation; the pectin obtained by purification is degraded by hydrogen peroxide as a reactant and ascorbic acid as a catalyst, optimizing the radical degradation (H 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, the HILIC-MS/MS and bioinformatics software GlycReSoft 2.0 are adopted for combined analysis, so that automatic qualitative and quantitative analysis of mass spectrum data is realized, and a pectin fine structure is analyzed.
Description
Technical Field
The invention relates to the field of plant pectin polysaccharide oligosaccharides, in particular to a method for purifying and degrading pectin and analyzing the structure.
Background
Pectin (Pectin) is a class of galacturonic acid (GalA) -rich heteropolysaccharides that are widely found in the intercellular layer and primary wall of plant cells. Prior studies have roughly divided pectin molecules into 4 classes of domains, depending on their backbone and branched structure: homogalacturonan (HG), rhamnogalacturonan I (Rhamngalacturonan I, RGI), rhamnogalacturonan II (RGII), and Xylogalacturonan (XG). Pectin is complex in structure (50-1000 kDa) and relatively viscous, and pectin is also diverse in its composition depending on source, extraction conditions, plant development stage, plant species, cell wall location and environmental factors, as yet there is a question about the relative positions of pectin domains and how these domains are linked in pectin. The prior research shows that the structure of pectin is closely related to the functions of pectin, and the pectin with different structures has obvious differences in aspects of gel composition, intestinal flora regulation, colitis resistance and the like. Therefore, analyzing the fine structure of pectin is a precondition for utilizing abundant pectin resources.
The structural analysis of the complete pectic polysaccharide is very difficult. Since the extracted pectin crude polysaccharide contains more impurities (proteins, polyphenols, starch, some small molecular substances and the like) to influence the subsequent analysis, the pectin crude polysaccharide must be separated and purified. The traditional method combines the methods of multistage separation and purification, periodic acid-Smith degradation, methylation, nuclear magnetic resonance spectrum and the like to solve the problems of time consumption, complex operation and fine structure loss of pectolysaccharide analysis. Therefore, development of a rapid, efficient and trace pectic polysaccharide fine structure analysis method is an inevitable trend at present.
Currently, more and more studies use controlled methods to degrade pectin into oligosaccharides and analyze the oligosaccharides using liquid phase mass spectrometry techniques. Common polysaccharide degradation methods include acid degradation, enzymatic degradation, free radical degradation, and the like. Wherein, the acid degradation reaction is severe and difficult to control, and the fine structure of the polysaccharide is lost; the enzymatic degradation has the advantages of strong specificity, mildness and controllability, but the related enzymes are few in types, and complex pectic polysaccharide cannot be completely degraded. Based on Fenton system (H) 2 O 2 ) The free radical degradation is different from the specificity of enzymolysis and the reaction of acid method is intense, the free radical degradation can be applied to all kinds of polysaccharide, the reaction can be controlled by the related conditions, the branch fracture or the loss of sulfuric acid group is not caused, the reproducibility is good, and the method generally needs to use metal Cu 2+ And Fe (Fe) 2+ Auxiliary ultrasonic is carried out, but the two metal ions are required to be removed by adopting a certain method after the reaction, so that the reaction time is prolonged, and the analysis efficiency is reduced. Therefore, there is a need for improvements in the radical degradation process to create a nonmetallic controlled radical degradation process.
Disclosure of Invention
Aiming at the defects of the prior pectin structure analysis method in the background technology, the invention applies the Ji Anjiang anion exchange chromatographic column to the purification and separation of pectin for the first time, and can obviously improve the purification efficiency; the method for degrading the pectin by the free radicals after purification is established, so that the pectin is thoroughly degraded, meanwhile, the loss of the structure is reduced, and the method is particularly suitable for LC-MS/MS detection, and the analysis of the fine structure of the pectin is realized; and the rapid processing (10 min) of the mass spectrum data is realized by combining biological information software, so that the analysis efficiency is remarkably improved.
The invention provides a method for rapidly purifying pectin, degrading nonmetal Fenton of pectin and performing HILIC-MS/MS analysis on pectin oligosaccharide obtained by degradation, which can be used for preparing pectin oligosaccharide and analyzing pectin fine structure, and specifically comprises the following steps:
1. purification and degradation of pectin
(1) Loading the crude polysaccharide solution to a chromatographic column for eluting and separating, 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 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 effectively collected by a collection method commonly used in the art. For example, 1 tube of eluent is collected per minute, the component of the eluent is detected by analyzing the peak position of the eluent, and the same components are screened out and combined.
The analytical method can be a trace phenol-sulfuric acid method: after adding 20. Mu.L of a 5% (v/v) phenol aqueous solution to 40. Mu.L of the sample, 200. Mu.L of concentrated sulfuric acid was added thereto, and the mixture was allowed to stand for 15 minutes to develop color, and the mixture was detected at a wavelength of 490 nm. The micro m-hydroxy biphenyl method can also be adopted: 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 for 20min at 90 ℃ in an oven, cooling to room temperature, adding 4 mu L of m-hydroxybiphenyl, mixing uniformly, and detecting at 525nm wavelength.
Further, the concentration of the crude polysaccharide solution is 5-6 mg/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 50-60 min and 1mol/L, deionized water for 60-70 min, and the flow rate of eluent is 2.5mL/min.
Further, in the step (3), the constant temperature reaction time is 6 hours.
Further, the quaternary ammonium type anion exchange packing in the step (1) uses agarose gel as a matrix.
2. Performing structure identification on the pectin oligosaccharide obtained above
The invention adopts a structure analysis method based on HILIC-MS/MS combined bioinformatics software, and the specific method is as follows:
a. dissolving pectin oligosaccharide obtained in the step (3) in acetonitrile water solution with the volume fraction of 50%, wherein the concentration is 1-2 mg/ml;
b. and c, separating the oligosaccharide from the solution obtained in the step a by using an ultra-high performance liquid phase system and combining with a time-of-flight Q-TOF mass spectrum for mass spectrum detection.
Liquid phase detection conditions: ultra-high performance liquid chromatograph Waters Acqtity UPLC classic; chromatography column UPLC-Amide (2.1X105 mm,1.7 μm); mobile phase a phase-20% acetonitrile +10mM ammonium formate +0.2% formic acid (v/v), B phase-80% acetonitrile +10mM ammonium formate +0.2% formic acid (v/v); the flow rate is 0.25mL/min; column temperature is 35 ℃; time gradient: 0- & gt 35- & gt 50- & gt 55- & gt 56; concentration gradient: 100% →70% →20% →100% phase b, sample volume: 5. Mu.L.
Mass spectrometry conditions: triple-TOF 5600 + Time-of-flight liquid chromatography-mass spectrometry; an anion scanning mode; scanning range: m/z is 100-1500; atomizing gas: 55psi; atomizing gas: 55psi; curtain gas (CUR): 35psi; ion source Temperature (TEM): 550 ℃; ion source voltage (IS): -4500V; primary scanning: de-clustering voltage (DP): 100V; focus voltage (CE): 10V; and (3) secondary scanning: and acquiring mass spectrum data by using a TOF MS-Product Ion-IDA mode, wherein CID energy is 40+/-20 eV, and performing mass axis correction by using a CDS pump before sample injection to ensure that the mass axis error is less than 2ppm.
c. The UPLC Q-TOF MS data is automatically acquired and quantified directly using GlycReSoft 2.0 software, comprising two steps: firstly, establishing a pectin oligosaccharide database; then searching the oligosaccharide conforming to the database structure in the sample, wherein the specific parameters are set as follows: minimum abundance: 1.0; minimum number of scans: 1, a step of; lower molecular weight boundary: 200; upper limit of molecular weight: 2,000; mass transfer: CHOOH; matching error: 5.0ppm; grouping error: 80ppm; and adduct tolerance: 5.0ppm.
The invention has the following beneficial effects:
(1) Compared with the prior art, the separation and purification method adopts the novel strong anion column chromatography, can ensure the high binding capacity and high flow rate of the filler, purify the viscous sample at the high flow rate, has the advantages of high efficiency and rapidness, and simple operation, and can shorten the purification time from 1 day to 1 hour.
(2) The degradation method of the invention adopts improved 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, ensures complete degradation of fructose, reduces structure loss, and is beneficial to subsequent LC-MS fine structure analysis.
(3) According to the invention, the HILIC-MS/MS and bioinformatics software GlycReSoft 2.0 are combined to analyze the structure of the pectin oligosaccharide, so that the automatic qualitative and quantitative of mass spectrum data are 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 illustration of an embodiment of the present invention;
FIG. 2 shows a rapid separation and purification diagram of citrus pectin;
FIG. 3 gel permeation chromatogram after citrus pectin purification;
FIG. 4 shows a Superdex Peptide 30 chromatogram after Fenton degradation of a citrus pectin purification sample;
FIG. 5 HILIC-MS/MS diagram of citrus pectin oligosaccharide;
FIG. 6 is a quantitative result of citrus pectin oligosaccharides calculated from GlycReSoft;
FIG. 7 shows a schematic structural formula of citrus pectin;
FIG. 8 is a rapid separation and purification diagram of apple pectin;
FIG. 9 gel permeation chromatogram after apple pectin purification;
FIG. 10 shows a Superdex Peptide 30 chromatogram after Fenton degradation of an apple pectin purification sample;
FIG. 11 shows a HILIC-MS/MS diagram of apple pectin oligosaccharides;
FIG. 12 is a quantitative result of apple pectin oligosaccharides calculated from GlycReSoft;
FIG. 13 is a diagram of a conventional strong anion chromatography separation and purification of citrus pectin;
the nuclear magnetic spectrum of fig. 14SCP0.4M; a: 1 H NMR；b： 13 C NMR；c： 13 C NMR(160～185ppm)；d： 13 C NMR(160～185ppm)(D2O，25℃)。。
Detailed Description
In order to further describe the technical means and effects adopted by the present invention for achieving the intended purpose, the following detailed description will refer to the specific implementation, structure, characteristics and effects according to the present invention with reference to the accompanying drawings and preferred embodiments.
The implementation of the invention is illustrated in fig. 1, and the invention is further described below with reference to examples.
Example one: preparation and structure identification of citrus pectin oligosaccharide
The HiTrap Capto Q strong anion exchange column was used for separation and purification. The concentration of the sample is 10mg/mL, the sample is centrifuged at 12000rpm for 20min, the loading is 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: 0-10 min,0mol/L NaCl;10 to 50min,0 to 0.8mol/L NaCl; 50-60 min,1mol/L NaCl; 60-70 min,0mol/L NaCl. One tube was collected every 1min and the total sugar and uronic acid content was determined and an elution profile was drawn. Samples of the same peak on the combined elution curve are respectively collected, dialyzed and freeze-dried at 10kDa, elution conditions are shown in figure 2, and are respectively named SCPW, SCP1, SCP2, SCP3, SCP4 and SCP5 according to the peak outlet sequence, the recovery rate (mass fraction) of the SCP5 is respectively 4.3%, 8.5%, 10.3%, 10.7%, 20.6% and 6.2%, the total recovery rate is up to 60.6%, and the purification efficiency is remarkably improved. The gel chromatogram of the purified citrus pectin (SCP 4) is shown in fig. 3, the molecular weight is 76.18kda, mw/mn=2.61, and compared with the SCP (Mw/mn=4.65) before purification, the purification significantly improves the uniformity of polysaccharide samples.
6mg of purified SCP4 is taken and completely dissolved in 6ml of newly prepared 10mmol/L ascorbic acid aqueous solution, 60 mu L of 30% hydrogen peroxide solution is added, the reaction system is placed at a constant temperature of 100 ℃ for reaction for 6 hours, and then freeze drying is carried out, so that citrus pectin (SCP 4) oligosaccharide is obtained, the Superdex Peptide 30 chromatogram of the citrus pectin (SCP 4) oligosaccharide is shown as figure 4, and after Fenton degradation, the SCP4 is almost completely degraded into oligosaccharide with a polymerization Degree (DP) of 2-10. The oligosaccharides were redissolved in 50% (v/v) acetonitrile aqueous solution, after membrane filtration, were sampled in HILIC-MS/MS system, and after deconvolution of the data using Decon Tools, quantitative analysis was performed using GlycReSoft 2.0, and the analysis results are shown in FIG. 5 and FIG. 6. As can be seen from the data analysis results, SCP4 is mostly HG-containing with small amount of RG-I domain, only small amount of monosaccharides was detected, further demonstrating that nonmetallic Fenton degraded SCP4 thoroughly, and the structure loss was small. From the analysis results, the result of the SCP4 is presumed to be as shown in FIG. 7.
Example two: preparation and structure identification of apple pectin oligosaccharide
The HiTrap Capto Q strong anion exchange column was used for separation and purification. The concentration of the sample is 10mg/mL, the sample is centrifuged at 12000rpm for 20min, the loading is 2.5mL, the solution with the highest gradient elution concentration is 1mol/L NaCl solution, the peristaltic pump controls the flow rate to be 2.5mL/min, and the gradient elution is carried out: 0-10 min,0mol/L NaCl;10 to 50min,0 to 0.8mol/L NaCl; 50-60 min,1mol/L NaCl; 60-70 min,0mol/L NaCl. One tube was collected every 1min and the total sugar and uronic acid content was determined and an elution profile was drawn. Samples of the same peak on the combined elution curve are respectively collected, dialyzed against 10kDa and freeze-dried, elution conditions are shown in FIG. 8, the samples are respectively named SAPW, SAP1, SAP2, SAP3, SAP4, SAP5 and SAP6 according to the peak-out sequence, recovery rates (mass fraction) are respectively 8.4%, 6.3%, 14.6%, 18.9%, 10.7% and 3.3%, the total recovery rate is up to 62.2%, and the purification efficiency is remarkably improved. The gel chromatogram of the apple pectin (SAP 3) after purification, as shown in fig. 9, has a molecular weight of 45.82kda, mw/mn=1.78, and significantly improved the homogeneity of the polysaccharide sample compared to the SAP before purification (Mw/mn=4.65).
6mg of purified SAP3 is taken and completely dissolved in 6mL of newly prepared 10mmol/L ascorbic acid water solution, 60 mu L of 30% hydrogen peroxide solution is added, the reaction system is placed at a constant temperature of 100 ℃ for reaction for 6 hours, and then the apple pectin (SAP 3) oligosaccharide is obtained after freeze drying. The Superdex Peptide 30 chromatogram of apple pectin (SAP 3) oligosaccharides is shown in FIG. 10, and it can be seen that SAP3 was almost completely degraded to DP 2-10 oligosaccharides after Fenton degradation. The oligosaccharide is redissolved in 50% acetonitrile water solution, and after passing through the membrane, the sample is injected in an HILIC-MS/MS system, and the results are shown in FIG. 11, the FIG. 11 shows the SAP3 oligosaccharide structure, and the pectin is thoroughly degraded and has little structure loss. The data were quantitatively analyzed using GlycReSoft after deconvolution using Decon Tools, and the analysis results are shown in FIG. 12. As can be seen from the data analysis results, SAP3 possesses more RG-I domain than SCP4, arabinan and galactosan are linked to RG-I through O-4, and HG and RG-I are in a 3:1 ratio.
Comparative example 1: preparation and structural analysis of citrus pectin oligosaccharides
The citrus pectin is separated and purified by loading 300mL of QFF filler into a 2.6x30 mL glass chromatographic column. Because the chromatographic column has different filling materials, experimental parameters need to be correspondingly adjusted. The concentration of the sample is 16mg/mL, the centrifugation is carried out for 20min at 8000r/min, the loading is 50mL, the solution with the highest gradient elution concentration is 0.6mol/L NaCl solution, the peristaltic pump controls the flow rate to be 3mL/min, and the gradient elution is carried out: respectively using distilled water for 0-120 min; 120-240 min,0.2mol/L;240 to 360min,0.4mol/L; 360-480 min,0.6mol/LNaCl, one tube was collected every 5min, and the total sugar and uronic acid content were determined, and elution curves were plotted. Samples were collected from the same peak on the pooled elution curves, dialyzed against 10kDa and lyophilized. The elution conditions are shown in fig. 13, and are named SCP0M, SCP0.2M, SCP0.4M according to the peak-out sequence thereof, the recovery rates (mass fraction) are 4.5%, 19.75% and 21.24%, respectively, the total recovery rate is 44.99%, which is significantly lower than that of the HiTrap Capto Q strong anion exchange column, and the purification takes 1 day.
Because the sample is not degraded, the nuclear magnetic technology is adopted to analyze the SCP0.4M structure, which takes 3 days, as shown in FIG. 14, the structure information is partially lost compared with the mass spectrum, so that partial neutral sugar (1-4 linked Gal,1-5linked Ara) and galacturonic acid main chain information (1-4 linked GalA) can be revealed, and the data processing takes 2 days, which hinders the pectin structure analysis efficiency.
The present invention is not limited to the above embodiments, but is capable of modification and variation in detail, and other modifications and variations can be made by those skilled in the art without departing from the scope of the present invention.
Claims (1)
1. A method for purifying and degrading pectin, which is characterized by at least comprising the following steps:
(1) Loading the crude polysaccharide solution to a chromatographic column for eluting and separating, wherein the chromatographic column is a HiTrap Capto Q strong anion exchange column; the concentration of the crude polysaccharide solution is 5-6 mg/mL; the elution procedure was: deionized water for 0-10 min, naCl solution for 10-50 min and 0-0.8 mol/L, naCl solution for 50-60 min and 1mol/L, deionized water for 60-70 min, and the flow rate of eluent is 2.5mL/min;
(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 hydrogen peroxide solution, and reacting at a constant temperature of 80-100 ℃ for 6 hours; 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.
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