Preparation Of HG-type Pectin And Distribution Of Methyl-esterification

Homogalacturonan（HG）is a kind of pectic polysaccharide existing in plant cell walls,with diverse biological activities and wide application fields.HG-type pectin is a linear-（1,4）-linked galacturonic acid（Galp A）homopolymer,and the Galp A residue can form methylester substitution on the C-6 carboxyl group.Factors such as the degree of methylesterification（DM）and the distribution of methylester groups are important fine structure features.At present,the research on the fine structure and methylester group distribution of HG-type pectin is not thorough enough,which limits the research on its structure-activity relationship and the development and application of related products.This paper takes HG-type pectins from different plant sources as the research object,and systematically analyzes the structural characteristics of these HG-type pectins,and focuses on the analysis of distribution pattern of methylester structural units in HG pectin.The specific research results are as follows:Eighteen HG pectins with uniform charge and molecular weight distribution were isolated and prepared from ten plants,including Panax japonicus,Pseudostellaria heterophylla and Panax notoginseng by means of hot water extraction,ethanol precipitation,ion exchange chromatography and gel chromatography.The molecular weight distribution of these HG-type pectins ranged from 10 k Da to 60 k Da,the content of Galp A in monosaccharide composition was above 65%.The results of specific optical rotation showed that these HG-type pectins were all dextrorotatory polysaccharides,and the specific optical rotation was positively correlated with the content of Galp A in HG-type pectins.The characteristic signal peaks of→4)-α-Galp A-(1→can be clearly observed in the ¹³C nuclear magnetic resonance spectrum(¹³C-NMR),which proved that these pectin molecules mainly contain HG-type pectin.At the same time,the signal peaks of methylester groups with different intensities were observed in these NMR spectra,indicating that these HG molecules were modified with different degrees of methylesterification;No obvious signal peak of acetyl group was observed,indicating these HG molecules contain only a small amount of acetylation.In order to accurately determine the DM of the above HG-type pectins,this paper prepared standard products of HG pectins with different DM,and comprehensively evaluated the accuracy,advantages and disadvantages of titration,infrared spectroscopy（FT-IR）and high performance liquid chromatography（HPLC）methods for the determination of DM.Due to the large amount of samples required for the determination of DM by titration,The DM of eighteen HG-type pectins was further determined by FT-IR and HPLC.The results showed that the DM values of these HG-type pectins ranged from 6%to 40%,and they were all low esterified pectin（DM<50%）.The values of the two methods to determine the DM of the same HG-type pectin were relatively close,and there was no significant difference between the DM values determined by these two methods.Both FT-IR and HPLC methods can use a small amount of samples to determine the DM of HG pectin.The FT-IR method is relatively simple and does not damage the pectin structure.The samples can be reused after recovery,but the determination results are easily affected when the samples contain more acetyl groups.The DM determination of HG pectin by HPLC method is more complicated than FT-IR method,which will destroy the structure of HG pectin.However,the acetylation degree can be determined simultaneously when the sample contains acetyl group.In order to further analyze the distribution of methylester groups in HG-type pectin,endopolygalacturonase（Endo-PG）was used to degrade HG-type pectins to obtain oligogalacturonides（OGA）structural units.Combining with the pre-column fluorescent labeling（Anthranilamide,2-AB）technology,a high-efficiency analytical method of hydrophilic chromatography-fluorescence detector-electrospray mass spectrometry（HILIC-FLR/ESI-MSn）was established.On-line separation,structure analysis and content determination of OGA structural units were carried out,and the fingerprint of HG-type pectin hydrolysis products was established.A total of 21 OGA monomers were detected from the OGA structural units of 18 HG-type pectins.The number of OGA monomers of different HG-type pectins was proportional to their DM value,ranging from 7 to 21.The degree of polymerization（DP）of these OGA monomers was between 1 and 10,and the number of methylester groups they contained was 1 to 6.In the methylester substituted OGA monomer,the substitution number of methylester groups increased with the increase of the degree of polymerization of the monomer.The MSⁿanalysis results showed that the number of continuous distributions of methylester groups in these OGA monomers was between 2 and 5,and there was a gap of 1 to 2 Galp A residues between the two continuous distributions of methylester groups.Quantitative analysis showed that in different HG-type pectins,the content of OGA monomer decreased with the increase of the number of methylester substitution and the degree of polymerization;The proportion of OGA monomer unesterified or mono-methylester substitution was the highest,accounting for 54.8%～97.1%of the total structural units.According to the molecular weight of HG-type pectins,the type and content of OGA structural unit monomers produced by enzymatic hydrolysis and other parameters,the number of OGA structural unit monomers(N_oligo)and methylester group substituted OGA structural unit module（Block）(N_Block)can be calculated.To characterize the distribution pattern of methylesterification structural units in HG pectin,we assumed that the"Blocks"were evenly distributed,and defined the average number of Galp A residues spaced between"Blocks"as"AVE DP_unesterified",which was an important parameter to reflect the distribution pattern of methylesterification structural units.According to the above parameters,the possible distribution of methylesterification structural units in HG-type pectins was inferred.The"AVE DP_unesterified"values of 18 HG-type pectins decreased with the increase of DM value,indicating that the interval of methylesterification structural unit decreased gradually,between 4 and 15.According to the above three parameters,the pectin structure models of four classes of DM were constructed,which more intuitively reflected the distribution of methylesterification structural units in different DM pectins.Taken together,this paper prepared HG-type pectins from different plants,characterized their overall structure and measured the degree of methylesterification.These HG-type pectins were degraded by glycosidase to obtain different methylesterified oligogalacturonic acid structural units.The strategies and methods for the fine structure analysis of oligogalacturonic acid structural units and the distribution of methylesterification structural units were established,and the distribution characteristics of methylesterification structural units in 18 HG-type pectins were systematically analyzed.These findings enrich the structural information of HG-type pectin,provide a theoretical basis for further structure-activity relationship research,and provide new ideas and methods for the structural analysis of HG-type pectin.