The Study On The Controlled Degradation Of Citrus Pectic Polysaccharide And The Purification And Activity Of Oligogalacturonic Acid Fragments

Pectin is a family of complex polysaccharides present in all plant primary cell walls and widely used in the food industry as a gelling agent and in the health care industry as a immunomodulator. The results of many studies on pectin suggested that pectic oligosaccharides, especially oligogalacturonic acids (oligo-GalAs), possessed many biological activities which were different from that of pectic polysaccharide. In this study, the physico-chemical properties and degradation of Citrus pectic polysaccharides and the isolation, purification, structure characterization and antioxidant activity of oligosaccharides were investigated. The results were summarized as follows:1. The citrus pectic polysaccharide (CPPS) consists of galacturonic acid, galactose, rhamnose, arabinose, glucose and xylose with the molar ratios of 42.8:7.0:2.4:1.2:1.0:0.3 by gas chromatography assay. The content of total sugar was assayed to be 80.5% by phenol-sulphuric acid method and the content of uronic acid was 71.6% by m-hydroxydiphenyl method. With high-performance liquid chromatography assay, degrees of methylation and acetylation were assayed to be 78.5% and 28.5% in citrus pectic polysaccharide, respectively. Therefore, CPPS is a kind of acidic heteropolysaccharide with high degree of esterification.2. The degradation is an ideal approach to manufacture oligosaccharides from polysaccharide. Firstly, different pectin hydrolysis procedures were carried out and the optimal parameter values were achieved. A high productivity of oligo-GalAs was obtained under emzymatic hydrolysis condition using pectic acid as the substrate, so this method was suitable for preparation of oligo-GalAs with DP 1-5. Secondly, combined chemical and enzymatic hydrolysis (0.5 mol/L trifluoroacetic acid at 80℃for 5 h and 0.05 mg/mL pectinase at 40℃for 1 h) could offer more oligosaccharide fragments, so this method could be employed to prepare the oligo-GalAs with DP>5.Thirdly, based on aniline stable isotopic labeling and relative quantification in ESI-MS analysis, the optimization of the preparation of active pentogalacturonic acid was investigated. The yield of pentogalacturonic acid reached 20% under the optimized condition.3.For a long time, the detection of oligosaccharides is one of the difficulties in the study on saccharide. The two analytical methods for pectic oligosaccharides were established in this study. Firstly, thin layer chromatography (TLC) was applied to the analysis of acidic and neutral oligosaccharides and the conditions were optimized. The results showed that it is an effective and convenient method for the analysis of saccharides. The separation of neutral oligosaccharides was improved by multiple development with n-butanol-acetic acid-water (3:3:2) and acidic oligosaccharides with n-butanol-formic acid-water (4:7:1) on TLC silica gel-100 UV254 plates. On that basis, a TLC scanning method was developed for qualitative analysis of oligosaccharides accurately. Secondly, the simultaneous determination of aldoses and uronic acids was available by high-performance liquid chromatography. The procedure involves acid hydrolysis followed by derivatization with 3-amino-9-ethyl-carbazole, which was first employed for aldoses and uronic acids derivatization. The usefulness of this method was seen in the ability to analyze citrus pectin. Compared to the GC method. The described method was suitable for routine analysis of pectin or other polysaccharides containing uronic acids.4. Firstly, an efficient and inexpensive method for large-scale preparation of oligo-GalAs (DP 1-5) from pectic acid was described. The mixture of oligo-GalA was obtained after pectic acid was digested with an available pectinase, and this mixture was effectively isolated by the anion exchange resin (Dowex-1×4-400, HCOO-) to obtain pure oligo-GalA of DP 1-5 with the yielding rate of 3.5%,12.7%,8.0% and 8.2% for DP 2-5, respectively. Secondly, another efficient method for large-scale preparation of the neutral pectic oligosaccharides was described. The mixture of pectic oligosaccharides was obtained after pectin was hydrolysed by trifluoroacetic acid. Then the mixture was effectively isolated by a combination of anion exchange resin (Dowex-1×4-400, HCOO-) and silica gel column chromatography to obtain pure neutral pectic oligosaccharides of DP 1-5, which were deduced to be probably present as (Hex)n (n=2-10) based on the analysis of GC and ESI-MS assay. Thirdly, a preparation method of rhamnogalacturonans I (RG-I) fragment was investigated. Pectin was hydrolysed by hydrochloric acid followed by trifluoroacetic acid. The hydrolysate was composed of galacturonic acid, galactose and rhamnose with the molar ratio of Rha to GalA as 0.17, which indicated the presence of RG-Ⅰfragment in the hydrolysates.5.The results of the biological activity experiments for oligo-GalA with different degree of polymerization (DP) showed that higher DP of oligo-GalA had stronger biological activities. Only oligo-GalA with DP more than 5 showed apparent inhibitory effect on the putrefying bacteria in food, antitumor effect on human hepatoma cell line SMMC7721 and scavenging effect on DPPH free radicals.