Studies On Pectin Degradation And Pectin Oligosaccharides

Pectin,one kind of structural component of vegetal cell wall,is an extremely complex heteropolysaccharide which is made up of neutral and acidic units.It can be used as thickening and gelling agent in food industry.Many studies indicate that pectin oligosaccharides,especially oligogalacturonic acid have many biological activities,such as inducing plant resistance,refraining fatty accumulate in rat liver cell and so on.In order to elevate polysaccharides' biological activity,structural modification and reshaping which include degradation are carried out frequently.In this paper,we combined chemical and enzymatic method to degrade pectin.The results were summarized as fellows:1.Physicochemical properties of pectin from citrus fruits:High-performance gel-permeation chromatography(HPGPC)analysis showed that the pectin molecular mass exceed 400 kDa;Gas chromatography(GC)assay showed that it was composed of GalA,Gal,Rha, Ara,Glc,Xyl,with a relative molar ratio of 78.25∶12.79∶4.37∶2.18∶1.83∶0.58; Phenol-sulphuric acid analysis indicated that the total sugar content of pectin was 95.2%;Its Uronic acid content was 77.3%(used m-hydroxydiphenyl method);Titration analysis indicated that the degree of acetylation(DAc) and the degree of methylation(DM) were 29.55%and 77.27%respectively.2.Condition optimization of Fluorophore-assisted carbohydrate electrophoresis(FACE): FACE was used to analyze pectin oligosaccharides.The influence of different factors on the separation of oligosaccharides by FACE was investigated.The condition including the type and the content of fluorescent reagent,label time and temperature,salt,acid and the concentration of resolving gel was optimized as follows:1.2 mg of pectin oligosaccharides (salty-free and acid-free) were labeled by 3.75μL of 0.2 mol·L^-1 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) solution and 5μL of 1.0 mol·L^-1 NaBH₃CN solution.The derivative reaction was accomplished at 40℃for 16 h.The electrophoresis conditions were:8% polyacrylamine in the stacking gel and 38%polyacrylamine in the resolving gel respectively, permanent electric current of 15 mA.Under the optimized condition,we got clear electrophoresis strips and a well separation result.3.The establishment of a novel high performance liquid chromatogram(HPLC) precolumn derivatization method for pectin composition analysis:We analyzed AEC-derivatitized monosaccharide standards by HPLC,optimized the chromatographic condition and validated the analysis method.Under the optimized Chromatographic condition and validated the analysis method.Under the optimized Chromatographic conditions,mobile phase was 28∶72 of acetonitrile-water(0.1 mol·L^-1ammonium acetate buffer,pH=4.3),at a flow rate of 1 mL·min^-1,with an UV detector at 235 nm, monosaccharides in pectin could be well separated.Detection limits for the samples was 0.05 ng·μL^-1.We applied this method on the pectin composition analysis and compared with gas chromatography(GC) method.The result showed that HPLC result was consistent with that of GC,which indicateded that the established method is applicable to pectin composition analysis.4.Pectin degradation and preparation of oligogalacturonic acid:we optimized the degradation condition by orthogonal method.Finally,we combined acidic hydrolysis(0.5 mol·L^-1TFA,80℃,5 h) with enzymolysis(0.05 mg·mL^-1 enzyme solution,60 min) method to prepare oligogalacturonic acid.5.Activities assay of pectin and pectin hydrolysates:The bacteriostatic activities of pectin and pectin hydrolysates were investigated.The results showed that only oligogalacturonic acid could inhibit bacteria(Escherichia coli,Staphylococcus) proliferation. In addition,we studied the anti-tumor(human hepatoma carcinoma cell SMMC-7721) activities of pectin and pectin hydrolysates.The results indicateded that only oligogalacturonic acid had the anti-tumor activity.