[en] A new method for the determination of the main neutral sugars in pectin has been developed. The sample preparation involves a mild chemical attack followed by an enzymatic hydrolysis. The completeness and nondestructive character of the method are demonstrated by comparison of the results obtained with different acids such as H2SO4, HCI, and trifluoroacetic acid (TFA) at different concentrations (2, 1, or 0.2 M) at two temperatures (80 or 100 degreesC). The chemical hydrolysis of pectin neutral sugar chains with strong acid (1 or 2 M) and high temperature (100 degreesC) shows that the liberation of the pectin sugars is not realized at the same rate for each sugar. Different optimum conditions are thus obtained. However, the chemical pectin hydrolysis with 0.2 M TFA at 80 degreesC is characterized by the liberation of pectin neutral sugar side chains without any degradation within 72 h of hydrolysis. Under these conditions, the liberation of some pectin sugars, essentially galactose, glucose, and rhamnose, was not complete. An enzymatic hydrolysis is necessary to obtain a complete release of all the sugars. The combination of the two treatments, a chemical hydrolysis realized with diluted acid (0.2 M) for 72 h at low temperature (80 degreesC) on one hand and an enzymatic hydrolysis on the other hand, allow a total liberation of pectin sugars. The quantitative analysis of the carbohydrates is realized with accuracy, high selectivity, and sensitivity with high-performance anion-exchange chromatography with pulsed-amperometric detection. The sugars can be analyzed without any derivatization with a limit of quantification of 0.1 mM.
Disciplines :
Agriculture & agronomy
Author, co-author :
Garna, H.
Mabon, N.
Wathelet, Bernard ; Université de Liège - ULiège > Gembloux Agro-Bio Tech > Gembloux Agro-Bio Tech
Paquot, Michel ; Université de Liège - ULiège > Gembloux Agro-Bio Tech > Gembloux Agro-Bio Tech
Language :
English
Title :
New method for a two-step hydrolysis and chromatographic analysis of pectin neutral sugar chains
Publication date :
2004
Journal title :
Journal of Agricultural and Food Chemistry
ISSN :
0021-8561
eISSN :
1520-5118
Publisher :
American Chemical Society, Washington, United States - District of Columbia
Matsuhashi, S.; Inoue, S. I.; Hatanaka, C. Simultaneous measurement of the galacturonate and neutral sugar contents of pectic substances by an enzymic-HPLC method. Biosci., Biotechnol., Biochem. 1992, 56, 1053-1057.
Dutton, G. G. S. Adv. Carbohydr. Chem. Biochem. 1973, 28, 11-160.
Biermann, C. J. Hydrolysis and other cleavages of glycosidic linkages in polysaccharides. Adv. Carbohydr. Chem. Biochem. 1988, 46, 251-271.
De Ruiter, G. A.; Schols, H. A.; Voragen, A. G. J.; Rombouts, F. M. Carbohydrate analysis of water-soluble uronic acid containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods. Anal. Biochem. 1992, 207, 176-185.
Saeman, J. F.; Moore, W. E.; Mitchell, R. L.; Millett, M. A. Techniques for the determination of pulp constituents by quantitative paper chromatography. Tappi 1954, 37, 336-343.
Englyst, H. N.; Cummings, J. H. Simplified method for the measurement of total non-starch polysaccharides by gas-liquid chromatography of constituent sugars as alditol acetates. Analyst 1984, 109, 937-942.
Quemener, B.; Lahaye, M.; Thibault, J.-F. Studies on the simultaneous determination of acid and neutral sugars of plant cell wall materials by HPLC of their methyl glycosides after combined methanolysis and enzymatic pre-hydrolysis. Carbohydr. Polym. 1993, 20, 87-94.
Albersheim, P.; Nevins, D. J.; English, P. D.; Karr, A. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 1967, 5, 340-345.
Chambers, R. E.; Clamp, J. R. An assessment of methanolysis and other factors used in the analysis of carbohydrate-containing materials. Biochem. J. 1971, 125, 1009-1018.
Cheetham, N. W. H.; Sirimanne, P. Methanolysis studies of carbohydrates using HPLC. Carbohydr. Res. 1983, 112, 1-10.
Quemener, B.; Thibault, J.-F. Assessment of methanolysis for the determination of sugars in pectins. Carbohydr. Res. 1990, 206, 277-287.
Roberts, E. J.; Godshall, M. A.; Clarke, M. A.; Tsang, W. S. C.; Parrish, F. W. Methanolysis of polysaccharides: a new method. Carbohydr. Res. 1987, 168, 103-109.
Marga, F.; Freyssac, V.; Morvan, H. Rapid gas liquid chromatography microanalysis of carbohydrates in woody plant tissues. J. Trace Microprobe Tech. 1995, 13, 473-478.
Bertaud, F.; Sundberg, A.; Holmbom, B. Evaluation of acid methanolysis for analysis of wood hemicelluloses and pectins. Carbohydr. Polym. 2002, 48, 319-324.
Ip, C. C. Y.; Manam, V.; Hepler, R.; Hennessey, J. P. High-performance anion exchange chromatography for carbohydrate analysis. Anal. Biochem. 1992, 31, 343-349.
Selvendran, R.; March, J. F.; Ring, S. Determination of aldose and uronic content of vegetable fiber. Anal. Biochem. 1979, 18, 282-292.
Blakeney, A. B.; Harris, P. J.; Henry, R. J.; Stone, B. A. A simple and rapid preparation of alditol acetates for monosaccharides analysis. Carbohydr. Res. 1983, 113, 291-299.
Garleb, K. A.; Bourquin, L. D.; Fahey, G. C. Neutral monosaccharide composition of various fibrous substrates: A comparison of hydrolytic procedures and use of anion-exchange high-performance liquid chromatography with pulsed amperometric detection of monosaccharides. J Agric. Food Chem. 1989, 37, 1287-1293.
Quigley, M.; Englyst, H. N. Determination of the uronic acid constituents of non-starch polysaccharides by high-performance liquid chromatography with pulsed amperometric detection. Analyst 1994, 119, 1511-1518.
Salvador, L. D.; Suganuma, T.; Kitahara, K.; Tanoue, H.; Ichiki, M. Monosaccharide composition of sweetpotato fiber and cell wall polysaccharides from sweetpotato, cassava and potato analysed by the high-performance anion exchange chromatography with pulsed amperometric detection method. J. Agric. Food Chem. 2000, 48, 3448-3454.
Le Goff, A.; Renard, C. M. G. C.; Bonnin, E.; Thibault, J.-F. Extraction, purification and chemical characterization of xylogalacturonans from peal hulls. Carbohydr. Polym. 2001, 45, 325-334.
Churms, S. C. Recents developments in the chromatographic analysis of carbohydrates. J. Chromatogr. 1990, 500, 555-583.
Hicks, K. B. High performance liquid chromatography of carbohydrates. Adv. Carbohydr. Chem. Biochem. 1988, 27, 17-72.
Inoue, S.; Miyawaki, M. quantitative analysis of iduronic acid and glucuronic acid in sulfated galactosaminoglycuronans by gas chromatography. Anal. Biochem. 1975, 14, 164-174.
Lampio, A.; Finne, J. Sugar analysis of glycoproteins and glycolipids after methanolysis by high performance liquid chromatography with pulsed amperometric detection. Anal. Biochem. 1991, 30, 132-136.
Rocklin, R. D.; Pohl, C. A. Determination of carbohydrates by anion exchange chromatography with pulsed amperometric detection. J. Liquid Chromatogr. 1983, 6, 1577-1590.
Martens, D. A.; Frankenberger, W. T. Determination of glucuronic acids by high performance anion chromatography with pulsed amperometric detection. Chromatographia 1990, 30, 651-656.
Lee, Y. C. High-performance anion-exchange chromatography for carbohydrate analysis. Anal. Biochem. 1990, 189, 151-162.
Clarke, A. J.; Sarabia, J.; Keenleyside, W.; MacLachlan, P. R.; Whitfield, C. The composional analysis of bacterial extracellular polysaccharides by high-performance anion-exchange chromatography. Anal. Biochem. 1991, 30, 68-74.
De Vries, J.; Rombouts, F. M.; Voragen, A. G. J.; Pilnik, W. Enzymic degradation of apple pectins. Carbohydr. Polym. 1982, 2, 25-33.
Guillon, F.; Thibault, J.-F. Methylation analysis and mild acid hydrolysis of the hairy fragments of sugar-beet pectins. Carbohydr. Res. 1989, 190, 85-96.
Renard, C. M. G. C.; Thibault, J.-F. Degradation of pectins in alkaline conditions kinetics of demethylation. Carbohydr. Res. 1996, 286, 139-150.
Micard, V.; Renard, C. M. G. C.; Thibault, J.-F. Enzymatic saccharification of sugar beet pulp. Enzyme Microb. Technol. 1996, 19, 162-170.
Levigne, S.; Ralet, M. C.; Thibault, J.-F. Characterization of pectins extracted from fresh sugar beet under different conditons using an experimental design. Carbohydr. Polym. 2002, 49, 145-153.
Thomas, M.; Tibault, J.-F. Cell-wall polysaccharides in the fruits of Japanese quince (Chaenomeles japonica): extraction and preliminary characterization. Carbohydr. Polym. 2002, 49, 345-355.
Selvendran, R. R.; Dupont, M. S. In Developments in Food Analysis Techniques; King, R. D., Ed.; Elsevier Applied Science: New York, 1984; Vol. 3, pp 1-68.
Thibault, J.-F.; Renard, C. M. G. C.; Axelos, M. A. V.; Roger, P.; Crepeau, M. J. Studies of the length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydr. Res. 1993, 238, 271-286.
Massiot, P.; Thibault, J.-F.; Rouau, X. Degradation of carrot (Daucus carota) fibers with cell-wall polysaccharide-degrading enzymes. J. Sci. Food Agric. 1989, 49, 45-57.
Jones, T.; Albersheim, P. A gas chromatographic method for the determination of aldose and uronic acid constituents of plant cell. Plant Physiol. 1972, 49, 926-936.