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Fructooligosaccharides (FOS) are short-chain fructans with a terminal glucose moiety and are found naturally in many plant species. Besides their wide use as an alternative sweetener in food and beverage industry, FOS have shown great potential as neutraceuticals against diabetes, colon cancer and bowel disease. The uses of FOS are dependent on the degree of polymerisation that they exhibit. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex®Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and size-exclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Analysis of various mixtures of FOS by mass spectrometry, HPLC and 1H-NMR was undertaken. Results indicated that MS with electrospray ioniza-tion and 1H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures.