Identification of the fructo-oligosaccharides common to beet medium invert sugar and pyrolysed sucrose

Chromatography and enzymolysis have been used to identify the primary oligosaccharides common to beet medium invert sugar and pyrolysed sucrose. Anion exchange and reversed phase HPLC showed the two materials to contain a similar spectrum of primary oligomers, although at a much higher level in pyrolysed sucrose. Confirmation of the similarity was obtained by reversed phase fractionation and anion exchange HPLC analysis of the individual fractions. Gel filtration chromatography was used to estimate the size of the oligomers with gel filtration fractionation of the sample of beet medium invert sugar and subsequent anion exchange HPLC analysis of the fractions, confirming that the primary oligosaccharides elute in the same position as the starch trisaccharide maltotriose. Enzymolysis, using invertase EC 3.2.1.26, of beet medium invert sugar and pyrolysed sucrose, and subsequent analysis of the digest using anion exchange HPLC, showed that four of the oligosaccharides were cleaved by the enzyme, indicative of a terminal beta-D-fructofuranoside residue in each. Another two oligosaccharides were stable to the enzyme, and therefore do not possess the terminal beta-D-fructofuranoside feature. One additional oligosaccharide appeared during the enzymolysis. From the enzymolysis and chromatographic data and by comparison of the elusion positions of the primary oligosaccharides with those of reference standards it was concluded that the primary oligosaccharides present in both beet medium invert sugar and pyrolysed sucrose are the kestoses, 1-kestose, 6-kestose, and neokestose and their alpha-anomers, iso-1-kestose, iso-6-kestose, and iso-neokestose. Although the kestoses may be the products of enzymic action on sucrose the iso-kestoses are not produced and therefore must originate through a thermal process. These oligosaccharides used as indicators for the detection of beet medium invert sugar extension of orange juice are unlikely to occur naturally in oranges as orange invertase has alpha-D-glucosidase activity and therefore oligosaccharides produced in situ would have glucose linked to sucrose rather than the kestoses and iso-kestoses, produced by beta-D-fructosidase or thermolysis, where fructose is linked to sucrose.