Structure of Fructan Prepared from Onion Bulbs ( Allium cepa L.)

Abstract

The occurrence of fructan in several Allium species has been known since 1894, as reported by Archbold. 1) The content, distribution, and structure of fructan in Allium species were first investigated, during the 1970s, by Bacon, 2) Darbyshire, 3,4) and Henry. 3,4) Later, its content and distribution were studied by Jaime et al. 5) and Campbell et al. 6) These studies were characterized by a relative lack of data because chemical and/or enzymatic methods were used to assess and deduce the content and distribution of fructan, and the techniques used for these analyses did not allow the separation or identification of fructan. Recently, new techniques for separating fructan determining their structural composition in onions have been developed. However, apart from the identification of the saccharide using only HPAEC, the structure of fructan in onion bulbs has been paid little attention. Therefore, we attempted to clarify the structure of fructan obtained from onion bulbs (Allium cepa L.) by high resolution 13 C-NMR spectroscopy and MALDI-TOF MS of the native saccharide as well as gas-liquid chromatography of the methyl derivatives of the methanolyzed monosaccharides. MATERIALS AND METHODS Materials. Onion (Allium cepa L.) bulbs were harvested in October at the experimental farm of Rakuno Gakuen University and stored at 30C. Jerusalem artichoke tubers (Helianthus tuberosus L.) and timothy (Phleum pratense L.) roots were obtained from the Hokkaido National Agricultural Experiment Station in November. Standard sugars were prepared as follows: crystalline 1-kestose (l F-β-D-fructofuranosylsucrose) and nystose [l F (l-β-D-fructofuranosyl)2 sucrose] were prepared from sucrose using the Scopulariopsis brevicaulis enzyme. 7) Neokestose and l F (l-β-D-fructofurano-syl)m-6 G (l-β-D-fructofuranosyl)n sucrose (4b: m = 0, n = 2; 4c: m = 1, n = 1; 5a: m = 3, n = 0; 5b: m= 0, n = 3; 5c: m = 2, n = 1; 5d: m= 1, n = 2; 6a: m = 4, n = 0; 6b: m= 0, n = 4; 6c: m = 3, n = 1; 6d1: m= 1, n = 3; 6d2: m= 2, n = 2) were isolated from asparagus roots. 810) l F (l-β-D-Fructofuranosyl)5 sucrose (7 a) and l F (l-β-D-fructofuranosyl)n sucrose (n ≥ 5) were prepared from Jerusalem artichoke tubers. Timothy levan was obtained from the roots of Phleum pratense L. 11) Inulin was purchased from Nakalai Tesque inc. (Kyoto, Japan). Quantitative determination of sugar. Total sugars were determined by the anthrone method. 12) Reducing sugars were Abstract: Fructan was prepared from an extract of onion bulbs by preparative paper and gel permeation chromatographies. Fructan was a mixture of saccharides with degree of polymerization (DP) and MW range of approximately 713 and 1,1522,124, respectively, as determined by matrix-assisted laser desorp-tion ionization/time of flight mass spectrometry. It yielded fructose and glucose upon hydrolysis with acid or yeast β-fructofuranosidase. The ratio of D-fructose to D-glucose in the enzyme hydrolysate was 8.7:1, determined by high performance anion-exchange chromatography analysis. Structural determinations were made, based on 13 C-nuclear magnetic resonance spectroscopy. Numerous signals corresponding to carbon-1 (C-1), C-2, C-3, C-4, C-5, and C-6 of the D-fructose residues of fructan were observed at δ 61.0461.81, 103.70104.41, 77.4678.10, 74.5075.15, 81.77, and 62.8562.96, respectively. These chemical shifts were similar to those of inulin. Moreover, weaker signals were detected at δ 92.90, 71.75, 73.10, 69.76 and 72.22 due to C-1, C-2, C-3, C-4, and C-5, respectively of the D-glucose residue. The chemical shifts are almost identical to those of the D-glucose carbons of neokestose, 6 G (1-β-D-fructofuranosyl)2 sucrose, and 1 F , 6 G-di-β-D-fructofuranosyl sucrose. These findings were supported by analysis of the methanolysate from permethylated fructan using gas-liquid chromatography. The fructan from onion bulbs was composed of saccharides possessing approximately 612 D-fructose residues, linked by β-2,1 bonds, and a non-terminal D-glucose residue bound with D-fructose residues at the C-1 and C-6 positions.). Abbreviations: HPAEC, high performance anion-exchange chromatography ; 13 C-NMR, 13 C-nuclear magnetic resonance; MALDI-TOF MS, matrix-assisted laser desorption ionization/time of flight mass spectrometry; GLC, gas liquid chromatography.