Dominguez, Ana

Ferreira, E. C.

Nobre, C.

Rocha, I.

Rodrigues, L. R.

Santos, Maria João

Teixeira, J. A.

Torres, D.

Universidade do Minho: RepositoriUM

Fractionation of fructo-oligosaccharides by gel filtration

Nobre, Clarisse

Ferreira, Eugénio C.

Book  of  Abstracts  of  the  10th  International  Chemical  and Biological Engineering Conference – CHEMPOR 2008 Integration of life sciences& engineering  882 Fractionation of fructo-oligosaccharides by gel filtration  C. Nobre1*, M.J. Santos1, A. Dominguez1, D. Torres1,2, L. Rodrigues1,2, I. Rocha1,2, J.A. Teixeira1, E.C. Ferreira1 1IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710–057 Braga, Portugal 2Biotempo Lda., AVEPARK, Zona Industrial da Gandra, Apartado 4152,  4806 - 909 Caldas das Taipas, Portugal  Keywords: FOS, Gel filtration, Sugar fractionation  Nowadays, production of fructo-oligosaccharides (FOS) has gained large commercial interest due to its beneficial properties in the human health as prebiotics. Fermentative processes appear to be a good technique for large scale production of FOS, namely kestose (GF2), nistose (GF3) and fructo-furanosilnistose (GF4). However, the sugars mixture that results from the fermentations contains high levels of fructose (F), glucose (G) and sucrose (GF) which have to be eliminated. Thus, the aim of the present study was to separate FOS from those components. The separation was performed by gel filtration chromatography using Bio-Gel P2 polyacrylamide. The elution of sugars from the column was based on the molecular weight with the largest sugars eluting first and the smallest later. The molecular weights of sugars present in the fermentative broth are respectively: 180 (F, G), 342 (GF), 504 (GF2), 666 (GF3) and 825 g/mol (GF4). Bio-gel P2 appears to be an efficient gel for the purification of these sugars since it allows the fractionation of molecules with molecular weight between 100 and 1800 g/mol. Two different samples were used in this study; one from the end of fermentation (Sample 1) consisting of 16.1 (F), 47.0 (G), 64.84 (GF), 84.37 (GF2), 13.18 (GF3) and 0 g/L (GF4); and the other (Sample 2)  previously purified in a charcoal column to concentrate FOS consisting of 0.92 (F), 1.64 (G), 2.68 (GF), 18.06 (GF2), 46.02 (GF3) and 7.96 g/L (GF4). Two hundred microliters of sample were loaded onto the Bio-Gel P2 column (100 X 1.6 cm) and eluted with distilled water at a flow rate of 0.3 mL/min. Fractions were collected and analysed by ELSD (Evaporative Light Scattering Detector) and HPLC. Results achieved from the fractionation of sample 1 showed that it is possible to obtain pure kestose, and that sucrose and monosaccharides (G/F) are also well separated. Nevertheless, and although its presence was detected by ELSD (elution volume: 104-112 mL) it was not possible to quantify nistose by HPLC as it was much diluted. Kestose and nistose were obtained from the fractionation of sample 2. As the concentrations of fructo-furanosilnistose, sucrose and monosaccharides in the initial sample were very low it was not possible to quantify them by HPLC. However, the presence of GF4 (elution volume: 92 - 102 mL) and sucrose (elution volume: 125-132 mL) was detected by ELSD. In sum, although gel filtration with Bio-Gel P2 provides a good fractionation of FOS from fermentative broths, the process is time consuming (8h) and the amounts of final product achieved are very small. Therefore, unless the final product has a high commercial value in its fractionated form, the implementation of this procedure at an industrial scale is not economically viable. However, the achieving of pure sugars makes the chemical, clinical and nutritional characterization of each single sugar possible.  References Diplock, A.T., Aggett, P.J., Ashwell, M., Borne, F., Fern, E.B., Roberfroid, M.B. (1999) Scientific Concepts of Functional Foods in Europe Consensus Document. British Journal of Nutrition 81(4): S1-S27. Gibson, G.R., Roberfroid, M.B. (1995) Dietary Modulation of the Human Colonic Microbiota - Introducing the Concept of Prebiotics. Journal of Nutrition 125(6): 1401-1412. Nguyen, Q.D., Mattes, F., Hoschke,A., Rezessy-Szabo, J., Bhat, M. K. (1999) Production, purification and identification of fructooligosaccharides produced by fructofuranosidase from Aspergillus niger IMI 303386. Biotechnology Letters 21: 183–186. 

Fractionation of fructo-oligosaccharides by gel filtration

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