Síntese enzimática e separação cromatográfica de isomalto-oligossacarídeos de cadeia longa

Abstract

Prebiotic isomalto-oligosaccharides (IMOs) are carbohydrates able to reach the large bowels intact, where they are metabolized by bifidobacteria and lactobacillus, stimulating their growth and providing beneficial health effects. They may be obtained by enzymatic synthesis using sucrose and maltose as precursors. However, non consumed substrates, fructose and dextran are present together with the produced isomalto-oligosaccharides. This work aimed the optimization of the enzymatic synthesis and the separation of prebiotic isomalto-oligosaccharides by ion exchange chromatography on semipreparative scale. The first part of the thesis investigated the effect of different feeding strategies of sucrose and maltose to the reaction medium on the chain elongation of isomalto-oligosaccharides. The synthesis was performed by adding 1UI/mL of enzyme to reaction medium containing sucrose and maltose as substrate. In the first strategy, only sucrose was added to the reaction medium every 2 hours for 8 hours. In the second strategy, a mixture of sucrose and maltose was added to the reaction medium every 2 hours for 8 hours. In the third strategy, at every hour, a mixture of sucrose and maltose was added for six hours. Then, only sucrose was added at every hour for more six hours. The latter strategy resulted of isomalto-oligosaccharides chain elongation up to degree of polymerization of 9. The concentration of IMOs (79.04 g/L) was also observed. In the second part of the thesis, the influence of the cationic form of the chromatographic stationary phase in the isomalto-oligosaccharides separation was evaluated. Adsorption isotherms of isomalto-oligosaccharides, maltose, glucose and fructose were determined in preparative columns packed with ion-exchange resin in different cationic forms (K+, Ca2+, H+ and Na+) at 25°C. All stationary phases adsorbed the sugars with variable capacities, following the same order of affinity: fructose > glucose > maltose > IMOs with increasing degrees of polymerization. It was found that the resin in H + form presented the best performance to be used in simulated moving bed units, allowing for the separation of prebiotic isomalto-oligosaccharides with lower dilution and higher productivity. Nevertheless, the resin K+ had only a slighty inferior performance and this cation is more compatible with buffer requirements of the enzymatic synthesis.