Formation of new oligosaccharides with potential prebiotic character from lactulose and Pectinex Ultra sp-l

Abstract

Since the introduction of the prebiotic concept as a non-digestible oligosaccharide that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, there is an increasing interest in the use of prebiotics for manipulating the intestinal microflora composition in order to improve the activity of the gastrointestinal tract. Quantitative changes in bacterial groups, fermentation and products and the place where fermentation mainly occurs (proximal or distal colon) are important factors in assessing the prebiotic effect of non-digestible carbohydrates. The relative resistence of oligosaccharides to digestion in the intestine depends on several factors such as the type of hexoses, types of glycosidic linkages, extent of polymerization, etc. Galactooligosaccharides (GOS) are commercially available prebiotics produced from lactose by transfer of galactose residues onto the galactose moiety of lactase catalyzed by b-galactosidases. Lactulose is a well-known disaccharide with excellent prebiotic activity; however, a drawback of this carbohydrate is the production of gas during its fermentation, due to the fact that it is mainly consumed by the bacteria of proximal colon. Therefore, it is reasonable to assume that lactulose-derived oligosaccharides originated during enzymatic hydrolysis of lactulose might be bioactive carbohydrates slowly fermented and therefore, with higher colonic persistence than lactulose. In this work, we have investigated the possible formation of new oligosaccharides derived from hydrolysis of lactulose by the b-galactosidase activity of the commercial enzymatic preparation Pectinex Ultra SP-L derived from Aspergillus aculeatus. The effect of transgalactosylation conditions such as time (1, 3, 5, 7 and 24 h) temperature (40, 50 and 60ºC), pH (0.1M acetate buffer at pH 4.5 or 5.5. and 0.1M phosphate buffer at pH6.5), initial lactulose (250, 450, 650 and 850 g/L) and enzyme concentration (8, 16 and 24units/mL) during the hydrolysis of lactulose as well as product inhibition has been exhaustively studied. Reaction mixtures were purified by HPLC-RI and characterization of GOS was carried out by Mass Spectrometry in electrospray positive mode (APl-ES) amd 1H amd 13CNMR. High performance anion-exchange chromatography with pulsed amperometric detection (HPAEC.PAD) analysis of reaction mixtures of lactulose and Pectinex Ultra SP-L showed the presence of unreacted lactulose, fructose and galactose amd the formation of different GOS, di- and trisaccharides and other oligosaccharides with degree of polimerization (DP)>=3. Main formed GOS were indentified as galactobiose (Gal-beta(1-6) Gal) and 6' galactosyl lactulose (beta-D-Gal-(1-6)-beta-D-Gal-(1-4) beta-D-Fru). Disaccharide formation was maximal at SO oc amd pH of 4.5 reaching levels up to 12% after 24h of reaction. The optimal conditions for the formation of 6'galactosyl lactulose were 60 °C, pH 6.5, 450g/L of lactulose, 16 U/mL of enzyme and 7 hours of reaction, reaching yields of 15% (about 30% of total GOS). lnhibition studies adding galactose and fructose to reaction mixtures of lactulose and Pectinex Ultra SP-L, showed that fructose did not inhibit lactulose hydrolysis whereas galactose acts as a competitive inhibitor for the enzyme active sites. The results here presented show that galactosidase from Pectinex Ultra allows the production of GOS with high content of disaccharides or trisaccharides, with potential prebiotic character, depending on the operating conditions.