Optimisation of synthesis of oligosaccharides derived from lactulose (fructosyl-galacto-oligosaccharides) with b-galactosidases of different origin Cecilia Guerrero, Carlos Vera, Andrés Illanes ⇑ School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2147, Valparaíso, Chile article info Article history: Received 24 August 2012 Received in revised form 20 October 2012 Accepted 24 October 2012 Available online 10 November 2012 Keywords: Lactulose Prebiotic b-Galactosidase Fructosyl-galacto-oligosaccharides Transgalactosylation Galacto-oligosaccharides abstract Batch synthesis of fructosyl-galacto-oligosaccharides from lactulose was performed with commercial b-galactosidase preparations from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. The enzyme from A. oryzae produced the highest yield and specific productivity of synthesis, being selected for further studies. Optimization of fructosyl-galacto-oligosaccharides synthesis was carried out using response surface methodology, considering temperature and initial sugar concentration as variables and yield and specific productivity as response parameters. Maximum yield of 0.41 g g 1 fructosyl- galacto-oligosaccharides was obtained at 70 °C and 60% w/w lactulose concentration, while maximum specific productivity of 1.2 g h 1 mg 1 was obtained at 70 °C and 40% w/w lactulose concentration. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Prebiotics are increasingly being considered as health-pro- moting food components (Wang, 2009). Most prebiotics are non-digestible oligosaccharides (NDO) and, among them, galac- to-oligosaccharides (GOS), fructo-oligosaccharides (FOS), inulin and lactulose have been conclusively proven to exert prebiotic effects (Rycroft, Jones, Gibson, & Rastall, 2001), while other NDO are considered as health-promoting agents complying with some, but not all, of the requirements to be considered as prebi- otics (Gänzle, Haase, & Jelen, 2008; Klewicki, 2007; Li et al., 2009). Prebiotic effects on the colonic microbiota depend on the chemical structure of the oligosaccharides (number and type of monomers; type, position and conformation of the glycosidic linkages) (Hernández-Hernández, Montañes, Clemente, Moreno, & Sanz, 2011; Martínez-Villaluenga et al., 2008; Olano & Corzo, 2009). Therefore, evaluating new NDO structures is an open field of research as some of them may lead to better prebiotics (Cardelle-Cobas et al., 2011). b-Galactosidases are important biocatalysts for industry, tradi- tionally used for their hydrolytic activity to reduce the lactose con- tent in foods and process wastewaters (Illanes, 2011; Tuure & Korpela, 2004), and, more recently, as catalysts for transgalactosy- lation reactions leading to the synthesis of GOS, lactulose and lactosucrose (Albayrak & Yang, 2002; Guerrero, Vera, Plou, & Illanes, 2011; Kim, Park, & Oh, 2006; Lee, Kim, & Oh, 2004; Li et al., 2009). The mechanism of the reaction catalyzed by b-galactosidase was described more than 50 years ago as a transglycosylation reac- tion in which the enzyme catalyzes the transfer of a galactose moi- ety in a non reducing b-galactoside (donor) to an acceptor containing a hydroxyl group (Prenosil, Stuker, & Bourne, 1987). However, the potential technological value of such a reaction be- gan to be explored more than three decades later for the synthesis of transgalactosylated oligosaccharides, once these compounds ac- quired interest as potential prebiotics. Among them, GOS and lac- tulose stand out for their scientifically proven prebiotic condition. GOS are composed of a variable number of galactose units (usually from two to ten) and a terminal glucose unit, mostly b-(1 ? 4)- and b-(1 ? 6)- linked; lactulose is a disaccharide (4-O-b-D-galacto- pyranosyl-D-fructose). In the synthesis of GOS, lactose plays both the role of donor and acceptor of the galactosyl residue, so forming trisaccharides which in turn can act as acceptors forming tetrasac- charides and so on (Albayrak & Yang, 2002; Vera, Guerrero, & Ill- anes, 2011). In the enzymatic synthesis of lactulose, lactose is the galactose donor, fructose acting as acceptor, but since lactose can also act as acceptor, a mixture of lactulose and GOS will be pro- duced (Guerrero et al., 2011; Kim et al., 2006; Lee et al., 2004). The synthesis of GOS and lactulose is strongly determined by the origin of the b-galactosidase (Guerrero et al., 2011; Kim et al., 2006; Lee et al., 2004; Sanz-Valero, 2009), so that product compo- sition, yield and specific productivity will vary accordingly. Most studies have been focused in increasing yield that, as said above, is strongly dependent on the enzyme source, since it results from 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.10.128 ⇑ Corresponding author. Tel.: +56 32 2273642; fax: +56 32 2273803. E-mail address: aillanes@ucv.cl (A. Illanes). 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