The enzymatic production of lactulose via transglycosylation in conventional and non-conventional media Seyyedhadi Khatami a , Farzin Zokaee Ashtiani a, b, * , Babak Bonakdarpour a, b , Mahsa Mehrdad a a Department of Chemical Engineering, Amirkabir University of Technology, No. 424, Hafez Ave., Tehran, Iran b Food Process Engineering and Biotechnology Research Centre, Amirkabir University of Technology, No. 424, Hafez Ave., Tehran, Iran article info Article history: Received 12 February 2013 Received in revised form 8 July 2013 Accepted 8 July 2013 abstract In this study, lactulose production with b-galactosidase from Kluyveromyces lactis in aqueous and non- conventional media was studied. The optimisation of the rate of enzymatic lactulose production in aqueous media was carried out using factorial design methodology. The results showed a statistically significant effect of both lactose and fructose levels e as well as their interaction e on the rate of lac- tulose production. Maximum lactulose production rate was obtained at fructose and lactose concen- trations of 25e30% (w/w) and 9e12% (w/w), respectively. The addition of acetone had a negative effect on lactulose production, whereas the incorporation of triethyl phosphate up to a concentration of 30% (w/w) caused an increase of up to 20% in the lactulose production. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Lactulose (4-O-b-D-galactopyranosyl-D-fructose) is a synthetic ketose disaccharide with greater sweetness and better solubility than lactose, and is known as a bifido-factor and prebiotic in nutrition (Mizota, Tamura, Tomita, & Okonogi, 1987). Alkaline iso- merisation and enzymatic reaction are two well-known methods of lactulose synthesis (Schuster-Wolff-Bühring, Fischer, & Hinrichs, 2010). The yield of chemical isomerisation is higher than that of enzymatic production (Zokaee, Kaghazchi, Zare, & Soleimani, 2002). However, according to previous researchers, the advan- tages of the enzymatic lactulose production include product puri- fication (Kim & Oh, 2012), waste management, less cost (Hua et al., 2010) and the valuable status of ‘natural product’ in the food in- dustry (Mayer et al., 2004). Three different enzymes or combination of enzymes have been reportedly used for the production of lactulose. The first one is b- galactosidase, which either catalyses the reaction between lactose and fructose to form lactulose via transgalactosylation, which is kinetically controlled, or between galactose and fructose by reverse hydrolysis, which is equilibrium controlled (Ajisaka, Nishida, & Fujimoto, 1987). Second, b-galactosidase has been used in conjunction with glucose isomerase with lactose and fructose as the substrates (Hua et al., 2010). Finally, a thermostable cellobiose 2-epimerase has been used to enzymatically convert lactose as the sole source of substrate to lactulose (Kim & Oh, 2012). Glycosyl- transferases are another class of enzymes that can be considered for the enzymatic production of lactulose, but, since they need a cofactor which increases the cost of reaction, have never been used (Mayer et al., 2004). The results of previous studies on enzymatic lactulose production have been summarised in Table 1 . b-Galactosidase catalyses the hydrolysis of lactose to glucose and galactose and is used extensively in food and pharmaceutical products to eliminate lactose. However, under appropriate condi- tions, b-galactosidase can catalyse the galactosyl transfer of a b- galactoside (lactose) to a hydroxyl containing acceptor moiety (fructose, lactose) other than water, and synthesis instead of hy- drolysis occurs (Fig. 1)(Guerrero, Vera, Plou, & Illanes, 2011). The yield of lactulose for the transgalactosylation is, however, still lower than that reported for the chemical isomerisation and this was commonly attributed to the low transgalactosylation ac- tivity of b-galactosidase (Hua et al., 2013). Theoretically, two stra- tegies can be envisaged for increasing the enzymatic lactulose production: 1) protein engineering and 2) the use of non- conventional media. The former has been attempted by some re- searchers (Hansson, Kaper, Oost, Adlercreutz, & Vos, 2001) who increased the oligosaccharide yield (45%) compared with the wild- type CelB (40%) by an exchange of one phenylalanine to tyrosine (F426Y). The term non-conventional media in biocatalysis alludes to systems that take advantage of solvents other than water, or use * Corresponding author. Tel.: þ98 21 64543124. E-mail address: zokaee@aut.ac.ir (F. Zokaee Ashtiani). Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj 0958-6946/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2013.07.010 International Dairy Journal 34 (2014) 74e79