Production of galacto-oligosaccharides from lactose by immobilized beta-galactosidase and posterior chromatographic separation

Galacto-oligosaccharides (GOS) are non-digestible sugars containing two to five molecules of galactose and one molecule of glucose connected through glycosidic bonds. They are classified as prebiotic food because they can selectively stimulate the growth of bifidobacteria and lactobacilli in the lower intestine. The addition of GOS as a functional food ingredient has great potential to improve the quality of many foods. GOS can be produced from lactose, which is abundant in cheese whey, by enzymatic transgalactosylation with beta-galactosidase present in either free or immobilized form. The goal of this research was to evaluate the feasibility of using various microbial lactases immobilized on cotton cloth for GOS production from lactose and posterior purification by chromatographic technique using a commercial cation exchange resin Dowex 50W. The production of GOS from lactose was first studied with lactases from Aspergillus oryzae, Bacillus circulans, and Kluveromyces lactis. The total amount, types, and size of GOS produced were affected by the enzyme type and the initial lactose concentration in the reaction media. In general, more GOS can be produced when the initial lactose concentration was higher. With 400 g/L of lactose solution, a maximum GOS content of 40% (w/w) was achieved with B. circulans lactase, followed by K. lactis with 31% GOS and A. oryzae with 27% GOS. The addition of galactose inhibited the enzyme, reducing both GOS yield and the reaction rate.;The method of immobilizing enzyme on cotton cloth via adsorption with the binding support of polyethyleneimine (PEI) and cross linking with glutaraldehyde (GA) was developed for beta-galactosidase from B. circulans and A. oryzae. A high enzyme loading of 250 mg/g support with 35% and 90% activity yield was achieved for B. circulans and A. oryzae lactase, respectively. Thermal stability of B. circulans and A. oryzae lactase increased by 12 and 25-fold, respectively, upon immobilization on the cotton cloth. The immobilized enzyme showed the same GOS formation kinetics as that of the free enzyme, indicating that there was no diffusion limitation in the catalytic cotton cloth for the reaction.;Continuous production of GOS with immobilized enzyme in packed-bed reactors was studied. A high volumetric productivity of 4300 g/L/h was attained at 50% conversion with A. oryzae lactase and 180 g/L/h at 60% conversion with B. circulans lactase. Higher overall GOS productivity and conversion can be achieved with two enzyme reactors in sequence, with the first one containing A. oryzae lactase and second one containing B. circulans lactase.;Commercial gel-type cation exchange resins (Dowex 50W) with different salt forms (Na+, K+, and Ca++) and degrees of cross-linking (2%, 4%, and 8%) were evaluated for the separation of GOS from the multicomponent sugar mixture. Among all the factors studied, the degree of cross linking had the most significant effect on sugar adsorption capacity and selectivity, with 4% cross linking being the optimal. Other factors such as temperature and counter ion showed minimal effect. A high degree of removal of monosaccharides (95%) from the GOS mixture was achieved using an elution chromatographic column packed with Dowex 50W resins of 4% cross linking and in the Na+ form at room temperature.