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Synthesis of prebiotic oligosaccharides derived from lactose and lactulose using immobilized β-galactosidase from Lactobacillus plantarum

AutorRuiz-Matute, Ana I. ; Cardelle-Cobas, Alejandra ; Curiel, José Antonio ; Benavente, Rocio ; Rivas, Blanca de las ; Muñoz, Rosario ; Pessela, Benevides C. ; Corzo, Nieves
Fecha de publicación2012
CitaciónSECyTA 2012
ResumenIn the food industry an increasing aitention is being paid to the synthesis of prebiotic new oligosaccharides derived from lactose (GOS) or with improved prebiotics properties using pgalactosidases since their chemical production is very tedious. β-Galactosidases from different sources are wmmercially availabk, however, in rnany cases their use is limited by a low specific activity, low thermostability or high inhibition by products. Therefore, the search and characterization of new and improved β-galactosidases in GRAS microorganisms is a mandatory subject. Lactobacillus plantamrn, a very interesting source for the production of new enzymes in food. has been described to have tanase activity. However, to the best of our knowledge, its p-galactosidase activity has not been studied yet. Thus, the aim of this work was to evaluate the activity of a β-galactosidase isolated from L. plantamrn (LPG) previously purified, characierized and immobilized in our laboratory. Lactose and lactulose were used as substracts and two different pHs were assayed for production of GOS and oligosaccharides derived from lactulose (OsLu) along the incubation time. A β-galactosidase from L. plantamrn (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC supports as previously described. Production of GOS and OsLu was carried out using 300 g/L of lactose or 450 g/L of lactulose in 50 mM of sodium phosphate and sodium acetate buffer at two different pHs (5 and 7) and 1 g of glyoxyl-LPG during incubation at 45% up to 24 h. Samples of 200 pL were withdrawn from the reaction mixtures at different times. GOS and OsLu were determined by HPAEC-PAD on a CarboPac PA-1 column (250 x 4 mrn) connected to a CarboPac PA-1 (50 x 4 mm) guard column. The elution, at a flow rate of 1 mL min>, was in gradient using 100% of 100 mM NaOH solution from O to 20 min and 0-100% of 100 mM NaOH and 50 mM NaOAc solution from 20 to 70 min. After each run, the column was washed for 10 min with 100% 100 rnM NaOH and 1 M NaOAc. Quantification of carbohydrates was períormed by extemal calibration and the rernaining amount of lactose and lactulose and the yield of GOS and OsLu were expressed as % by weight of the total carbohydrate content in the reaction mixtures. All experiments were períormed in duplicate.
Different GOS were formed by transgalactosylation during lactose hydrolysis, 6-galactobiose, 3'- and 6'-galactosyl-lactose being identified. Both pH values tested (5 and 7) gave similar results. It was obsewed that synthesis of trisaccharides predominated over other oligosaccharides. Among the GOS formed, the main trisaccharide was 3'- galactosyl-lactose, showing a maximum value of 10 gllOOg of total carbohydrates after 3 h of incubation at pH5. Total GOS formation gave a maximum value of 34 and 32 g/100g of total carbohydrates for 4h of incubation at pH 7 and 5, respectively. Formation of OsLu was also observed by the LPG tested, 6-galactobiose, 6'-galactosyl-lactulose, 1- galactosyl-lactulose being found in the reaction mixture. An unknown trisaccharide was also detected and was tentatively assigned as 3'- galactosyl-lactulose. This trisaccharide showed a maximum formation at 3h of incubation (9 g/100g) for both pH studied. The highest yield of OsLu was produced after 24 h of reaction at pH 5 and pH 7, reaching values of 32 and 24 g/l00g of total carbohydrates. respectively. The present study shows the feasibility of β-galactosidases L. plantarum strains to hydrolyze and transglywsylate lactose and lactulose producing high yields of prebiotic oligosaccharides.
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