Prebiotic galacto-oligosaccharides (GOS), which are non-digestible carbohydrates and are produced by transgalactosylation of lactose catalyzed by ?–galactosidase, have been found to modulate the colonic micro flora by stimulating the growth of probiotics and suppressing adverse microbial activities and have received much attention in last few years. The prebiotic efficacy of GOS is largely dependent on glycosidic linkages among monosaccharides and GOS mixtures typically result in better growth of that strain that had served as ?–galactosidase source. In the present book, ?–galactosidases from different lactobacilli and other sources have been characterized in detail for various biochemical properties and their transgalactosylation abilities have been evaluated. To overcome the problem of less activity, genes of ?–galactosidases were cloned and expressed in different expression systems. The mixtures of GOS were characterized using different analytical techniques including HPLC and capillary electrophoresis.