␤-Galactosidase enzymes were extracted from pure cultures of Bifidobacterium angulatum, B. bifidum BB-12, B. adolescentis ANB-7, B. infantis DSM-20088, and B. pseudolongum DSM-20099 and used in glycosyl transfer reactions to synthesize oligosaccharides from lactose. At a lactose concentration of 30% (wt/wt) oligosaccharide yields of 24.7 to 47.6% occurred within 7 h. Examination of the products by thin-layer chromatography and methylation analysis revealed distinct product derived spectra from each enzyme. These were found to be different to that of Oligomate 55, a commercial prebiotic galacto-oligosaccharide. Fermentation testing of the oligosaccharides showed an increase in growth rate, compared to Oligomate 55, with products derived from B. angulatum, B. bifidum, B. infantis, and B. pseudolongum. However B. adolescentis had a lower growth rates on its oligosaccharide compared with Oligomate 55. Mixed culture testing of the B. bifidum BS-4 oligosaccharide showed that the overall prebiotic effect was equivalent to that of Oligomate 55.Oligosaccharides are increasingly being recognized as useful dietary tools for the modulation of the colonic microflora toward a healthy balance (8). This usually involves selectively increasing the levels of gut bifidobacteria and lactobacilli at the expense of less-desirable organisms such as Escherichia coli, clostridia, and proteolytic bacteroides. This selective fermentation is known as the prebiotic concept defined by Gibson and Roberfroid (9).Although many oligosaccharide preparations are used in functional foods in Japan (17), two general classes are widely used in Europe. These are fructans, such as inulin and fructooligosaccharides, and ␤-galacto-oligosaccharides (17, 18, 22). The latter are manufactured from lactose by glycosyl transfer catalyzed by ␤-galactosidase and occur as complex mixtures with various glycosidic linkages (7). The commercial products are made using ␤-galactosidases isolated from several sources such as bacteria and fungi (5, 7). The prebiotic properties of these galacto-oligosaccharides have been established in several studies, both in vitro (21) and in vivo (11). The consensus is that the substrates have a selective stimulatory effect on bifidobacteria.Despite the interest in galacto-oligosaccharides as prebiotics, there has been very little effort made to study the relative effects of products synthesized by different glycosidases. Given that the ␤-galactosidase enzymes from different micro-organisms display differing rate constants for hydrolysis for specific glycosidic linkages and that synthesis of galacto-oligosaccharides is kinetically controlled, synthetic product mixtures made with different enzymes are likely to contain differing profiles of FIG. 1. pH optima of cell-associated ␤-galactosidase and lactase activities of Bifidobacterium angulatum. Cells were harvested after 18 h of growth on 10 g of lactose liter Ϫ1 ; lactase activity (s) was determined using lactose as the substrate (1 mol of glucose released min Ϫ1 mg of protein Ϫ1 ), an...

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Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, alpha-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 degrees C ( K(M) 0.55 mM, V(max) 0.80 micromol min(-1) mg(-1) of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6' alpha-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.

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The dairy industry produces large quantities of whey as a by-product of cheese production and is increasingly looking for new ways to utilize this waste product. Gellan gum is reliably produced bySphingomonas paucimobilis in growth media containing lactose, a significant component of cheese whey, as a carbon source. We studied and compared polysaccharide biosynthesis by S. paucimobilis ATCC 31461 in media containing glucose, lactose (5 to 30 g/liter), and sweet cheese whey. We found that altering the growth medium can markedly affect the polysaccharide yield, acyl substitution level, polymer rheological properties, and susceptibility to degradation. Depression of gellan production from lactose compared with gellan production from glucose (approximately 30%) did not appear to occur at the level of synthesis of sugar nucleotides, which are the donors of monomers used for biosynthesis of the repetitive tetrasaccharide unit of gellan. The lactose-derived biopolymer had the highest total acyl content; the glucose- and whey-derived gellans had similar total acyl contents but differed markedly in their acetate and glycerate levels. Rheological studies revealed how the functionality of a gellan polysaccharide is affected by changes in the acyl substitution.

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