Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates

Abstract

Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22 isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes encoding β-galactosidases, and three of them were induced during bacterial growth on commercial galactooligosaccharides. Recombinant β-galactosidases from B. infantis ATCC 15697 displayed different preferences for β-galactosides such as 4' and 6'-galactobiose, and four β-galactosidases in this strain released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent evidence for physiological differences in the metabolism of prebiotics that might have a differential impact on the host.

Copyright © 2012 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

In vitro growth of strains of B. infantis using 0.5% commercial GOS as the sole media carbon source.

Fig. 2

Analysis of the consumption of GOS by B. infantis isolates by TLC. Supernatants represent strains in Fig. 1, and a control prepared under the same conditions but with no bacteria added was included (GOS lane). Plates were run in n-butanol-acetic acid-water 2:1:1 and developed with α-naphthol. Numbers correspond to strains in Supplementary Table 1.

Fig. 3

Relative quantification of the gene expression of B. infantis β-galactosidases after logarithmic growth on the substrates indicated in the x-axis. Dashed lines indicate a two-fold change in gene expression.

Fig. 4

Determination of the substrate specificities of B. infantis β-galactosidases for different galactosyl linkages by TLC indicated in the bottom of the figure. (A) Coincubations of Blon_0268 and Blon_2334 for 5, 20 and 60 min with different β-galactosides. Lanes 1 and 8: standards; lanes 2–4: Blon_0268 with 4-galactobiose; lanes 5–7: Blon_2334 with 4-galactobiose, 5-20–60 min; lanes 9–11: Blon_0268 with 6-galactobiose; lanes 12–14: Blon_2334 with 6-galactobiose. Lane 15: Lactose and galactose; lane 16: glucose; lanes 17–19: Blon_0268 with lactose; lanes 20–22: Blon_2334 with lactose. Lane 23: Commercial GOS; lane 24: lactose and galactose; lanes 25–27: Blon_0268 with GOS; lanes 28–30: Blon_2334 with GOS. (B) Coincubations of Blon_2016, Blon_2123 and Blon_2416 with 4′ or 6′ galactobiose for 20′ and 60′. Lanes 1 and 8: galactose and 4′ or 6′ galactobiose standards; lanes 2–3: Blon_2016 on 4-galactobiose; lanes 4–5: Blon_2123 on 4-galactobiose; lanes 6–7: Blon_2416 on 4-galactobiose; lanes 9–10: Blon_2016 on 6-galactobiose; lanes 11–12: Blon_2123 on 6-galactobiose; lanes 13–14: Blon_2416 on 6-galactobiose. (C) Glycolytic activity on 3′ galactosyl lactose. Enzymes were incubated with this substrate for 60′. Lane 1: 3′ galactosyl lactose; lane 2: galactose and lactose; lane 3: Blon_0268; lane 4: Blon_2016; lane 5: Blon_2123; lane 6: Blon_2334; lane 7: Blon_2416. (D) Coincubations of Blon_2016, Blon_2123 and Blon_2416 with commercial GOS for 20′ and 60′. Lane 1: GOS; lane 2: lactose and galactose; lane 3: glucose; lanes 4–5: Blon_2016; lanes 6–7: Blon_2123; lanes 8–9: Blon_2416. Gal: galactose, Glc: glucose, Lac: lactose.