Structural Identity of Galactooligosaccharide Molecules Selectively Utilized by Single Cultures of Probiotic Bacterial Strains

Abstract

Various β-galactosidase enzymes catalyze the trans-glycosylation reaction with lactose. The resulting galactooligosaccharide (GOS) mixtures are widely used in infant nutrition to stimulate growth of beneficial gut bacteria. GOS consists mainly of compounds with a degree of polymerization (DP) varying from 2-8 and with diverse glycosidic linkages. In recent years, we have elucidated in detail the composition of several commercial GOS mixtures in terms of DP and the structural identity of the individual compounds. In this work, 13 (single) probiotic strains of gut bacteria, belonging to 11 different species, were grown to stationary phase with a Vivinal GOS-derived sample purified to remove lactose and monosaccharides (pGOS). Growth among the probiotic strains varied strongly between 30 and 100% of OD600nm relative to positive controls with glucose. By identifying the components of the pGOS mixture that remain after growth, we showed that strains varied in their consumption of specific GOS compounds. All strains commonly used most of the GOS DP2 pool. Lactobacillus salivarius W57 also utilized the DP3 branched compound β-d-Galp-(1 → 4)-[β-d-Galp-(1 → 2)]-d-Glc. Bifidobacterial strains tended to use GOS with higher DP and branching than lactobacilli; Bifidobacterium breve DSM 20091, Lactobacillus acidophilus W37, and Bifidobacterium infantis DSM 20088 were exceptional in using 38, 36, and 35 compounds, respectively, out of the 40 different structures identified in pGOS. We correlated these bacterial GOS consumption profiles with their genomic information and were able to relate metabolic activity with the presence of genome-encoded transporters and carbohydrate-active enzymes. These detailed insights may support the design of synbiotic combinations pairing probiotic bacterial strains with GOS compounds that specifically stimulate their growth. Such synbiotic combinations may be of interest in food/feed and/or pharmacy/medicine applications.

Keywords: bifidobacteria; catabolic pathways; galactooligosaccharides; glycosidic linkages; lactic acid bacteria; synbiotics.

Figure 1

(A) Bifidobacterial strains grown with 5 mg/mL pGOS; glucose (5 mg/mL) and modified MRS-medium served as positive and negative controls (Neg. control), respectively. Growth was followed by measuring ΔOD manually in anaerobic glass tubes (s. Material and Methods). (B) Strains from LAB grown with 5 mg/mL pGOS; glucose (5 mg/mL) and modified MRS-medium served as positive and negative controls (Neg. control), respectively. OD600nm values were acquired by growing strains in microtiter plates and following ΔOD using a microtiter plate reader. All the values shown are means from 3 biological replicates. Most standard deviations are smaller than the size of the symbols and therefore not apparent.

Figure 2

HPAEC–PAD chromatograms of pGOS (control, first line) and pGOS after the growth of probiotic strains. For each strain, pGOS composition was analyzed in n = 3 biological replicates (numbers 1, 2, and 4 indicate Gal, Glc, and lactose, respectively). Other numbers indicate single pGOS compounds that were not utilized by the strains at the stationary growth phase (Figures 1 and 3). Bifidobacterium strains were grown in a carbon source-free Bifidobacterium medium with 5 mg/mL pGOS added for 25–32 h. LAB strains were grown in modified MRS-medium with 5 mg/mL pGOS added for 18 h. Peaks marked * are non-GOS peaks stemming from the growth medium.

Figure 3

Differential utilization of pGOS components for growth as observed for Bifidobacterium and LAB strains highlighting their diverse capabilities to consume GOS of a specific DP level and different glycosidic linkages present.

Figure 4

Candidate genes involved in GOS catabolism in probiotic bacterial strains. (A) Candidate genes in known pathways for LacEF/LacG, LacS/LacZ, GosDEC/GosG, and GalA/GalCDE/GalG retrieved from BLAST searches of reference genes against bacterial genomes. The family and number of GHs annotated with dbCAN2. (B) Signal sequences for extracellular secretion searched by dbCAN2, PSORTb 3.0 (1, 2).

Figure 5

Catabolic routes identified in Bifidobacterium and LAB to degrade β-galactooligosaccharide compounds from pGOS. Lactobacillus strains employ genes of (i) LacEF/LacG pathway to utilize mostly pGOS compounds with a similar structure to lactose (labelled as fraction “1”) or (ii) LacS/LacZ pathway to utilize mostly DP2 compounds of pGOS and certain DP3 compounds (labelled as fraction 1 and 2, respectively). Bifidobacterial strains employ (i) also the LacS/LacZ pathway to utilize DP2 compounds from pGOS and in addition (ii) GosDEC/GosG and/or the GalA/GalCDE/GalG pathway(s) to utilize GOS compounds with a higher DP.