Glycoprofiling bifidobacterial consumption of galacto-oligosaccharides by mass spectrometry reveals strain-specific, preferential consumption of glycans

Abstract

Galacto-oligosaccharides (GOS) are versatile food ingredients that possess prebiotic properties. However, at present there is a lack of precise analytical methods to demonstrate specific GOS consumption by bifidobacteria. To better understand the role of GOS as prebiotics, purified GOS (pGOS) without disaccharides and monosaccharides was prepared and used in bacterial fermentation experiments. Growth curves showed that all bifidobacteria assayed utilized and grew on pGOS preparations. We used a novel mass spectrometry approach involving matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) to determine the composition of oligosaccharides in GOS syrup preparations. MALDI-FTICR analysis of spent fermentation media demonstrated that there was preferential consumption of selected pGOS species by different bifidobacteria. The approach described here demonstrates that MALDI-FTICR is a rapid-throughput tool for comprehensive profiling of oligosaccharides in GOS mixtures. In addition, the selective consumption of certain GOS species by different bifidobacteria suggests a means for targeting prebiotics to enrich select bifidobacterial species.

Figures

FIG. 1.

Positive-mode MALDI-FTICR spectra of GOS syrup obtained under two different experimental conditions. The voltage of the MALDI-FTICR quadruple ion guide was adjusted to detect small (A) or larger (B) oligosaccharides. Major peaks at m/z 527, 689, 851, 1013, 1175, 1337, 1499, 1662, and 1824 represent sodium-coordinated ([M+Na]+) GOS with DP ranging from 3 to 11. Minor signals observed at 18 mass units less could correspond to B-type fragments.

FIG. 2.

Positive-mode MALDI-FTICR spectra of GOS Bio-Gel P-2 fractions. (A) Forty-five-milliliter fraction; (B) 56-ml fraction; (C) 67-ml fraction; (D) 74-ml fraction; (E) 82-ml fraction. Signals at m/z 527, 689, 851, 1013, 1175, 1337, 1499, 1662, 1824, 1966, 2148, 2310, and 2473 represent sodium-coordinated GOS with DP ranging from 3 to 15.

FIG. 3.

IRMPD MALDI-FTICR spectra of GOS. (A) GOS with a DP of 5; (B) GOS with a DP of 4; (C) GOS with a DP of 3. Fragment ions corresponding to glycosidic bond cleavages (Hex) and cross-ring cleavages (60, 90, and 120) were obtained.

FIG. 4.

Positive-mode MALDI-FTICR spectrum of pGOS with selected DP used in bifidobacterial fermentation experiments.

FIG. 5.

Growth curves of B. adolescentis ATCC 15703, B. breve ATCC 15700, B. longum subsp. infantis ATCC 15697, and B. longum subsp. longum DJO10A for growth on modified MRS containing (A) 0.5% (wt/vol) pGOS, (B) 1% (wt/vol) pGOS, (C) 1.5% (wt/vol) pGOS, and (D) 2% (wt/vol) pGOS. Growth studies were carried out in triplicate, and a representative data set is shown. Erratic curves for B. breve were observed when it began to aggregate.

FIG. 6.

Absolute ion intensities of pGOS remaining after bacterial fermentation obtained from MALDI-FTICR mass spectra. Four spectra were acquired and averaged for each biological replicate. The error bars indicate standard deviations. A.U, arbitrary units.