Broad conservation of milk utilization genes in Bifidobacterium longum subsp. infantis as revealed by comparative genomic hybridization.

Human milk oligosaccharides (HMOs) are the third-largest solid component of milk. Their structural complexity renders them nondigestible to the host but liable to hydrolytic enzymes of the infant colonic microbiota. Bifidobacteria and, frequently, Bifidobacterium longum strains predominate the colonic microbiota of exclusively breast-fed infants. Among the three recognized subspecies of B. longum, B. longum subsp. infantis achieves high levels of cell growth on HMOs and is associated with early colonization of the infant gut. The B. longum subsp. infantis ATCC 15697 genome features five distinct gene clusters with the predicted capacity to bind, cleave, and import milk oligosaccharides. Comparative genomic hybridizations (CGHs) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations. Multilocus sequence typing provided taxonomic subspecies designations and grouped the strains between B. longum subsp. infantis and B. longum subsp. longum. CGH analysis determined that HMO utilization gene regions are exclusively conserved across all B. longum subsp. infantis strains capable of growth on HMOs and have diverged in B. longum subsp. longum strains that cannot grow on HMOs. These regions contain fucosidases, sialidases, glycosyl hydrolases, ABC transporters, and family 1 solute binding proteins and are likely needed for efficient metabolism of HMOs. Urea metabolism genes and their activity were exclusively conserved in B. longum subsp. infantis. These results imply that the B. longum has at least two distinct subspecies: B. longum subsp. infantis, specialized to utilize milk carbon, and B. longum subsp. longum, specialized for plant-derived carbon metabolism.

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

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.

Daily variations in oligosaccharides of human milk determined by microfluidic chips and mass spectrometry.

Human milk is a complex biological fluid that provides not only primary nourishment for infants but also protection against pathogens and influences their metabolic, immunologic, and even cognitive development. The presence of oligosaccharides in remarkable abundance in human milk has been associated to provide diverse biological functions including directing the development of an infant's intestinal microflora and immune system. Recent advances in analytical tools offer invaluable insights in understanding the specific functions and health benefits these biomolecules impart to infants. Oligosaccharides in human milk samples obtained from five different individual donors over the course of a 3 month lactation period were isolated and analyzed using HPLC-Chip/TOF-MS technology. The levels and compositions of oligosaccharides in human milk were investigated from five individual donors. Comparison of HPLC-Chip/TOF-MS oligosaccharides profiles revealed heterogeneity among multiple individuals with no significant variations at different stages of lactation within individual donors.

Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation.

The molecular basis by which human breast milk supports the development of a protective intestinal microbiome in infants is unknown. After lactose and lipids, human milk oligosaccharides (HMOs) are quantitatively the third largest and most diverse component of breast milk. In this work, glycomic profiling of HMO consumption by bifidobacteria using Fourier transform ion cyclotron resonance mass spectrometry reveals that one species, Bifidobacterium longum biovar infantis ATCC 15697, an isolate from the infant gut, preferentially consumes small mass oligosaccharides, representing 63.9% of the total HMOs available. These HMOs were detected in human breast milk at the onset and constantly through the first month of lactation by use of high performance liquid chromatography-chip time-of-flight mass spectrometry. Further characterization revealed that strain ATCC 15697 possesses both fucosidase and sialidase activities not present in the other tested strains. This work provides evidence that these small mass HMOs are selectively metabolized by select bifidobacterial strains and represent a potential new class of bioactive molecules functioning as prebiotics to facilitate a protective gut colonization in breast-fed newborns.

A versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides.

Human milk contains approximately 200 complex oligosaccharides believed to stimulate the growth and establishment of a protective microbiota in the infant gut. The lack of scalable analytical techniques has hindered the measurement of bacterial metabolism of these and other complex prebiotic oligosaccharides. An in vitro, multi-strain, assay capable of measuring kinetics of bacterial growth and detailed oligosaccharide consumption analysis by FTICR-MS was developed and tested simultaneously on 12 bifidobacterial strains. For quantitative consumption, deuterated and reduced human milk oligosaccharide (HMO) standards were used. A custom software suite developed in house called Glycolyzer was used to process the large amounts of oligosaccharide mass spectra automatically with (13)C corrections based on de-isotoping protocols. High growth on HMOs was characteristic of Bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains tested, B. longum bv. longum, B. adolescentis, B. breve and B. bifidum, showed low or only moderate growth ability. Total oligosaccharide consumption ranged from a high of 87% for B. infantis JCM 7009 to only 12% for B. adolescentis ATCC 15703. A detailed analysis of consumption glycoprofiles indicated strain-specific capabilities towards differential metabolism of milk oligosaccharides. This method overcomes previous limitations in the quantitative, multi-strain analysis of bacterial metabolism of HMOs and represents a novel approach towards understanding bacterial consumption of complex prebiotic oligosaccharides.

Analysis and quantitation of fructooligosaccharides using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry.

Inulin is a class of fructooligosaccharide (FOS) derived from plants, which is often used as a natural food ingredient. Inulin is currently used as an additive in baked goods, dairy products, infant formula, and dietary supplements as a result of its purported health-promoting properties. The growth of health-promoting lactobacilli and bifidobacteria is supported by FOS, giving it the classification of a prebiotic; however, its ability to selectivity stimulate only beneficial bacteria has not been demonstrated. In order to better understand the role of inulin and FOS as prebiotics, matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry has been used for qualitative and quantitative analysis on bacterial growth. A method using an internal standard has been developed to quantify the consumption of FOS by Bifidobacterium longum bv. infantis using a calibration curve. Due to the differential consumption of FOS, the calibration curve was modified to include intensity components for each polymer unit in order to achieve more accurate quantitation. The method described was designed to be more rapid, precise, and robust for quantitative analysis when compared to existing methods.

Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry.

Oligosaccharides are the third most abundant component in human milk. In the past decades, it became apparent that they would be able to protect against pathogens and participate in the development of the gut microflora for infants. However, their role in infants' nutrition and development remains poorly understood. To better understand this function, it is extremely important to have a quantitative tool for profiling oligosaccharides. In this article, we show the development of a method to quantitatively differentiate the relative amounts of oligosaccharides fermented by different intestinal bacteria. To determine the oligosaccharide consumption, bacteria were grown in a medium using human milk oligosaccharides (HMOs) as the only carbon source purified from breast milk and further analyzed by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS). A method using an internal deuterium-labeled standard was developed and compared with an external standard method, with the internal standard method giving better precision and unambiguous measurements than the external standard method and providing to be a novel and robust tool for following bacterial fermentation of milk oligosaccharides.