Oligosaccharides in human milk represent a group of bioactive molecules that have evolved to be an abundant and diverse component of human milk, even though they have no direct nutritive value to the infant. A recent hypothesis proposes that they could be substrates for the development of the intestinal microflora and the mucosal immune system. The inability to determine the exact composition of these oligosaccharides limits research and the ability to understand their biological functions. Oligosaccharides isolated from the lipids and proteins of individual human milk samples were analyzed by a combination of techniques including microchip liquid chromatography mass spectrometry (HPLC-Chip/MS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS). Accurate mass measurements obtained using an orthogonal time-of-flight (o-TOF) mass spectrometry provided oligosaccharide composition for approximately 200 individual molecular species. Comparison of HPLC-Chip/MS profiles from five different women revealed variations in milk oligosaccharide compositions. HPLC-Chip/MS profiling provides a method for routinely identifying milk oligosaccharides. Tandem MS in combination with exoglycosidase digestion provides unambiguous differentiation of structural isomers.

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.

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.Human milk is unique because of the high concentration and diversity of human milk oligosaccharides (HMO). HMO are the third most abundant component of human milk (9), and at least 130 different masses have been identified (14). In vitro, HMO are resistant to catabolism by host hydrolases (3, 4), and based on the mass balance between consumption and excretion, Chaturvedi et al.(1) calculated that 97% of HMO pass through infants undigested, while Coppa et al. (2) estimated that 40 to 50% of HMO pass through infants undigested.A prebiotic function was attributed to HMO based on studies done in the 1950s with Bifidobacterium bifidus subsp. pennsylvannicus (6, 7), yet the measurements were based on growth enhancement, not fermentability, as the media contained lactose. Direct fermentation of HMO by Bifidobacterium spp. and/or Lactobacillus spp. has not been demonstrated yet, and there are many questions about the metabolic fate of these molecules. Do subpopulations mediate specific functions, such as pathogen binding or prebiotic activity? Are specific bacterial species required for catabolism in the gut? The goals of this investigation were to determine the fermentability of HMO by two representative species of breast-fed infant microbiota, Bifidobacterium infantis and Lactobacillus gasseri (8,10) and to characterize the changes in the HMO after bacterial growth. Determination of the biological basis underlying the HMO abundance in human milk should be of general interest in human nutrition (15).Pooled milk was provided by the Mother's Milk Bank of San Jose, CA, and the Mother's Milk Bank of Austin, TX. Oligosaccharides were extracted as described by Gnoth et al. (5), with modifications. One liter of milk was centrifuged at 5,000 ϫ g for 30 min at 4°C, and the fat was removed. Ethanol (2 liters) was added, and the solution was incubated overnight at 4°C. The precipitate was removed by centrifugation at 5,000 ϫ g for 30 min at 4°C, and the solvent was removed by rotary evaporation. The concentration of the solution was adjusted to 0.05 M with phosphate buffer (pH 6.8), 3,000 U ␤-galactosidase (Kluyveromyces fragilis) was added, and the solution was incubated for 1 h at 37°C. Then the solution was extracted with 4 volumes of chloroform-methanol (2:1, vol/vol), and the aqueous layer was collected.As described by Redmond and Packer (12), monosaccharides and disaccharides were removed by selective adsorption of HMO, using solid-phase extraction with graphitized nonporous graphitized carbon cartridges (Supelco Inc., Bellefonte, PA). The oligosaccharides that were retained were eluted wit...