Touching the High Complexity of Prebiotic Vivinal Galacto-oligosaccharides Using Porous Graphitic Carbon Ultra-High-Performance Liquid Chromatography Coupled to Mass Spectrometry

Abstract

Galacto-oligosaccharides (GOS) are used in infant formula to replace the health effects of human milk oligosaccharides, which appear to be dependent upon the structure of the individual oligosaccharides present. However, a comprehensive overview of the structure-specific effects is still limited as a result of the high structural complexity of GOS. In this study, porous graphitic carbon (PGC) was used as the stationary phase during ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS). This approach resulted in the recognition of more than 100 different GOS structures in one single run, including reducing and non-reducing GOS isomers. Using nuclear magnetic resonance-validated structures of GOS trisaccharides, we discovered MS fragmentation rules to distinguish reducing isomers with a mono- and disubstituted terminal glucose by UHPLC–PGC–MS. UHPLC–PGC–MS enabled effective recognition of structural features of individual GOS components in complex GOS preparations and during, e.g., biological conversion reactions. Hence, this study lays the groundwork for future research into structure-specific health effects of GOS.

Structure‐Specific Fermentation of Galacto‐Oligosaccharides, Isomalto‐Oligosaccharides and Isomalto/Malto‐Polysaccharides by Infant Fecal Microbiota and Impact on Dendritic Cell Cytokine Responses

“…Recently, we discovered that Vivinal GOS is more structurally diverse than anticipated by using a newly developed high‐throughput characterization method based on UHPLC‐PGC‐MS. [ 20 ] More than 100 structures were identified, from which the most abundant reducing (20) and non‐reducing (11) DP3 isomers representing 44% of the total GOS preparation, were monitored to demonstrate the structure‐specific fermentability.…”

“…This characterization methodology originally developed for GOS, [ 20 ] also tremendously increased our understanding of the complexity of the IMO preparation Vitafiber. In total, nine reducing and six non‐reducing DP3 isomers were detected, which represented 44% of the total IMO preparation.…”

“…To study the fate of individual isomers with different degrees of polymerization, GOS and IMO were analyzed on an Accela ultra‐high‐performance liquid chromatography (UHPLC) system (Thermo Scientific, San Jose, CA, USA) coupled to a mass spectrometer (LTQ Velos Pro ion trap MS, Thermo Scientific) as described elsewhere with minor modification of the eluent profile. [ 20 ] For the analysis of GOS, mobile phase A (ULC−MS water + 0.1% v/v formic acid) and B (acetonitrile + 0.1% v/v formic acid) were eluted as follows: 0–2 min, 3% B; 2–51.7 min, 3–11% B; 51.7–58.6 min, 11–15% B; 58.6–60.2 min, 15–100% B; 60.2–68.0 min, 100% B; 68.0–69.5, 100–3% B and 69.5–77.3 min, 3% B. For the analysis of IMO, mobile phase A and B were eluted as follows: 0–2 min, 2% B; 2–23.7 min, 2–9% B; 23.7–49.6 min, 9–14% B; 49.6–51.1 min, 14–100% B; 51.1–58.9 min, 100% B; 58.9–60.5, 100–2% B and 60.5–68.2 min, 2% B.…”

“…[ 16 ] Besides reducing GOS with a free anomeric carbon at the terminal position, also non‐reducing GOS containing a (1↔1) linkage have been reported. [ 17 , 18 , 19 , 20 ] A recently developed characterization method showed that the commercial Vivinal GOS is more structurally diverse than anticipated. [ 20 ] Over 100 different structures were identified in Vivinal GOS, including a large variety of non‐reducing GOS isomers with different location and anomeric configuration of the (1↔1) linkage.…”

A novel class of human milk oligosaccharides based on 6’- galactosyllactose and containing N-acetylglucosamine branches extended by oligogalactoses

“…Distinct from the new HG-HMO, however, is that these components, e. g. in the Tammar Wallabys neither contains the GlcNAc branching nor the fucosylation at either branching arm. To differentiate the new HG-HMO from the commercially used GOS it is important to underline that GOS contains a variable number of galactose moieties as the only monosaccharide in their galacto-chains 27,28 . Hence, GOS do not have any similarity neither to classical HMOs nor to HG-HMOs…”