Building a fructan LC–MS2 library and its application to reveal the fine structure of cereal grain fructans
Introduction
Fructans are defined as fructose-based oligomers and polymers (Lewis, 1993) and are accumulated by a diverse array of algae, flowering plants and bacteria (Hendry, 1993). They vary widely in chemical structure and five classes of structurally different fructans are known to occur in nature. Inulin-type fructans and levan-type fructans have a linear fructose backbone consisting mainly of Fruf(β2-1) and Fruf(β2-6) units, respectively (Fig. 1).
When a terminal glucose is present in inulin-type or levan-type fructans, the anomeric carbon of glucose is linked to the anomeric carbon of fructose as in sucrose (Fruf(β2-1α)Glc). When fructans are branched with both Fruf(β2-1) and Fruf(β2-6) units in one molecule, they are classified as graminan-type fructans. Fructans with an internal glucose unit belong to the neo-inulin and neolevan-type fructans and have mainly Fruf(β2-1) and Fruf(β2-6) units, respectively (Van den Ende, 2013).
Fructans have been studied intensively as they can serve multiple functions in plants (Van den Ende, 2013) and because their consumption affects human health (Roberfroid et al., 2010). Fructans are dietary fibers and inulin-type fructans are recognized as prebiotics (Roberfroid et al., 2010). However, for some sensitive individuals, fructan consumption causes unwanted gastro-intestinal side effects as well (Kelly, 2009). Patients suffering from irritable bowel syndrome appear to benefit from following a diet low in FODMAPs (fermentable oligo-, di- and monosaccharides and polyols), a specific group of carbohydrates that includes fructans (Gibson, 2017). FODMAPs are defined as poorly digested short-chain carbohydrates and polyols that are rapidly fermented in the colon (Gibson & Shepherd, 2005). Fructan structure is important in this context because it affects the fermentation rate (Hernot et al., 2009; Stewart, Timm, & Slavin, 2008). Insight in fructan structure is also useful from a food technological point of view as it affects the extent of fructan degradation during food processing (Verspreet, Dornez, Van den Ende, Delcour, & Courtin, 2015). In addition, fructan structure is under scrutiny in fundamental research aiming to comprehend the role of fructans in plants under stressful conditions (Hincha et al., 2007; Peshev, Vergauwen, Moglia, Hideg, & Van den Ende, 2013). Structural analysis is hence becoming instrumental in the understanding of the nutritional effects of fructans, their fate during food processing and their physiological role in plants.
The most promising technique to study fructan structure is liquid chromatography – mass spectrometry (LC–MS). LC–MS can provide detailed structural information of individual fructan structures even when present in complex mixtures. It furthermore allows high-throughput screenings as no preceding purification or derivatization steps are required (Verspreet, Hansen, Dornez, Courtin, & Harrison, 2014). Dr. Harrison was one of the first to apply this technique to study the chemical structure of fructans and described the MS2 fragmentation of inulin-type, levan-type (Harrison, Xue, Lane, Villas-Boas, & Rasmussen, 2012) and neo-inulin-type fructans (Harrison, 2012). More recently, the MS2 fragmentation of graminan-type fructans was documented (Verspreet, Hansen, Dornez, Harrison, & Courtin, 2014), leaving the neolevan-type fructans as the only remaining fructan class for which the MS2 fragmentation has not yet been described.
The first aim of this study is to analyze the MS2 fragmentation of neolevan-type fructans. Their MS2 fragmentation will be compared with that of fructans from other structural classes and all data combined to establish a fructan LC–MS2 library. We hypothesize that creating such a library will pave the way for rapid structural identification of fructans in any type of sample. The second aim of this study is to demonstrate the wide applicability of the LC–MS2 library by characterizing fructans in a set of cereal flours. Although cereal grains are staple foods worldwide, their fructan structure has not yet been closely examined. Only the structure of wheat grain fructans was recently investigated (Verspreet, Hansen et al., 2015).
Section snippets
Materials
All chemicals, solvents and reagents were purchased from Sigma-Aldrich (Bornem, Belgium) and were of analytical grade unless specified otherwise. 1-Kestotriose (1-K) was purchased from Megazyme (Bray, Ireland) and 1,1-kestotetraose (1,1-KT) along with 1,1,1-kestopentaose (1,1,1-KP) from Elicityl (Crolles, France). Sucrose labeled at specific positions with 13C isotopes was bought from Campro Scientific (Veenendaal, The Netherlands). Universally labelled (UL) sucrose ([UL-13C6 Fru] sucrose)
Enzymatic synthesis and LC–MS analysis of neolevan-type fructans
Neolevan-type fructans were produced in vitro by incubating barley 6-SFT with 6G-K and either normal sucrose or universally labelled sucrose. The reaction mixtures were examined by LC–MS to determine the molecular structure of the produced fructans. Inulin-type fructans, levan-type fructans and graminan-type fructans were identified by injection of earlier produced fructan standards (Harrison et al., 2012; Verspreet, Hansen, Dornez, Harrison et al., 2014). Identification of the remaining
Conclusions
Neolevan-type fructans were made by incubation of barley 6-SFT with sucrose and 6G-K and the formed structures were identified with LC–MS2. Barley 6-SFT had a strong tendency to extend 6G-K by adding β-(2,6)-linked fructose units to the β-(2,6)-linked fructose of 6G-K. Surprisingly, the enzyme also added fructose units via β-(2,1)-linkages to the existing fructan chain. The newly formed fructan structures together with standards containing fructans from other structural classes, were analyzed
Acknowledgements
JV acknowledges “Vlaams Agentschap Innoveren en Ondernemen” (Vlaio, former IWT) for financial support (IWT-SBO-130028). WVdE is supported by FWO Vlaanderen.
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