Topochemical Design of Cellulose-Based Carriers for Immobilization of Endoxylanase.

Xylooligosaccharides (XOSs) gained much attention for their use in food and animal feed, attributed to their prebiotic function. These short-chained carbohydrates can be enzymatically produced from xylan, one of the most prevalent forms of hemicellulose. In this work, endo-1,4-ß-xylanase from Thermotoga maritima was immobilized on cellulose-based beads with the goal of producing xylooligosaccharides with degrees of polymerization (DPs) in the range of 4-6 monomeric units. More specifically, the impact of different spacer arms, tethers connecting the enzyme with the particle, on the expressed enzymatic activity and oligosaccharide yield was investigated. After surface functionalization of the cellulose beads, the presence of amines was confirmed with time of flight secondary ion mass spectrometry (TOF-SIMS), and the influence of different spacer arms on xylanase activity was established. Furthermore, XOSs (DPs 2-6) with up to 58.27 mg/g xylan were obtained, which were greatly enriched in longer oligosaccharides. Approximately 80% of these XOSs displayed DPs between 4 and 6. These findings highlight the importance of topochemical engineering of carriers to influence enzyme activity, and the work puts forward an enzymatic system focusing on the production of longer xylooligosaccharides.

Aqueous phase extractable protein of wheat bran and germ for the production of liquid and semi-solid foods.

To achieve a more sustainable global food production, a shift from animal to plant protein based food is necessary. At the same time, these plant proteins are preferentially derived from side-streams of industrial processes. Wheat bran and germ represent two major side-streams of the wheat milling industry, and contain aqueous-phase soluble proteins with a well-balanced amino acid composition. To successfully use wheat bran and germ proteins in novel plant-based liquid and semi-solid foods, they need to (i) be rendered extractable and (ii) contribute functionally to stabilizing the food system. Prior heat treatment and the occurrence of intact cell walls are important barriers in this regard. Several strategies have been applied to overcome these issues, including physical processing and (bio)chemical modification. We here present a comprehensive, critical overview of the aqueous-phase extraction of protein from (modified) wheat bran and germ. Moreover, we discuss the functionality of the extracted protein, specifically in the context of liquid (foam- and emulsion-type) and semi-solid (gel-type) food applications. In each section, we identify important knowledge gaps and highlight several future prospects that could further increase the application potential of wheat bran and germ proteins in the food industry.

Arabinoxylan, ß-glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo-SEM.

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of ß-glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo-SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with ß-glucan depositions on the inside. During germination, arabinoxylan and ß-glucan are hydrolysed, but unlike ß-glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.

Health benefits of whole grain: effects on dietary carbohydrate quality, the gut microbiome, and consequences of processing.

Grains are important sources of carbohydrates in global dietary patterns. The majority of these carbohydrates, especially in refined-grain products, are digestible. Most carbohydrate digestion takes place in the small intestine where monosaccharides (predominantly glucose) are absorbed, delivering energy to the body. However, a considerable part of the carbohydrates, especially in whole grains, is indigestible dietary fibers. These impact gut motility and transit and are useful substrates for the gut microbiota affecting its composition and quality. For the most part, the profile of digestible and indigestible carbohydrates and their complexity determine the nutritional quality of carbohydrates. Whole grains are more complex than refined grains and are promoted as part of a healthy and sustainable diet mainly because the contribution of indigestible carbohydrates, and their co-passenger nutrients, is significantly higher. Higher consumption of whole grain is recommended because it is associated with lower incidence of, and mortality from, CVD, type 2 diabetes, and some cancers. This may be due in part to effects on the gut microbiota. Although processing of cereals during milling and food manufacturing is necessary to make them edible, it also offers the opportunity to still further improve the nutritional quality of whole-grain flours and foods made from them. Changing the composition and availability of grain carbohydrates and phytochemicals during processing may positively affect the gut microbiota and improve health.

Modifying wheat bran to improve its health benefits.

Consumption of wheat bran (WB) has been associated with improved gastrointestinal health and a reduced risk for colorectal cancer, cardiovascular diseases and metabolic disorders. These benefits are likely mediated by a combination of mechanisms, including colonic fermentation of the WB fiber, fecal bulking and the prevention of oxidative damage due to its antioxidant capacities. The relative importance of those mechanisms is not known and may differ for each health effect. WB has been modified by reducing particle size, heat treatment or modifying tissue composition to improve its technological properties and facilitate bread making processes. However, the impact of those modifications on human health has not been fully elucidated. Some modifications reinforce whereas others attenuate the health effects of coarse WB. This review summarizes available WB modifications, the mechanisms by which WB induces health benefits, the impact of WB modifications thereon and the available evidence for these effects from in vitro and in vivo studies.

In vitro prebiotic potential of agricultural by-products on intestinal fermentation, gut barrier and inflammatory status of piglets.

The inclusion of fibre-rich ingredients in diets is one possible strategy to enhance intestinal fermentation and positively impact gut ecology, barrier and immunity. Nowadays, inulin-type fructans are used as prebiotics in the feed of piglets to manipulate gut ecology for health purposes. Likewise, some by-products could be considered as sustainable and inexpensive ingredients to reduce gut disorders at weaning. In the present study, chicory root and pulp, citrus pulp, rye bran and soya hulls were tested in a three-step in vitro model of the piglet's gastro-intestinal tract combining a pepsin-pancreatin hydrolysis (digestion), a dialysis step using cellulose membranes (absorption) and a colonic batch fermentation (fermentation). The fermentation kinetics, SCFA and microbiota profiles in the fermentation broth were assessed as indicators of prebiotic activity and compared with the ones of inulin. The immunomodulatory effects of fermentation supernatant (FS) were investigated in cultured intestinal porcine epithelial cells (IPEC-J2) by high-throughput quantitative PCR. Chicory root displayed a rapid and extensive fermentation and induced the second highest butyrate ratio after inulin. Citrus pulp demonstrated high acetate ratios and induced elevated Clostridium clusters IV and XIVa levels. Chicory root and pulp FS promoted the intestinal barrier integrity with up-regulated tight and adherens junction gene expressions in comparison with inulin FS. Chicory pulp FS exerted anti-inflammatory effects in cultured IPEC-J2. The novel approach combining an in vitro fermentation model with IPEC-J2 cells highlighted that both chicory root and pulp appear to be promising ingredients and should be considered to promote intestinal health at weaning.

Introducing insoluble wheat bran as a gut microbiota niche in an in vitro dynamic gut model stimulates propionate and butyrate production and induces colon region specific shifts in the luminal and mucosal microbial community.

The spatial organization of gut microorganisms is important with respect to their functional role in the gut ecosystem. Regional differences in the longitudinal and lateral direction are, however, not frequently studied, given the difficulty to sample these human gut regions in vivo. Particularly the insoluble food particle-associated microbiota is poorly studied. Therefore, the long-term effects of insoluble wheat bran supplementation on the composition and functionality of the gut microbial community derived from six individuals were explored in the Dietary Particle-Mucosal-Simulator of the Human Intestinal Microbial Ecosystem in vitro model. Wheat bran stimulated propionate and butyrate production and induced shifts in the luminal and mucosal microbial community composition. The insoluble wheat bran residue and the mucus layer were identified as crucial platforms in sustaining diversity by selectively enriching species, which are not thriving in the luminal environment, including Lactobacillus, Bifidobacterium and Dialister species, Roseburia faecis, Prevotella copri and Bacteroides ovatus. Despite the evident habitat preference, some parallels could be drawn between the enrichment of taxa on bran platforms and their stimulation in the luminal and mucosal communities. Removing wheat bran during the wash-out period reversed the functional effects and gave rise to a blooming of some taxa that are considered opportunistic pathogens.

Reduced-Particle-Size Wheat Bran Is Efficiently Colonized by a Lactic Acid-Producing Community and Reduces Levels of Enterobacteriaceae in the Cecal Microbiota of Broilers.

In the present study, we investigated whether reducing the particle size of wheat bran affects the colonizing microbial community using batch fermentations with cecal inocula from seven different chickens. We also investigated the effect of in-feed administration of regular wheat bran (WB; 1,690 µm) and wheat bran with reduced particle size (WB280; 280 µm) on the cecal microbial community composition of broilers. During batch fermentation, WB280 was colonized by a lactic acid-producing community (Bifidobacteriaceae and Lactobacillaceae) and by Lachnospiraceae that contain lactic acid-consuming butyric acid-producing species. The relative abundances of the Enterobacteriaceae decreased in the particle-associated communities for both WB and WB280 compared to that of the control. In addition, the community attached to wheat bran was enriched in xylan-degrading bacteria. When administered as a feed additive to broilers, WB280 significantly increased the richness of the cecal microbiota and the abundance of bacteria containing the butyryl-coenzyme A (CoA):acetate CoA-transferase gene, a key gene involved in bacterial butyrate production, while decreasing the abundances of Enterobacteriaceae family members in the ceca. Particle size reduction of wheat bran thus resulted in the colonization of the bran particles by a very specific lactic acid- and butyric acid-producing community and can be used to steer toward beneficial microbial shifts. This can potentially increase the resilience against pathogens and increase animal performance when the reduced-particle-size wheat bran is administered as a feed additive to broilers.IMPORTANCE Prebiotic dietary fibers are known to improve the gastrointestinal health of both humans and animals in many different ways. They can increase the bulking capacity, improve transit times, and, depending on the fiber, even stimulate the growth and activity of resident beneficial bacteria. Wheat bran is a readily available by-product of flour processing and is a highly concentrated source of (in)soluble dietary fiber. The intake of fiber-rich diets has been associated with increased Firmicutes and decreased Proteobacteria numbers. Here, we show that applying only 1% of a relatively simple substrate which was technically modified using relatively simple techniques reduces the concentration of Enterobacteriaceae This could imply that in future intervention studies, one should take the particle size of dietary fibers into account.

Kluyveromyces marxianus yeast enables the production of low FODMAP whole wheat breads.

There is evidence that a diet low in Fermentable Oligo-, Di-, Monosaccharides And Polyols (FODMAPs) alleviates symptoms in approx. 70% of the patients suffering from irritable bowel syndrome. Through fructans, wheat containing products are a major source of FODMAPs in the western diet. Although fructans are partially degraded during dough fermentation by Saccharomyces cerevisiae invertase, wheat bread contains notable fructan levels. In this study, it was shown that Kluyveromyces marxianus strain CBS6014 can degrade more than 90% of the fructans initially present in wheat whole meal during bread making, which can be attributed to its high inulinase activity. As K. marxianus CBS6014 was not able to consume maltose during fermentation, alternative sugars (sucrose) or glucose releasing enzymes (amyloglucosidase) had to be included in the bread making recipe to ensure sufficient production of CO2 and high bread quality. Five volatile flavor compounds were produced in significantly different levels when K. marxianus CBS6014 was used as starter culture compared with the conventional S. cerevisiae bakery strain. These differences were, however, not detected when sensory analysis of the crumb was performed. This study demonstrates the potential of inulinase-producing K. marxianus strains for the production of (whole meal) breads low in FODMAPs.

Impact of Preharvest Sprouting on Endogenous Hydrolases and Technological Quality of Wheat and Bread: A Review.

The cereal-based food industry faces the challenge to produce food of high and uniform quality to meet consumer demands. However, adverse weather conditions, including prolonged and repeated rainfall, before harvest time evoke germination of the kernels in the ear of the parent plant, which is known as preharvest sprouting (PHS). PHS results in the production of several hydrolytic enzymes in the kernel, which decreases the technological quality of wheat and causes problems during processing of the flour into cereal-based products. Therefore, wheat that is severely sprouted in the field is less suitable for products for human consumption, and is often discounted to animal feed. Up till now, most knowledge on PHS is obtained by research on laboratory-sprouted wheat as a proxy for field-sprouted wheat. Knowledge on PHS in the field itself is more scarce. This review gives a comprehensive overview of the recent findings on PHS of wheat in the field, compared to knowledge on controlled sprouting. The physiological and functional changes occurring in wheat during PHS and their impact on wheat and bread quality are discussed. This review provides a useful background for further research concerning the potential of field-sprouted wheat to be used as raw material in the food industry.

Systemic availability and metabolism of colonic-derived short-chain fatty acids in healthy subjects: a stable isotope study.

KEY POINTS: The short-chain fatty acids (SCFAs) are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fibre and prebiotics, and can mediate the interaction between the diet, the microbiota and the host. We quantified the fraction of colonic administered SCFAs that could be recovered in the systemic circulation, the fraction that was excreted via the breath and urine, and the fraction that was used as a precursor for glucose, cholesterol and fatty acids. This information is essential for understanding the molecular mechanisms by which SCFAs beneficially affect physiological functions such as glucose and lipid metabolism and immune function. ABSTRACT: The short-chain fatty acids (SCFAs), acetate, propionate and butyrate, are bacterial metabolites that mediate the interaction between the diet, the microbiota and the host. In the present study, the systemic availability of SCFAs and their incorporation into biologically relevant molecules was quantified. Known amounts of 13 C-labelled acetate, propionate and butyrate were introduced in the colon of 12 healthy subjects using colon delivery capsules and plasma levels of 13 C-SCFAs 13 C-glucose, 13 C-cholesterol and 13 C-fatty acids were measured. The butyrate-producing capacity of the intestinal microbiota was also quantified. Systemic availability of colonic-administered acetate, propionate and butyrate was 36%, 9% and 2%, respectively. Conversion of acetate into butyrate (24%) was the most prevalent interconversion by the colonic microbiota and was not related to the butyrate-producing capacity in the faecal samples. Less than 1% of administered acetate was incorporated into cholesterol and <15% in fatty acids. On average, 6% of colonic propionate was incorporated into glucose. The SCFAs were mainly excreted via the lungs after oxidation to 13 CO2 , whereas less than 0.05% of the SCFAs were excreted into urine. These results will allow future evaluation and quantification of SCFA production from 13 C-labelled fibres in the human colon by measurement of 13 C-labelled SCFA concentrations in blood.

Inter-individual differences determine the outcome of wheat bran colonization by the human gut microbiome.

Gut microbiota research reveals a vital role for the luminal and mucosal gut microbiota in human health. Fewer studies, however, have characterized the microbiome associated with undigested, insoluble dietary particles in the gut. These particles can act as a food source for bacteria and offer a physical platform to which they can attach. In this study, the microbiome colonizing wheat bran particles was analyzed. In a batch experiment, wheat bran particles were separately incubated with the faecal microbiota derived from 10 donors and washed after 48 h to remove loosely attached bacteria. The response of the luminal community to wheat bran and inulin, acting as a well-characterized control, was largely donor-dependent, both functionally, and with respect to the microbiome composition. Depending on the donor, wheat bran and inulin fermentation yielded proportionally higher propionate or butyrate production. Clostridium cluster XIVa and, depending on the donor, Prevotella, Roseburia, Megamonas, Bifidobacterium and Bacteroides species were enriched on the wheat bran particles. These genera include species with the documented ability to serve as primary degraders of wheat bran components and other species depending on cross-feeding to obtain their energy. Both functional groups were present in all donors, despite the large inter-individual differences.

Bread Dough and Baker's Yeast: An Uplifting Synergy.

Yeast-mediated dough fermentation is an important phase in the bread making process. The fermentative performance of yeast cells during fermentation is of critical importance for final bread quality, since yeast cells produce CO2 and other metabolites that have an influence on dough rheology and bread texture, volume, and taste. Different factors affect the fermentative performance of yeast cells during dough fermentation, including dough ingredients, fermentation conditions, the type of yeast strain used and yeast pregrowth conditions. Bread dough is a complex matrix that contains several ingredients that can affect the fermentation rate of yeast cells. Although the individual effects of sugar availability and salt level on the leavening ability of yeast have been studied extensively, a comprehensive overview of the relationship between bread dough constituents, fermentation conditions and yeast functionality is still lacking. Moreover, the dough environment is highly variable as several types of dough like lean, sweet or frozen doughs are currently produced by commercial bread producers. For optimal fermentation rates in different types of dough, the use of appropriate yeast strains with specific phenotypic traits is required. Therefore, many researchers have focused on the improvement of yeast strains for optimal fermentation in different types of dough like lean, sweet or frozen dough. Against this background, this review summarizes the current knowledge on the interaction between bread dough and baker's yeast and how to improve this interaction, thereby providing a useful background for further research concerning the functionality of yeast in bread dough.

Molecular Oxygen and Reactive Oxygen Species in Bread-making Processes: Scarce, but Nevertheless Important.

In bread making, O2 is consumed by flour constituents, yeast, and, optionally, some additives optimizing dough processing and/or product quality. It plays a major role especially in the oxidation/reduction phenomena in dough, impacting gluten network structure. The O2 level is about 7.2 mmol/kg dough, of which a significant part stems from wheat flour. We speculate that O2 is quickly lost to the atmosphere during flour hydration. Later, when the gluten network structure develops, some O2 is incorporated in dough through mixing-in of air. O2 is consumed by yeast respiration and in a number of reactions catalyzed by a wide range of enzymes present or added. About 60% of the O2 consumption in yeastless dough is ascribed to oxidation of fatty acids by wheat lipoxygenase activity. In yeasted dough, about 70% of the O2 in dough is consumed by yeast and wheat lipoxygenase. This would leave only about 30% for other reactions. The severe competition between endogenous (and added) O2-consuming systems impacts the gluten network. Moreover, the scarce literature data available suggest that exogenous oxidative enzymes but not those in flour may promote crosslinking of arabinoxylan in yeastless dough. In any case, dough turns anaerobic during the first minutes of fermentation.

Effects of wheat bran extract rich in arabinoxylan oligosaccharides and resistant starch on overnight glucose tolerance and markers of gut fermentation in healthy young adults.

PURPOSE: Specific combinations of dietary fiber (DF) have been observed to result in improved glucose tolerance at a subsequent standardized breakfast. Arabinoxylan oligosaccharides (AXOS) are considered as DF with prebiotic potential, but so far no studies have investigated their metabolic effects in humans. This randomized cross-over study evaluated the overnight impact of breads containing AXOS-rich wheat bran extract and resistant starch (RS, Hi-Maize), separately or combined, on glucose tolerance, related metabolic parameters and markers of gut fermentation in healthy subjects. METHODS: Evening reference and test products were: (1) reference white wheat flour bread (WWB), WWB supplemented with (2) AXOS and RS (WWB + AXOS + RS), (3) an increased content of either AXOS (WWB + hiAXOS) or (4) RS (WWB + hiRS). At the subsequent standardized breakfast, blood was sampled for 3 h to monitor glucose, insulin, nonesterified fatty acids, glucagon-like peptide (GLP)-1 and GLP-2. Breath hydrogen (H2) and short chain fatty acids (SCFA) were measured as markers of gut fermentation, and subjective appetite was rated using visual analog scales. RESULTS: Dose-dependent decreases in glucose responses were observed with increased AXOS over the duration of 3 h. Insulin sensitivity index was improved in the morning after the WWB + hiAXOS evening meal. An increase in breath H2 concentration and circulating SCFA was observed in the morning after both evening meals containing AXOS. CONCLUSION: The present study indicates that AXOS have the potential of improving glucose tolerance in an overnight perspective and suggested mechanisms are improved insulin sensitivity and increased gut fermentation.

The impact of yeast fermentation on dough matrix properties.

BACKGROUND: Most studies on dough properties are performed on yeastless dough to exclude the complicating, time-dependent effect of yeast. Baker's yeast, however, impacts dough matrix properties during fermentation, probably through the production of primary (CO2 and ethanol) and secondary (glycerol, acetic acid and succinic acid) metabolites. The aim of this study is to obtain a better understanding of the changes in yeasted dough behavior introduced by fermentation, by investigating the impact of yeast fermentation on Farinograph dough consistency, dough spread, Kieffer rig dough extensibility and gluten agglomeration behavior in a fermented dough-batter gluten starch separation system. RESULTS: Results show that fermentation leads to a dough with less flow and lower extensibility that breaks more easily under stress and strain. The dough showed less elastic and more plastic deformation behavior. Gluten agglomerates were smaller for yeasted dough than for the unyeasted control. CONCLUSION: These changes probably have to be attributed to metabolites generated during fermentation. Indeed, organic acids and also ethanol in concentrations produced by yeast were previously shown to have similar effects in yeastless dough. These findings imply the high importance of yeast fermentation metabolites on dough matrix properties in industrial bread production. © 2015 Society of Chemical Industry.

Wheat (Triticum aestivum L.) Bran in Bread Making: A Critical Review.

Wheat bran, a by-product of the industrial roller milling of wheat, is increasingly added to food products because of its nutritional profile and physiological effects. Epidemiological data and scientific studies have demonstrated the health benefits of consuming bran-rich or whole-grain food products. However, incorporation of wheat bran in cereal-based products negatively affects their production process. Furthermore, the organoleptic quality of the obtained products is mostly perceived as inferior to that of products based on refined wheat flour. This review summarizes the current knowledge on the impact of wheat bran on bread making, provides a comprehensive overview of the bran properties possibly involved, and discusses different strategies that have been evaluated up till now to counteract the detrimental effects of wheat bran on bread making.

A ¹H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates.

BACKGROUND: The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs). METHODS: Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs. RESULTS: The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups. CONCLUSIONS AND GENERAL SIGNIFICANCE: The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.

Liquid chromatography/mass spectrometry analysis of branched fructans produced in vitro with 13C-labeled substrates.

RATIONALE: Fructans are carbohydrates predominantly based on fructose which are generally considered to be soluble dietary fibers with health-promoting properties. It is known that the nutritional properties of fructans are affected by their structure. This study focused on structural determination of branched fructans, as the most important dietary fructans are branched graminan-type fructans. METHODS: Branched fructans were synthesized enzymatically by incubation of a heterologously expressed sucrose:fructan 6-fructosyltransferase (6-SFT) from Pachysandra terminalis with native or (13)C-labeled substrates. Liquid chromatography/mass spectrometry (LC/MS) was used for the structural identification of branched fructans. The MS(2) fragmentation of these compounds is described for the first time. Analytes were charged by electrospray ionization in negative mode and a quadrupole mass analyzer was used for MS(2) analysis. RESULTS: The MS(2) fragmentation patterns of branched and linear fructans were shown to differ and distinctive ion formation allowed differentiation between all branched fructan isomers formed. P. terminalis 6-SFT preferred extending the existing fructan branch rather than creating a new branch. CONCLUSIONS: The MS(2) fragmentation patterns described in the current paper now allow rapid screening of large sample sets for the presence of branched, graminan-type fructans. Furthermore, the data enables the characterization of fructan-metabolizing enzymes by identification of the fructan structures produced by in vitro reactions as described here for P. terminalis 6-SFT.

A new high-throughput LC-MS method for the analysis of complex fructan mixtures.

In this paper, a new liquid chromatography-mass spectrometry (LC-MS) method for the analysis of complex fructan mixtures is presented. In this method, columns with a trifunctional C18 alkyl stationary phase (T3) were used and their performance compared with that of a porous graphitized carbon (PGC) column. The separation of fructan isomers with the T3 phase improved clearly in comparison with the PGC phase, and retention times were lower and more stable. When the T3-based method was applied on a wheat grain extract, multiple fructan isomers could be discerned, even for fructans with a degree of polymerization of 10. This indicates that wheat grain fructans do not, or not only, have a simple linear structure. The presented method paves the way for elucidation of fructan structures in complex mixtures that contain many structural isomers.