Distribution and characterisation of fructan in wheat milling fractions

Structure and health effects of inulin-type fructans have been extensively studied, while less is known about the properties of the graminan-type fructans in wheat. Arabinoxylan (AX) is another important indigestible component in cereal grains, which may have beneficial health effects. In this study, the fructan content in milling fractions of two wheat cultivars was determined and related to ash, dietary fibre and AX contents. The molecular weight distribution of the fructans was analysed with HPAEC-PAD and MALDI-TOF MS using ¹H NMR and enzymatic hydrolysis for identification of fructans. The fructan content (g/100 g) ranged from 1.5 ± 0.2 in flour to 3.6 ± 0.5 in shorts and 3.7 ± 0.3 in bran. A correlation was found between fructan content and dietary fibre content (r = 0.93, P < 0.001), but with a smaller variation in fructan content between inner and outer parts of the grain. About 50% of the dietary fibre consisted of AX in all fractions. The fructans were found to have a DP of up to 19 with a similar molecular weight distribution in the different fractions.     

Genotypic variation in wheat grain fructan content revealed by a simplified HPLC method

Fructans are prebiotics, with potentially beneficial effects on human health. This study aimed to examine genetic variation in wheat grain fructan content using a simplified analytical method. The method involves extracting fructans from wheat grain followed by enzymatic hydrolysis to break down fructans into monosaccharides that can then be quantitatively measured by anion-exchange liquid chromatography coupled with pulsed amperometric detection. The modified procedure is reliable and allows the handling of large numbers of flour samples at a low cost, and could therefore be useful for assessing large numbers of wheat breeding lines. Using this method, grain samples taken from 19 bread wheat cultivars and breeding lines grown in both glasshouse and the field were analysed for grain fructan content. In addition, grain samples of 29 international wheat landraces and 14 new wheat breeding lines from the International Maize and Wheat Improvement Center (CIMMYT) were surveyed for their fructan contents. There was significant genotypic variation among these materials, with grain fructan content ranging from 0.7 to 2.9% of grain dry weight. There was no evidence of strong genotype-by-environment interaction; the fructan contents of field-grown grain samples were positively correlated (r = 0.83) with those of glasshouse-grown samples of the same cultivars. It should therefore be possible to investigate the genetic control of variation for this trait using the simplified HPLC method and to select effectively for increased grain fructan content in wheat breeding.     

Effects of Different Sources of Fructans on Body Weight,Blood Metabolites and Fecal Bacteria in Normal and Obese non-diabetic and Diabetic Rats

Fructans contribute significantly to dietary fiber with beneficial effects on gastrointestinal physiology in healthy individuals and offer a promising approach to treating some diseases. Two experiments (Experiment 1 = rats with normal weight; Experiment 2 = obese rats) were developed to compare the effects of three fructan sources (Cichorium intybus L. Asteraceae, Helianthus tuberosus L. Asteraceae and Agave angustifolia ssp. tequilana Haw, Agavaceae) on body weight change, blood metabolites and fecal bacteria in non-diabetic (ND) and diabetic (D) rats. In Experiment 1 total body weight gain and daily feed intake in D and ND rats decreased (P < 0.05) with supplements of fructan. Only in D rats, blood glucose concentrations, fecal Clostrodium spp. counts, and liver steatosis decreased, while blood HDL concentrations and fecal Lactobacillus spp. and Bifidobacterium spp. counts increased due to fructans. In Experiment 2, total body weight gain and feed intake in ND and D rats were also decreased by fructans. In ND rats, fructan decreased blood glucose concentrations. In D rats, fructans from A. angustifolia ssp. tequilana decreased blood cholesterol and LDL and liver steatosis. For both ND and D rats, fecal Lactobacillus spp. and Bifidobacterium spp. counts were higher (P < 0.05) with fructan supplements.     

Endogenous arabinoxylans variability in refined wheat flour and its relationship with quality traits

Arabinoxylans (AXs) are the major dietary fiber (DF) component in wheat and their consumption has been associated with several health benefits. Genetic improvement of the AX in refined wheat flour could be a good solution to improve the DF daily consumption while maintaining the flour desirable quality. In this study, 193 common wheat lines were analyzed for their AX content in refined flour and end-use quality. Wide variation in both the total arabinoxylan (TOT-AX) (10.8–16.5 mg/g) and water-extractable arabinoxylan (WE-AX) (3.2–7.6 mg/g) was identified and, in both cases, the genotype had the greatest impact on the observed phenotypes. Variation in the endogenous AX fractions appeared to have a moderate effect on wheat quality. The WE-AX, specifically, were positively correlated with gluten strength (r = 0.11 to 0.32) and bread loaf volume (r = 0.16), whereas the TOT-AX were negatively correlated with dough extensibility (r = −0.11) and bread making quality (r = −0.11). Overall, results of this study show that the genetic improvement of grain AX is feasible and that the AXs present in refined flour do not dramatically alter wheat quality indicating that it is possible to select varieties with high AX endosperm content end desired end-use quality.     

In vitro degradation of grass fructan by equid gastrointestinal digesta

Laminitis is a debilitating disease of the equid foot and appears to result from altered metabolism due to insulin resistance (IR) or hindgut acidosis. These disorders may be elicited by high intakes of simple sugars or fructans in pasture grasses. The aim was to determine in vitro (i) whether grass fructan is degraded in the equid foregut and (ii) the fermentation kinetics and organic acid production in cultures containing grasses differing in fructan content incubated with an equid hindgut microbial inoculum. When grass fructan was incubated with equid gastric and small intestinal digesta, polymeric fructan was partially degraded to oligo‐fructan. When incubated with small intestinal digesta, but not gastric digesta, there was limited net loss of total fructan (oligomeric + polymeric fructan). When Lolium perenne of elevated (PRGH) or moderate (PRGL) fructan content was incubated with an equine hindgut microbial inoculum, the rate of fermentation and accumulation of lactate 6 and 9 h post‐inoculation were significantly greater for PRGH than for PRGL (P < 0·05). This may suggest that grass fructan ingested by grazing horses is incompletely digested in the foregut and passes into the hindgut, resulting in elevated production of lactate, which may indirectly lead to metabolic disorders such as laminitis.     

A modified Megazyme fructan assay for rapidly screening wheat starch synthase IIa mutation populations reveals high fructan accumulation in mature grains of triple null lines

Cereal grains are the major source of fructan intake with potential health benefits for human. Measurement of fructan levels in cereal breeding populations is a great challenge and time-consuming. In this study, K-FRUCHK and K-FRUC kits commercialized by Megazyme International Limited (Bray, Ireland) were modified and optimized in a 96 deep well plate and a thermal block (BioShake iQ, Q.Instruments, Jena, Germany). The modified assays are able to measure up to 91 samples per day with a range of fructan concentrations (0.4–10.8% in this experiment). Of the two assays, the modified K-FRUC assay is more desirable for measuring fructan levels in cereal grains with high starch content. This assay was successfully used to screen three wheat starch synthase IIa (SSIIa) mutation populations with a total of 466 lines. All 21 SSIIa triple null mutants showed high fructan levels (3.1–10.8%) in wholemeal flours; while all single and double nulls were similar to wild types with ∼1% of fructans. This result indicates that lack of entire SSIIa activity changes carbon flux from starch synthesis into fructan pathway. The identified 21 triple null wheat lines may have potential use for the production of high fructan staple foods.     

Changes in bran structure by bioprocessing with enzymes and yeast modifies the in vitro digestibility and fermentability of bran protein and dietary fibre complex

Bran is a good source of dietary fibre, phytochemicals, and also protein, but highly insoluble and recalcitrant structure of bran hinders accessibility of these components for gastrointestinal digestion. In the present work, influence of bioprocessing on the microstructure and chemical properties of rye bran and wheat bread fortified with the rye bran were studied. In vitro protein digestibility, and release of short chain fatty acids (SCFA) and ferulic acid in a gut model were studied. Bioprocessing of rye bran was performed with subsequent treatments with cell-wall hydrolysing enzymes (40 °C, 4 h) and yeast fermentation (20 °C, 20 h). Bioprocessing of rye bran resulted in reduced total dietary fibre content, caused mainly by degradation of fructan and β-glucan, and increased soluble fibre content, caused mainly by solubilisation of arabinoxylans. Microscopic analysis revealed degradation of aleurone cell wall structure of the bioprocessed rye bran. Bioprocessing caused release of protein from aleurone cells, assessed as a larger content of soluble protein in bran and a higher hydrolysis rate in vitro. Bioprocessed bran had also faster SCFA formation and ferulic acid release in the colon fermentation in vitro as compared to native bran.     

Chain length of cereal fructans isolated from wheat stem and barley grain modulates in vitro fermentation

It is well established that inulin-type fructans, such as oligofructose derived from chicory root, improve gastrointestinal health via fermentation and proliferation of desirable bacteria in the large bowel. However, differences in fructan structure may contribute significantly to different fermentation and subsequent metabolic effects. Here we ascertain the chain length of two novel cereal fructans (graminan) isolated from wheat stem and barley grain, and investigate their fermentation capacity in vitro, in comparison to inulin and oligofructose. Fractions, representing three predominant fructo-oligosaccharide (FOS) size ranges from each source, were fermented and short chain fatty acids (SCFA) production was measured after 24 h of batch culture. Graminan FOS when fermented produced similar levels of total SCFA as oligofructose and inulin. The fermentation of high DP FOS fractions of barley grain and wheat stem (DP 5–15) produced more propionate than oligofructose. Graminan fructans may provide a useful alternative source of oligofructose with potential additional health benefits.     

Genetic responses in milling,flour quality,and wheat sensitivity traits to grain yield improvement in U.S. hard winter wheat

A rising global population necessitates continued genetic improvement of wheat (Triticum spp.), but not without monitoring of unintended consequences to processors and consumers. Our objectives were to re-establish trends of genetic progress in agronomic and milling traits using a generational meter stick as the timeline rather than cultivar release date, and to measure correlated responses in flour quality and human wheat-sensitivity indicators. Grain yield and kernel size showed stepwise increases over cycles, whereas wheat protein content decreased by 1.1 g/100 g. Reduced protein content, however, did not result in lower dough strength pertinent to bread baking. A novel method of directly testing gluten elasticity via the compression-recovery test indicated a general increase in gluten strength, whereas the ratio of total polymeric to total monomeric proteins remained stable. Also showing no change with genetic progress in yield were flour levels of gluten epitopes within the key immunotoxic 33-mer peptide. The oligosaccharide fructan, present in milled and wholemeal flours, increased with increasing grain yield potential. While yield improvement in U.S. bread wheat was not accompanied by a decline in gluten strength or systematic shift in a key wheat sensitivity parameter, the unanticipated rise in total fructans does implicate potentially new dietary concerns.     

Structural Characterisation of Water-extractable and Water-unextractable Arabinoxylans in Wheat Bran

Water-extractable arabinoxylan (WE-AX) in wheat bran, of which only a minority originates from adherent endosperm, amounted to 6% of the total arabinoxylan (AX) content in such bran. WE-AX had an arabinose to xylose (A/X)-ratio of 0·45. Graded ethanol precipitation (20–80% range) yielded AX with an A/X-ratio increasing from 0·31 to 0·85. A population of molecular weight (MW) of 50 kDa precipitated between 0 and 40% ethanol and one with much lower apparent MW precipitated at higher ethanol concentrations. Wheat bran unextractable cell wall material (UCM) was obtained as the residue withstanding thermostable α-amylase and protease treatments and consisted mainly of AX (ca 45%) and cellulose (30–35%). Two consecutive extractions of UCM with alkaline hydrogen peroxide (AHP; 2·0%, pH 11·5, 4 h, 60 °C) resulted in a cellulose rich residue (CRR) containing 33% of the AX originally present in UCM. The average A/X-ratio of AX in CRR was lower than that in UCM. The extracted AX polymers (ca 45% of the AX originally present in UCM) had a high A/X-ratio (0·82). Their elution profiles showed two polydisperse peaks with apparent MW of respectively 100–120 kDa and 5–10 kDa. Graded ethanol precipitation resulted in a lowly substituted fraction (A/X=0·40; 0–40% ethanol) of high MW and a fraction of highly substituted AX (A/X>0·95; 40–70% ethanol) containing both low molecular weight (LMW-) as well as high molecular weight (HMW-) AX. At an ethanol concentration of 70% or more, only LMW-AX precipitated.     

LC-MS analysis reveals the presence of graminan- and neo-type fructans in wheat grains

This is the first study describing the fine structure of the main, individual fructan oligosaccharides present in wheat grains. Wheat grain fructan structure was investigated in developing wheat grains and in different tissues of mature grains with liquid chromatography-mass spectrometry. Fructan oligosaccharides with a low degree of polymerization (<5) were mainly of the graminan- and inulin-type in developing wheat grains during the first week after anthesis. Starting from 14 days after anthesis, neo-type fructans, fructans with an internal glucose, were observed for the first time. Several neo-type fructan structures were identified and their portion in the total fructan pool gradually increased during grain development. In the mature kernel, almost no differences were noted between the fructan distributions of wheat flour and two wheat bran fractions enriched in either pericarp or aleurone tissue. Results are related to wheat fructan metabolizing enzymes and the nutritional implications are discussed.     

Effect of soluble dietary fibre addition on rheological and breadmaking properties of wheat doughs

The aim of this experimental work was to evaluate the effect of inulin addition on the rheological properties of common wheat doughs and bread quality. Three commercial fructan products of different number average degree of polymerisation (DPn) were used (DPn = 10 for inulin ST; DPn = 23 for inulin HP and HP-gel). Inulin contents from 2.5 to 7.5% on dry matter (wheat flour plus inulin) were used. Dough rheological properties were investigated using farinograph and dynamic rheological measurements. Upon addition of dietary fibre (DF), significant increase in mixing time and stability, and decrease in water absorption were recorded. Inulin ST exerted greater effect on water absorption than HP products.     

Dietary fiber in triticale grain: Variation in content, composition, and molecular weight distribution of extractable components

Triticale, a man made cereal is mainly used in animal feed but also in food and as a renewable crop for energy. In this study, eight triticale cultivars and reference rye and wheat were grown at two locations in Sweden (Svalöv and Kölbäck). The harvested grains were characterized for dietary fiber (DF) content, molecular weight distribution of extractable DF components, and some general quality parameters (bulk density, thousand kernel weight, and content of crude protein, crude fat, starch, maltose, sucrose, neutral detergent fiber, and acid detergent fiber). Total DF content (13-16%) was affected by both location and cultivar. Arabinoxylan (average 6.8%), fructan (average 2.3%), cellulose (average 2.1%), Klason lignin (average 1.6%), and β-glucan (average 0.7%) were main components of DF. Cultivars grown at Kölbäck contained extractable arabinoxylan with significantly lower weight-average and number-average molecular weights, probably due to rainy weather and sprouting in the ears. The Calcofluor average molecular weight of extractable β-glucan was highly reduced at this site. About 80% of the fructan had a low degree of polymerization (3-9). Our results show that modern triticale grain has a DF profile and molecular weight distribution of extractable DF components that are much more similar to wheat than rye.     

Glycaemic response to frozen stored wheat rolls enriched with inulin and oat fibre

The aim of this study was to examine the effect of dietary fibre addition to partially baked and frozen wheat rolls on the glycaemic index (GI).Healthy humans volunteers (n = 15) took part in the study. They were asked to attend six times in the early morning, over three weeks. Each tested four types of wheat rolls – two without dietary fibre addition: (1) fully baked, non-frozen (FBNF), (2) partially baked and frozen (PBF); and two with the addition of 10% dietary fibre: (3) fully baked, non-frozen (FBNF + F), (4) partially baked and frozen (PBF + F). Glucose solution was used as a reference food and tested twice. Blood glucose concentrations were measured before consumption, as well as at 15, 30, 45, 60, 90 and 120 min after the start of the meal. Dietary fibre consisted of oat fibre (75%) and of inulin (25%).It was concluded that both factors (freezing and fibre), applied to the wheat rolls at the same time, reduced statistically significantly (P ≤ 0.05) the glycaemic index by 34% – PBF + F (GI = 53 ± 7) compared to control – FBNF roll (GI = 87 ± 11). This effect was not observed when fibre supplementation or frozen storage were applied separately.     

Dynamic viscoelastic, calorimetric and dielectric characteristics of wheat protein isolates

Dynamic oscillatory rheology of two wheat protein isolate (Prolite 100 and Prolite 200) doughs (≈48% moisture content, wet basis) were studied over a frequency range of 0.1–10 Hz during temperature sweep from 20 to 90 °C at a heating rate of 2 °C/min. Both doughs behaved similarly during heating; showed a threshold value and increased sharply, thereafter. Prolite 200 dough had a higher elastic modulus (G′) and lower phase angle (δ) whereas Prolite 100 showed a distinct gel point at 52.2 °C followed by significant increase up to 90 °C. Rheological data of doughs after isothermal heating at 90 °C for 15 min followed by cooling to 20 °C resulted in strong mechanical strength. However, Prolite 100 dough showed more viscoelastic characteristics with significant transformation from liquid-like to solid-like behavior after heating than Prolite 200. Thermal analysis of isolates indicated distinct endothermic peaks in wider temperature range (50–130 °C) at various moisture levels. Lower temperatures could be associated with denaturation of various fractions of proteins whereas higher temperature linked to glass transition temperature of isolates. SDS–PAGE did not show any clear distinction among protein subunits between two isolates. Dielectric measurements of isolates at frequencies from 500 to 3000 MHz and temperature range between 30 and 80 °C indicated Prolite 200 had higher dielectric constant (ε′) and loss factor (ε″) than Prolite 100. Isolates showed significant changes in dielectric properties above 50 °C indicating protein denaturation and supported rheological and calorimetric data.     

A chromatographic survey of methods for extracting long‐chain grass fructans

To optimize yields of long‐chain fructans extracted from herbage, fructan concentrations and chromatographic profiles were compared in four cool‐season grasses extracted by different methods. In a preliminary study, extraction temperatures, pH prior to extract concentration, numbers of extractions, and tissue coarseness were varied to evaluate their effects on chromatographic profiles and quantities of total long‐chain fructan (degree of polymerization (DP) of ～7 or higher). Fructans were separated by anion‐exchange high‐performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD). The identity of the putative fructan peaks was confirmed by acid hydrolysis of fructans to fructose. Tall fescue and bluegrass fructans were below the detection limits used in this study. Pooled timothy and cocksfoot data indicated that two or three extractions at the same temperature yielded similar amounts of fructans and that adjusting pH from 6 to 7 did not affect recovery. Replicated extractions of cocksfoot by four methods chosen from the preliminary study demonstrated that the least fructan was extracted from chopped cocksfoot at ambient temperature (P = 0·019). One C₁₈ solid‐phase extraction cleanup step sufficed for analysis. The extraction method recommended is two boiling water extractions of either chopped or ground material. However, ground material extracted at ambient temperature is comparable in total fructan concentration and maximum DP.     

The molecular structure features-immune stimulatory activity of arabinoxylans derived from the pentosan faction of wheat flour

Recently, the immune stimulation properties of cereal arabinoxylans (AX) have been reported. The aims of this study were to identify the molecular features and potential immune stimulation activities of AX and enzymatic modified arabinoxylan (AXE) from the pentosan fraction of wheat flour.The results of molecular characterization of AX and AXE show that AXE contains a larger portion (85.7%) of low Mw arabinoxylans (≤25 kDa) and has a higher degree of branch substitution (0.81 ± 0.01) compared to AX (49.5% and 0.62 ± 0.02). In in vitro testing, the ability of AX and AXE to stimulate immune cells, as measured by NO₂⁻ production by U937 cells and IL-8 secretion by Caco-2 cells were found to be dose–dependent in the range tested (100–5000 μg/mL p ≤ 0.05). AXE showed a greater activity at each concentration (100–5000 μg/mML) than AX (p ≤ 0.05).In conclusion, the greater immune stimulating activity of AXE may be associated with its low Mw (≤25 kDa) and a higher degree of branch substitution (0.81 ± 0.01).     

Effects of fermentation and heat treatments on bound-ferulic acid content and total antioxidant capacity of bread crust-like systems made of different whole grain flours

In this study, bread crust-like systems were prepared by heating (5, 15 and 30 min at 200 °C) freeze-dried fermented doughs obtained using different cereal grains. The flours of whole wheat, refined wheat, whole einkorn, whole rye, whole oat and whole maize were used. The effects of fermentation and heating on bound ferulic acid concentration and total antioxidant capacity were determined. Besides, high molecular weight fractions, containing also melanoidins formed in bread crust-like systems during heating were analyzed for their bound ferulic acid content and total antioxidant capacity. Increasing heating time at 200 °C also increased the amounts of high molecular weight fractions in the bread crust-like systems for all cereals. The total antioxidant capacity of bread crust-like samples increased significantly with heating time (p < 0.05). However, the opposite was true for the high molecular weight fractions of bread crust-like samples. Mean bound ferulic acid concentrations of different cereal flours were found to range between 78.3 mg/kg (for refined wheat) and 667.7 mg/kg (for whole einkorn). Heating had no significant effect on bound ferulic acid contents of bread crust-like systems. For all cereals, the highest concentrations of bound ferulic acid were detected in the high molecular weight fractions of bread crust-like systems heated at 200 °C for 5 min. However, bound ferulic acid concentrations significantly decreased in high molecular weight fractions as heating time increased (p < 0.05).     

Characteristics of enzymatic hydrolysis of thermal-treated wheat gluten

Effect of thermal treatment at 50–90 °C on wheat gluten hydrolysis by papain was evaluated in this study. Thermal treatment decreased the amount of sodium dodecyl sulfate (SDS) extractable protein. The treatments at 80 and 90 °C had a strong impact on protein extractability. Thermal treatment for 30 min resulted in a significant reduction in SDS extractable glutenin level in wheat gluten. A significant drop in free sulphydryl level was found in wheat gluten treated at 70 °C for 30 min. It indicated that cross-linking of glutenin through S–S occurred during thermal treatment. The treatments at 70–90 °C led to significant decreases in soluble and nitrogen level, while significant increases in peptide nitrogen amount in the hydrolysates from treated gluten were found. A time-dependent effect was observed for the changes in soluble forms of nitrogen and PN. Thermal treatment resulted in molecular mass distribution change according to gel permeation chromatography analysis. Thermal treatment significantly increased the amount of fractions with molecular mass beyond 10 K (67.2%) in the hydrolysates and greatly decreased the amounts of fractions with MM of 10–5 K and below 5 K in hydrolysates.     

Impact of genetic and environmental factors on einkorn wheat (Triticum monococcum L. subsp. monococcum) polysaccharides

To evaluate the effect of genotype, year and location on 1000 kernel weight, falling number, alpha- and beta-amylase activity, pasting properties, β-glucan, fructan, total starch, amylose, resistant starch and dietary fibre contents of wholemeal flour, four einkorns and one control bread wheat were cropped in Italy for two years in four different locations.