Wheat bran tissue fractionation using biochemical markers

Phenolic acid analysis of hand-isolated outer grain layers and endosperm led to the identification of markers of pericarp and aleurone layers, respectively. A new dehydrotrimer of ferulic acid (DHT) was found to be concentrated in the outer pericarp of wheat bran whereas p-coumaric (p-CA) acid was mainly in the aleurone layer. Phytates were also used as a marker of aleurone layer and starch as a marker of starchy endosperm. Biochemical markers constitute an original method for determining the histological composition of any technological bran fractions. A pin milling process was applied to coarse bran produced by a conventional milling process. Three different fractions (B1, B2 and B3) were obtained by sieving the bran products and then the smallest bran particle fraction (B3) was air-classified to obtain two particle size fractions (B3a and B3b with a D50 of 83 and 7 μm, respectively). The biochemical composition of these fractions was used to calculate the distribution of tissues according to the sieving process. The dissociation behavior of individual bran tissues upon mechanical fractionation was investigated in relation to particle size and discussed according to their mechanical properties.     

Biochemical markers: Efficient tools for the assessment of wheat grain tissue proportions in milling fractions

To produce safe and healthy whole wheat food products, various grain or bran dry fractionation processes have been developed recently. In order to control the quality of the products and to adapt these processes, it is important to be able to monitor the grain tissue proportions in the different milling fractions produced. Accordingly, a quantitative method based on biochemical markers has been developed for the assessment of grain tissue proportions in grain fractions. Grain tissues that were quantified were the outer pericarp, an intermediate layer (including the outer pericarp, the testa and the hyaline layer), the aleurone cell walls, the aleurone cell contents, the endosperm and the germ, for two grain cultivars (Tiger and Crousty). Grain tissues were dissected by hand and analysed. Biochemical markers chosen were ferulic acid trimer, alkylresorcinols, para-coumaric acid, phytic acid, starch and wheat germ agglutinin, for outer pericarp, intermediate layer, aleurone cell walls, aleurone cell contents, endosperm and germ respectively. The results of tissue quantification by hand dissection and by calculation were compared and the sensitivity of the method was regarded as good (mean relative errors of 4% and 8% for Crousty and Tiger outer layers respectively). The impact of the analytical variability (maximum 13% relative error on coarse bran) was also regarded as acceptable. Wheat germ agglutinin seems to be a promising marker of wheat germ: even if the quantification method was not able to quantify the germ proportions in milling fractions, it was able to classify these fractions according to their germ content. The efficiency of this method was tested, by assessing the grain tissue proportions of fractions exhibiting very different compositions such as flour, bran and aleurone-rich fractions obtained from three different grain or bran dry fractionation processes (conventional milling, debranning process, production of aleurone-rich fractions from coarse bran). By calculation of the composition of the different products generated, it was possible to study the distribution of the different tissues among fractions resulting from the different fractionation processes. This quantitative method is thus a useful tool for the monitoring and improvement of processes, and allows the effects of these processes to be understood and their adaption to reach the objectives.     

Potential of dry fractionation of wheat bran for the development of food ingredients,part II: Electrostatic separation of particles

Wheat bran is a composite material made of several layers, such as pericarp, testa and aleurone. It could be fractionated into purified fractions, which might either be used as food ingredients, or serve as a starting material for extraction of bioactive compounds. The aim of this work was to evaluate the potential of using electrostatic separation as a way to obtain purified fractions from wheat bran. Ultrafine-ground bran obtained either by cryogenic grinding or by grinding at ambient temperature was used as starting material. The ultrafine bran was then charged by tribo-electrification and introduced in a chamber containing two high voltage electrodes, where bran particles were separated depending on their acquired charge, allowing positively and negatively charged fractions to be collected separately. The particle size distribution, microstructure and biochemical composition of the obtained fractions were studied. The charge of the particles was influenced by their biochemical composition: particles rich in highly branched and cross-linked arabinoxylans (pericarp) were separated from particles rich in β-glucan, ferulic acid and para-coumaric acid (aleurone cell walls). The testa and the intracellular compounds from aleurone were not highly charged, neither positively nor negatively. The most positively charged fraction represented 34% of the initial bran, and contained 62% of the ferulic acid present in the initial bran. The yield of the separation process was good (5.4% loss), and could be further increased.     

Ultra-fine grinding increases the antioxidant capacity of wheat bran

In order to study the influence of wheat bran particle size on its antioxidant capacity, the wheat bran was ground under normal and cryogenic conditions with variable intensity to produce ten fractions with different physical structures. The high energy grinding increased 3-fold the specific surface of the bran fractions and also the proportion of particles smaller than 50 μm assimilated to the proportion of disrupted aleurone cells. All the ground bran fractions presented similar total ferulic acid concentration and chemical form (free, conjugated, linked). A positive effect of the grinding on the antioxidant capacity of the bran fractions was noticed. The antioxidant capacity increased from 30 to 45 mmol TEAC/kg when the specific surface increased from 0.09 to 0.26–0.30 m²/g. The antioxidant capacity of the bran fractions was linearly correlated with the specific surface, with the D₅₀ values and with the proportion of particles smaller than 50 μm. In in vitro gastric conditions, the finely ground bran inhibited the accumulation of conjugated dienes more efficiently than coarse bran. In conclusion, the bran structure affects its antioxidant capacity. This effect remained in gastric conditions showing that grinding can be used to produce wheat bran fractions with higher nutritional value.     

Evaluation of Tissue Dissociation of Durum Wheat Grain (Triticum durum Desf.) Generated by the Milling Process

The three major botanical components (starchy endosperm, aleurone layer and pericarp) of eight durum wheat samples exhibited significantly different compositions and concentrations in phenolic acids. The starchy endosperm, the aleurone layer and the pericarp were respectively characterised by a low content in ferulic acid (FA), a high content intrans -sinapic acid (t -SA), and a high content in ferulic acid dehydrodimers (DHD). These three chemical markers can be exploited to differentiate the three grain botanical parts within milling fractions and to evaluate the milling efficiency, particularly the separation between bran and endosperm. The histological dissociation of the wheat grain generated by the milling process can be investigated further into details using the three phenolic acids markers. A separability index (S i) was proposed in order to quantify the ease of dissociation of endosperm from bran. Differences in S i values between wheat varieties grown under various agricultural conditions demonstrated the relevant variability of this character. The structural and molecular factors implied in the control of tissue dissociation are discussed in details.     

Wet grinding and microfluidization of wheat bran preparations: Improvement of dispersion stability by structural disintegration

The enrichment of liquid food matrix with wheat bran has not yet been explored. This study investigated the impact of disintegrating wheat bran preparations on their stability at high moisture content. Three wheat bran preparations – standard bran, peeled bran and aleurone rich fraction – were modified by dry grinding, enzymatic degradation, wet grinding and microfluidization. The sedimentation of processed preparations was evaluated in water solution and related to their physical structure, solubilized compounds and suspension viscosity. In dry ground preparations mixed in water (5% w/w), most of the particles sedimented already in 5 min. Wet grinding disintegrated the physical structure of bran preparations (d₅₀ = 10–16 μm), causing improvement of particle stability due to reduction of gravitational sedimentation. Enzymatic treatment with xylanase efficiently increased the total solubility of the bran preparations (from 18–24% to 40–50%), but the higher solubility was not related to the better stability of particles. Microfluidization of peeled bran and aleurone increased the viscosity and stability of dispersions. The higher viscosity of the microfluidized dispersions was likely correlated with the better homogenisation of the particles, and also with the modified microstructure of treated bran preparations. Disintegrated wheat bran preparations showed high potential for beverage applications.     

Effect of wheat bran ball-milling on fragmentation and marker extractability of the aleurone layer

Bran (bran_ml) obtained by roller milling of soft (Scipion) and hard (Baroudeur) wheat cultivars was further ball-milled for increasing times and the observed particle size distribution expressed as a dispersion index. Bran (bran_hi) and aleurone layers were also hand-isolated from the same grains and the pattern of size reduction during ball-milling were compared with bran_ml. Bran_ml and bran_hi were found to fracture more rapidly than isolated aleurone layers due to the presence of the highly friable pericarp and the possible mechanical constraints due to tissues surrounding the aleurone layer. Previously identified markers of the aleurone layer cell contents (phytates) and cell walls (p-coumaric acid) were used to determine their water extractabilities from ball-milled samples and the state and degree of dissociation of the aleurone layer, either as an isolated tissue or within bran_ml and bran_hi. The results suggest that ball-milling rapidly induces fractures in walls of cells in the aleurone layer. The partial opening of the cells in the aleurone layer allowed extraction of most (≈70%) of the water-extractable phytates, even though their mean particle size was much larger than the dimensions of the cells. A further increase in extractability of phytates was observed when the particle size was reduced below the aleurone cell dimensions. Although much less soluble, p-coumaric acid followed a similar trend to phytates. The different behaviour of bran_ml and bran_hi was consistent with a weakening effect of the tissues in the former, probably due to the previous milling process. The bran and aleurone layers from both wheat varieties exhibited a similar behaviour.     

Distribution and composition of phytosterols and steryl ferulates in wheat grain and bran fractions

Phytosterols and steryl ferulates are bioactive compounds accumulating in the bran and germ of wheat. However, little is known regarding their localisation and composition in the bran layers of the kernel. The aim of this study was to determine the distribution of phytosterols and steryl ferulates in the wheat grain and in the different layers of bran. The wheat fractions, produced by conventional debranning, aleurone separation and a novel electrostatic process, were analysed for phytosterol contents using GC–FID and for steryl ferulate contents using HPLC–UV. The compounds were identified by GC– and LC–MS. Phytosterols and steryl ferulates were concentrated in the bran layers. The steryl ferulates were accumulated in the intermediate layers, whereas the phytosterols were more evenly distributed in the intermediate layers and aleurone cell contents. The phytosterol composition varied within the wheat kernel, while the steryl ferulate composition was similar in different fractions. Sitosterol and campestanyl ferulate were the main compounds. The highest levels of phytosterols (up to 2117 μg/g) and steryl ferulates (up to 703 μg/g) were found in the pearling, aleurone and certain bran fractions. The phytosterol-rich fractions could be utilised in cereal foods to enhance the intake of health-promoting compounds from natural sources.     

Grain color development and the inheritance of high anthocyanin blue aleurone and purple pericarp in spring wheat (Triticum aestivum L.)

There is renewed interest in breeding for high anthocyanin content in wheat due to its antioxidant potential. A series of adapted spring wheat lines were developed with blue aleurone or purple pericarp. The development of anthocyanin concentration and color of these selected lines was measured during grain filling for two field seasons at Saskatoon, Canada. In addition, the inheritance of the blue aleurone and purple pericarp was studied. Anthocyanin concentration increased rapidly during grain development and then decreased before maturity. Anthocyanin concentration was highest in PIG03008, a purple pericarp wheat. For mature grain, genotypic variation for anthocyanin concentration was statistically significant while the year and genotype by year interaction were not, facilitating the breeding progress. Blue aleurone was shown to be controlled by a single dominant gene in BC populations whereas purple pericarp appeared to be controlled by two loci with a segregation ratio of 11 purple: 5 white in F₂ populations. The results indicate that breeding high anthocyanin blue or purple wheat is feasible.     

Isolation of cellulose with ionic liquid from steam exploded rice straw

Isolation of cellulose from straw is a bottleneck for exploiting such biomass resources. In recent years, considerable concerns have arisen over new efficient and environmentally friendly way for this purpose. A novel method for cellulose isolation has been proposed by dissolving steam exploded rice straw in 1-allyl-3-methylimidazolium chloride ionic liquid (IL), following regeneration of crude cellulose by diluting the cellulose-ionic liquid solution adequately after separation of insoluble residues. The crude cellulose was then bleached by 2% hydrogen peroxide aqueous solution with low-flux ozone blowing into. No acid-insoluble lignin and only 0.85% hemicelluloses were detected in the bleached cellulose. The isolated cellulose was analyzed by SEM, FT-IR, ¹³C CP/MAS solid state NMR, XRD spectroscopes, and the results indicated that high quality cellulose preparation could be isolated in this manner from rice straw.     

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.     

High-intensity ultrasound irradiated modification of sugarcane bagasse cellulose in an ionic liquid

This current work is concerned with the glutarylation of sugarcane bagasse cellulose in ionic liquid 1-butyl-3-methylimidazolium chloride by ultrasound irradiation without catalyst. The degree of substitution ranging from 0.22 to 1.20 was obtained in one-step homogeneous modification, which increased with ultrasound irradiation time, temperature, and the molar ratio of glutaric anhydride/anhydroglucose unit in cellulose. The structural characterization of the glutarylated cellulose was carried out by using FT-IR and CP/MAS ¹³C NMR and the results showed that the glutarylation reaction occurred. The thermal stability of the glutarylated cellulose decreased upon chemical modification.     

Preparation and characterization of aminobenzyl cellulose by two step synthesis from native cellulose

Synthesis and structural characterizations of nitro- and aminobenzyl cellulose were carried out. Cellulose derivatives were synthesized by etherification. Nitrobenzylation produced 80 % yield by treating a mixture of microcrystalline cellulose, 4-dimethyl aminopyridine, and 4-nitrobenzyl chloride at 80 °C for 10 h. Nitrobenzyl cellulose was then reduced to aminobenzyl cellulose with 93 % yield using indium metal in ethanol and saturated aqueous ammonium chloride. In addition to their structural characterizations by FT-IR and ¹³C CP/MAS NMR, TGA will also be described. These reactions serve as models for future cotton fiber finishing technology with applications in flame resistance.     

Application of LC–MS–MS to identify niacin in aleurone layers of yellow corn,barley and wheat kernels

Previous studies identified type II inclusions in-situ of aleurone layers as niacin reserves and quantified niacin using colorimetric methods. Such identification and quantification was based on the König reaction which lacks specificity because cyanogen bromide reacts with all pyridine derivatives. The aim of this investigation was to define the structure of niacin in aleurone layers and aleurone cell contents of yellow corn, wheat and barley using LC–MS/MS. Aleurone layers were manually separated and cell contents released by ultrasonic processing and the residue and pellets were examined microscopically. Niacin was extracted from the samples by autoclaving in alkali. The extracts were analysed using HPLC and the structural identity of niacin was confirmed using quadrupole time of flight mass spectrometry in positive mode. Niacin concentrations were highest in wheat aleurone and lowest in corn aleurone. The MS/MS spectra of pure niacin and the purported niacin peak in sample extracts showed similar fragmentation patterns. The major ion product occurred at m/z 80 representing loss of CO₂ and parent ion at m/z 124 (M + H)⁺. These findings define some of the structural characteristics of niacin in aleurone layer of cereal grains, and demonstrate the possibility of using an ultrasonic processor to release cell contents.     

Assessment of biochemical markers identified in wheat for monitoring barley grain tissue

The possible use of specific biochemical compounds identified in wheat grains was evaluated for monitoring barley grain tissues during fractionation. First barley grain anatomy was studied through microscopic observation and quantification of the relative proportion of each anatomical part in four distinct barley samples from both hulled and hulless genotypes. As expected from cereal phylogeny and irrespective of the possible presence of hull, common features were observed between barley and wheat grains, but the aleurone layer predominated in the outer layers. The specific location of the compounds identified in wheat was established. Phytic acid was specifically localized in the aleurone layer and alkylresorcinols in the composite layer containing the testa, even if their concentration differed from that observed in wheat grain tissues. Thus, these two markers identified in wheat can be used to monitor the corresponding barley tissues, independent of the presence of hulls. Conversely, phenolic compounds, either ferulic acid trimer or p-coumaric acid, cannot be used to monitor respectively the outer pericarp or the aleurone cell walls in barley grains. p-coumaric acid was identified as an efficient marker of the hull and could be used to distinguish hulled or hulless barley grains and to help monitor the dehulling process.     

Crystallinity changes in wheat starch during the bread-making process: Starch crystallinity in the bread crust

The crystallinity of starch in crispy bread crust was quantified using several different techniques. Confocal scanning laser microscopy (CSLM) demonstrated the presence of granular starch in the crust and remnants of granules when moving towards the crumb. Differential scanning calorimetry (DSC) showed an endothermic transition at 70 °C associated with the melting of crystalline amylopectin. The relative starch crystallinity, as determined by X-ray and DSC, from different types of breads was found to lie between 36% and 41% (X-ray) and between 32% and 43% (DSC) for fresh bread crust. Storage of breads in a closed box (22 °C) for up to 20 days showed an increase in crust crystallinity due to amylopectin retrogradation both by X-ray and DSC. However, DSC thermograms of 1-day old bread crust showed no amylopectin retrogradation and after 2 days storage, amylopectin retrogradation in the crust was hardly detectable. ¹³C CP MAS NMR was used to characterize the physical state of starch in flour and bread crumb and crust. The intensity of the peaks showed a dependence on the degree of starch gelatinization.     

Study on structure and molecular dynamics of starch/poly(sodium acrylate)-grafted superabsorbent by <Superscript>13</Superscript>C solid state NMR

The domain-structure of samples containing a series of starch/poly(sodium acrylate)-grafted superabsorbents, pure starch, pure poly(sodium acrylate), and blend of starch/poly(sodium acrylate) has been studied by high-resolution solid-state ¹³C NMR spectroscopy at room temperature. The result shows that the crystallinity of starch decreases greatly in the grafted and blended samples. The values of ¹H spin-lattice relaxation time in rotating frame T _1ρ and ¹H spin-lattice relaxation time T ₁ shows that starch and poly(sodium acrylate) components in both grafted and blended samples have good compatibility in nanometer scale. In the ¹³C cross-polarization/magic-angle-spinning (CP/MAS) spectra, the chemical shift of the carbonyl group of poly(sodium acrylate) depends on the composition of the grafting samples, which indicates that the starch and the poly(sodium acrylate) components of the grafting samples exhibit better compatibility with each other than that of blended samples at molecular level.     

Relationship between Bran Mechanical Properties and Milling Behaviour of Durum Wheat (Triticum durum Desf.). Influence of Tissue Thickness and Cell Wall Structure

Eight durum wheat samples, including four varieties grown in different environments, were employed in an investigation on the influence of mechanical properties of bran on milling behaviour. The anisotropic nature of bran due to the particular structure of the outer epidermis of the pericarp required investigation of its mechanical properties in two grain orientations. Rheological characteristics (strain and linear force to rupture, rigidity modulus and rupture energy) were determined using traction tests performed on isolated bran strips. The results revealed significant variability between samples with significant effects of wheat variety and crop site. Influences of tissue thickness on bran rigidity (E′), and the degree of arabinoxylan (AX) cross-linking in cell walls on bran strength and extensibility, respectively, were demonstrated. A strong influence of tissue extensibility on the degree of bran contamination of semolina generated during the milling process was established statistically.     

Wheat grain tissue proportions in milling fractions using biochemical marker measurements: Application to different wheat cultivars

Measurement of biochemical markers allows the quantification of wheat (Triticum spp.) grain tissue proportions in milling fractions. In order to evaluate the ability of extending this methodology to an unknown wheat grain batch, the variability of the markers in the different tissues was assessed on various wheat cultivars. Ferulic acid trimer amounts in the outer pericarp ranged from 0.97 to 1.67 μg mg⁻¹ (dm) with an average value equal to 1.31 μg mg⁻¹ (dm). Alkylresorcinols amounts in a composite layer, including the testa, the inner pericarp and the nucellar epidermis, ranged from 10.5 to 16.7 mg g⁻¹ (dm), with an average value equal to 14.0 mg g⁻¹ (dm). In the aleurone layer, phytic acid amounts ranged from 94.9 to 187.2 mg g⁻¹ (dm) with an average value equal to 152 mg g⁻¹ (dm) whereas, para-coumaric acid ranged from 0.08 to 0.29 μg mg⁻¹ with an average level of 0.18 μg mg⁻¹. In the embryonic axis, wheat germ agglutinin ranged from 879 μg g⁻¹ to 2086 μg g⁻¹ with an average value of 1487 μg g⁻¹. The impact of this variability on tissue proportion determination was evaluated and a strategy to decrease the prediction error was suggested. Percentages of the outer pericarp, intermediate layer (including the testa), aleurone layer and embryonic axis within grains were calculated and their variability discussed.     

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.