Study of grain cell wall structures by microscopic analysis with four different staining techniques

Four different grain cell wall staining techniques were compared. Two techniques specifically detected arabinoxylan (AX). The first technique used a xylanase probe, while the other one was based on immunolabeling of AX using monoclonal antibodies. The two other staining techniques, one based on Calcofluor and the other on immunolabeling using monoclonal antibodies, stained mixed-linkage β-glucan. Cell walls of wheat, barley, oat and rye grains, differing both in content and location of AX and β-glucan, were examined. The staining methods were complementary to each other in revealing the location and distribution of the major cereal dietary fiber components AX and β-glucan in the different grains. AX was mostly concentrated in nucellar epidermis and aleurone cells, whereas β-glucan was concentrated more in subaleurone cells. Furthermore, in the case of barley and rye, the endosperm cell walls also contained high amounts of β-glucan. Interestingly, β-glucan in rye and barley endosperm cell walls was located adjacent to the cell contents, suggesting that it is not evenly distributed in the endosperm cell walls. The results give new insight into the structure of the cereal dietary fiber complex. Further development of microscopic techniques will help in elucidating the cereal cell wall structure even in more detail.     

Role of aleurone cell walls in water diffusion and distribution within cereal grains

Wheat (Triticum aestivum L) and barley (Hordeum vulgare L) are two important cereals cultivated worldwide. The effect of aleurone cell wall structure on water diffusion and distribution within wheat and barley grains was evaluated at different relative humidity levels. Time domain nuclear magnetic resonance was used to measure the transverse relaxation time T₂ of grains. Two water states were distinguished within grains, namely W₁ (lower mobility) and W₂ (higher mobility). Grains with thicker aleurone layer cell walls had a higher W₂. The water-absorption and desorption rates were mainly determined by the thickness of the aleurone cell walls and decreased with increasing cell wall thickness. The higher W₂ values observed in grains with thicker aleurone cell walls with the a water content of 2.0% (w/w) and 12.1% (w/w) were probably related to a higher water motion within the grains, in response to higher porosity. Arabinoxylan and (1,3)(1,4)-β-glucan alternating multilayer films were prepared, each film had 20 layers, one layer was approximately 1 μm thick. The film was used as a model to simulate the aleurone layers. These results show that cell walls of the aleurone layer regulate the diffusion and distribution of water within grains.     

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.     

Spatial distribution of functional components in the starchy endosperm of wheat grains

The starchy endosperm of the mature wheat grain comprises three major cell types, namely sub-aleurone cells, prismatic cells and central cells, which differ in their contents of functional components: gluten proteins, starch, cell wall polysaccharides (dietary fibre) and lipids. Gradients are established during grain development but may be modified during grain maturation and are affected by plant nutrition, particularly nitrogen application, and environmental factors. Although the molecular controls of their formation are unknown, the high content of protein and low content of starch of sub-aleurone cells, compared to the other starchy endosperm cells types, may result from differences in developmental programming related to the cells having a separate origin (from anticlinal division of the aleurone cells). The gradients within the grain may be reflected in differences in the compositions of mill streams, particularly those streams enriched in the central and outer cells of the starchy endosperm, respectively, allowing the production of specialist flours for specific end uses.     

Endosperm Development in Autotetraploid Rice

By using the laser scanning confocal microscope and plastic （Leica 7022 historesin embedding kit） semi-thin sectioning technique, comparative studies on the development of endosperm were carried out between autotetraploid and diploid rices. About one third of the ovaries in the autotetraploid showed normal endosperm development as those in the diploid. In these ovaries, one of the polar nuclei would fuse with the sperm nucleus, and the primary endosperm nucleus formed and underwent the first division in 4 hours after pollination; the anticlinal wall began to grow centripetally between the free nuclei starting from the wall ingrowths of the embryo sac near the micropylar end, and some of the phragmoplasts formed transformed into periclinal walls. In addition, some of the cell wall situated in the middle of the endosperm appeared to originate from phragmoplasts, whereas others seemed to develop randomly without the obvious formation of phragmoplasts. Cellulose began to accumulate in the wall of aleurone cell layer at 6 days after pollination. The cellulose wall of the cells of the aleurone cell layer appeared to have completely formed within 7 to 8 days after pollination. On the other hand, about two thirds of the ovaries in the autotetraploid showed abnormality in endosperm development with various types, such as non-fertilization, abnormal fertilization, endosperm development-delay and non-synchronization in the development of cellulose wall of cells of the aleurone layer. These abnormalities usually resulted in decreased seed setting in autotetraploid rice.     

Effect of barley and oat flour types and sourdoughs on dough rheology and bread quality of composite wheat bread

The potential of sourdough to improve bread quality of barley and oat enriched wheat breads may depend on the characteristics of the added flour (cereal type, variety, extraction rate). We compared the effect of different barley flours and oat bran (substitution level 40%), unfermented and as sourdoughs (20% of total flour), on composite wheat dough and bread characteristics by combining empirical rheological analyses (DoughLab, SMS/Kieffer Dough and Gluten Extensibility Rig) with small-scale baking of hearth loaves. Whole grain barley flour sourdough increased resistance to extension (Rmax) of the dough and improved the form ratio of hearth loaves compared to unfermented whole grain barley flour. However, sourdough showed little effect on the breads prepared with sifted barley flour or oat bran. The breads made with oat bran showed highest bread volume, lowest crumb firmness and highest β-glucan calcofluor weight average molecular weight (MW). The heat treatment of oat bran inactivated endogenous enzymes resulting in less β-glucan degradation. High MW β-glucans will increase the viscosity of the doughs water phase, which in turn may stabilise gas cells and may therefore be the reason for the higher bread volume of the oat bran breads observed in our study.     

Barley grain β-glucan content as affected by nitrogen and irrigation

Barley (Hordeum vulgare L.) with high grain β-glucan content in the soluble dietary fiber fraction may be useful as a specialty crop for human food. In contrast, low β-glucan content is desirable for brewing or animal feed. The objective of this study was to evaluate the effect of nitrogen and irrigation on barley grain β-glucan content. Relationships between barley grain β-glucan content and some agronomic traits were examined. Generally, high nitrogen levels increased barley grain β-glucan content in both years. The N₂ treatment gave the best grain β-glucan contents. A negative effect of irrigation was observed for barley grain β-glucan content. Increased levels of irrigation tended to decrease grain β-glucan content in barley. Non-irrigated treatments commonly had the greatest values for grain β-glucan content in all treatments. Higher positive correlations between β-glucan and protein content were observed in both years. A negative correlation between β-glucan content and 1000 grain weight was significant in 1999 while a negative correlation between β-glucan content and sieve analysis was significant in 2000. It may be concluded that increased nitrogen is desirable for high grain β-glucan content in barley but not irrigation.     

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.     

Effect of processing on the beta-glucan physicochemical properties in barley and semolina pasta

A health claim linking the consumption of barley β-glucan and the lowering of blood cholesterol has been allowed in North America and Europe which resulted in increased interest in barley products. Waxy barley flour rich in β-glucan (10% d.b.) was used to produce barley functional spaghetti and compared to semolina spaghetti. The impact of processing (extrusion, drying and cooking) on the physicochemical properties of barley blends and pasta as the molecular characterization of β-glucan were investigated. Pasta processing did not significantly affect the amount of β-glucan, but it impacted the β-glucan physicochemical properties in the end products. In all pasta, extrusion and drying were detrimental to the β-glucan properties, while cooking significantly increased the extractability and molecular weight of β-glucan, and in turn its viscosity, which determines its physiological effectiveness. In general, replacing wheat semolina with barley flour rich in β-glucan resulted in improved barley pasta containing the recommended amount of β-glucan per serving and enhanced β-glucan properties.     

Physical aspects of the biopolymer matrix in wheat bran and its dissected layers

The objectives of this work were to 1) determine the physical structure of untreated wheat bran and the differences in physical structure between its dissected layers; 2) evaluate how bran hydration affected bran crystallinity and polymer order; and 3) determine how enzymatic treatment of wheat bran affected its physical structure. For the first time, X-ray diffraction (XRD), small angle X-ray scattering (SAXS), solid-state ¹³C cross-polarization magic-angle spinning nuclear magnetic resonance (¹³C CP/MAS NMR), and polarized light microscopy with a waveplate were used to study the physical structure of wheat bran and its dissected layers. The XRD and solid-state ¹³C CP/MAS NMR both confirmed the presence of crystalline cellulose in untreated bran, enzymatically treated bran, and dissected bran layers. The outer pericarp had the highest crystallinity of the dissected bran layers and showed negative birefringence. The aleurone layer was low in cellulose content and completely amorphous, yet the cell walls in the aleurone layer showed strong positive birefringence. The treatment of destarched and deproteinated bran with the Updegraff reagent removed amorphous material, leaving its crystalline cellulose structure intact. Hydration of the outer pericarp increased its crystallinity index and CP/MAS NMR resonance intensity, which indicated a possible increase in polymer order. The SAXS also confirmed that cell wall polymers, possibly aggregated cellulose microfibrils, increased in order as a result of hydration.     

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.     

Spatial Distribution of Phenolic Materials in Durum Wheat Grain as Probed by Confocal Fluorescence Spectral Imaging

Microspectrofluorometry has been employed to study the spatial distribution of phenolic material in cereal grain. Transverse sections of the grain were used for the spectral characterisation of different molecular species present inTriticum durumgrains. Auto-fluorescence emission spectra were recorded on micro regions of each section. The analysis of the whole set of spectra permitted the characterisation of three principal spectral features; indicators of phenolics in specific regions of wheat sections. The comparison with model reference spectra showed that spectral components could be attributed to ferulic and p-coumaric acids. Using these spectral components, spectral fluorescence imaging was performed allowing the relative fluorescence intensity of each phenolic feature to be mapped. Images generated were used for the generation of the 3D organisation of auto-fluorescent phenolic materials within the grain. This new and rapid method was further used for the spectral characterisation of the various aleurone cell walls with high sensitivity. Analysis of the data showed that outer and inner aleurone cell walls exhibited similar fluorescence profiles but with significantly different intensities.     

Investigation by Confocal Raman Microspectroscopy of the Molecular Factors Responsible for Grain Cohesion in theTriticum aestivum Bread Wheat. Role of the Cell Walls in the Starchy Endosperm

Confocal Raman microspectroscopy has previously been employed to investigate the protein content and composition of the starchy endosperm of the wheat grain. With the same objective, that is to determine the molecular basis of grain cohesion and more specifically of kernel hardness, the contribution of endosperm cell walls in the kernel structure and cohesion was explored. The technique showed that endosperm cell walls consist not only of arabinoxylan chains with ramifications of ferulic esters, but also of others components such as proteins and lipids that could play some role in the mechanical properties of the endosperm cell walls. A new model of interaction between ferulic ramifications and a phospholipid component was proposed. The investigation of cell wall composition at successive stages of grain development revealed a decrease in the protein to arabinoxylan ratio and simultaneously an increase of the ferulic acid to arabinoxylan ratio that could be associated with a strengthening of the cell wall structure. The study confirms the effectiveness of confocal Raman microspectroscopy to approach the structure of wheat grain at the micrometer scale and to identify specific molecular factors responsible for grain cohesion and involved in the fracture modes generated during the milling process.     

Effect of genotype, environment and agronomic management on β-glucan concentration of naked barley grain intended for health food use

(1,3:1,4)-β-d-Glucan is an important bioactive that contributes to the ability of barley foods to help prevent type-2 diabetes. Realisation of these benefits requires understanding of genotype and environment effects on β-glucan concentration and how this variation affects biological activity of barley foods. Field experiments showed genetic variation in β-glucan concentration (3.0-7.0 g/100 g DM), but also considerable variation between environments. β-Glucan concentrations were lower in the wet summer of 2007 than 2006 or 2009; and slightly less in the dry summer of 2006 than 2009. β-Glucan was not diluted by higher grain yields. The role of β-glucan as an assimilate buffer adds complexity to interpreting the effects of environment during grain filling. Autumn sowing and fungicide increased the duration of grain filling, decreased β-glucan concentration but increased environmental stability; possibly due to lower demand for assimilate buffering. Lodging and foliar disease decreased β-glucan concentration, by decreasing assimilate supply leading to remobilisation of carbohydrate from β-glucan, so that fungicide increased β-glucan in some disease-susceptible accessions. Sequential harvesting starting at GS 91 suggested an optimum harvest window for maximum β-glucan concentration. The variability in β-glucan reported here between genotypes and environments was sufficient to affect control of post-prandial blood glucose in healthy volunteers.     

The potential of naked barley sourdough to improve the quality and dietary fibre content of barley enriched wheat bread

The possibility of using naked barley for food products is gaining popularity due to its dietary fibre content, especially β-glucans. The technological process (dough preparation, fermentation and baking) influences bread quality but also may contribute to degradation or preservation of valuable grain components. The aim of the study was to investigate the effects of different wholemeal barley flour share and bread production method on the quality of bread and non-starch polysaccharides content and solubility.Barley enriched bread contained more both soluble and insoluble dietary fibre and β-glucans, products of 40% barley share contained 67% more total dietary fibre and 160% more β-glucans than control. However, barley incorporation decreased the amount of soluble arabinoxylans. High barley contents contributed to the breads’ volume reduction by 14% and change in their crumb and crust colour. However, barley enriched breads gained higher ratings of taste than wheat bread. Barley sourdough fermentation improved breads’ volume, colour and sensory properties. Sourdough fermentation also resulted in higher concentration of dietary fibre, arabinoxylans and β-glucans. The beneficial effect of barley addition to wheat bread may be successfully enhanced by using barley wholemeal sourdough fermentation.     

Release of Ferulic Acid from Cereal Residues by Barley Enzymatic Extracts

The use of esterase- and xylanase-rich extracts of barley for the release of ferulic acid (FA) from barley spent grain and wheat bran is proposed here. Esterase activity was determined in 11 barley varieties and enriched extracts were prepared from one of the varieties with higher activity, Halcyon (7·3 mU/g barley). The esterase(s) extract alone was able to release low amounts of FA from spent grain and wheat bran. However, the presence of the xylanase(s)-containing extract increased the release of FA and demonstrated synergistic reactions between these enzymes. Maximum FA released by the combination of esterase(s) and xylanase(s) extracts was 26% and 13·3% from spent grain and wheat bran respectively after 36 h incubation. FA from spent grain and wheat bran was also released by barley esterase(s) extract and Trichoderma viride xylanase. Reducing sugar release followed a pattern similar to that for the release of FA. However, barley xylanase(s) appear more specific than T. viride xylanase for spent grain. These results demonstrate the potential use of economical barley enzymes for the release of ferulic acid from cell wall materials.     

Proteins in Walls of Wheat Aleurone Cells

The protein content (1% w/w) of purified walls from aleurone layers is twice that of walls from the starchy endosperm but their amino acid compositions are comparable. Aprotic solvent extraction and specific enzymatic and chemical degradation of wall polysaccharides released some proteins, and treatment with specific proteases released peptides. The aleurone wall residue (18% of the original wall) after (1→3,1→4)-β-glucan and xylan hydrolase digestion contains 4·5% (w/w) protein associated with cellulosic glucan, glucomannan and highly-substituted arabinoxylan and remains autofluorescent. Wall residues after treatment with 1 M sodium hydroxide also remain autofluorescent indicating they retain a significant proportion of their original 1·84% (w/w) of hydroxycinnamic acids and that these are not exclusively attached to the wall polymers by ester linkages. Little protein was extracted from the walls using non-degradative solvents. However, significant quantities were recovered from whole walls and xylanase-treated wall residues by SDS/mercaptoethanol extraction and electroelution. The various protein fractions isolated were characterised by their amino acid compositions and, in some cases, by amino acid sequencing. Three classes of proteins were identified in wall fractions or from proteolysis fragments: glycine-rich proteins (37–86%), proline-rich proteins (11–39%) and proteins with up to 23% serine. Protein-polysaccharide cross-linking through tyrosine-hydroxycinnamic acid dimerisation may be responsible for the alkali-resistant autofluorescence and the insolubility of the protein may be due, in part, to cross-linking through tyrosine-tyrosine bridges. These associations may also contribute to the relative resistance of the inner aleurone wall layer to enzymic dissolution during germination.     

Effects of roasting on barley β-glucan,thermal, textural and pasting properties

Different hulled barley cultivars were subjected to roasting in hot sand (280 °C) and the effects on β-glucan extractability, physico-chemical, thermal and pasting properties were studied. The grain puffed upon roasting and grain hardness and colour difference ΔE were significantly lowered. The roasted barley flour had significantly higher water absorption and water solubility index. The cultivars DWR-28, RD-2503 and PL-172 had the highest total β-glucan content (up to 5.47%). Roasting brought about a decrease in the soluble β-glucan content in all the cultivars and this decrease ranged from 4.9 to 25.3%. The β-glucan extractability was not affected by the roasting process but roasting lowered the ratio of soluble to insoluble β-glucan content by 8.1–41.9%. Roasting significantly affected the pasting and thermal properties of the flours, together with an increase in the cooked starch content that ranged from 28.8 to 43.1%.     

Integration of β-Glucan Fibre Rich Fractions from Barley and Mushrooms to Form Healthy Extruded Snacks

β-glucan is a commonly researched plant cell wall component that when incorporated into food products has been associated with cholesterol and glycaemic response reductions. This study focusses on β-glucan rich fractions from barley and mushroom used in the production of extruded ready to eat snacks. Inclusion of barley β-glucan rich fractions and mushroom β-glucan fractions at 10 % levels increased the total dietary fibre content of extrudates compared to the control (P?<?0.05). Product expansion increased with the introduction of both barley and mushroom fraction (P?<?0.05) which in turn resulted in a reduction in product hardness (P?<?0.05). In vitro digestion protocol illustrated that inclusion of barley and mushroom β-glucan rich fractions manipulated the starch digestibility profile and hence rate of glucose release during digestion compared to the control sample. This in turn resulted in a significant (P?<?0.05) reduction in potential glycaemic response of the samples of between 20 and 25 % for barley β-glucan rich fractions and between 17 and 25 % for mushroom β-glucan rich fractions. We conclude that the inclusion of these fractions could be utilised by the food industry to manipulate the glycaemic response of extruded snack products.     

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.