Composition and molecular structure of polysaccharides released from barley endosperm cell walls by sequential extraction with water,malt enzymes,and alkali

Isolated and purified endosperm cell walls (CW), used in this study, were derived from a Canadian malting barley variety, AC Metcalfe, grown in three different environments in Canada in 2003, and varying in grain protein and β-glucan contents, as well as in grain hardness. The CW were initially extracted with water at 45 °C and subsequently digested with barley malt crude enzyme extract resulting in two fractions designated CW-WE45 and CW-MD, respectively. The remaining non-digested cell wall material (CW_ND) was further fractionated by sequential extraction with water at 95 °C (CW_ND-WE95), saturated barium hydroxide (CW_ND-BaE), and 1 N sodium hydroxide (CW_ND-NaE) at 25 °C. Composition and molecular structure analyses were carried out for all fractions including the remaining cell wall residue (CW_RES). Extraction of CW with water followed by digestion with malt crude enzyme extract solubilized the majority of β-glucans (∼55–70%) and glucomannans (∼60–80%) but only a small portion of arabinoxylans (∼20–30%) present in the intact CW. The CW-WE45 and CW_ND-WE95 fractions consisted mostly of β-glucans exhibiting high average molecular weights (M_w) (2–3 × 10⁶), whereas the CW_ND-BaE consisted mainly of arabinoxylans with M_w about 1–1.5 × 10⁶. The CW_ND-NaE contained almost equal amounts of β-glucans and arabinoxylans and a small amount of glucomannans, whereas the CW_RES contained approximately equal proportions of β-glucans, arabinoxylans and glucomannans. β-Glucans in CW_ND-WE95, CW_ND-NaE, and CW_RES exhibited a higher ratio of 3-O-β-d-cellobiosyl-d-glucose to 3-O-β-d-cellotriosyl-d-glucose (DP3/DP4) compared to β-glucans in CW-WE45 and CW-MD. β-Glucans in CW_ND-NaE showed the highest level of long cellulosic oligosaccharides with DP ≥ 5, whereas those in the CW_RES had the highest DP3/DP4 ratio. The CW-MD was fractionated by ultrafiltration into high (CW-MD_HMW) and low-molecular weight (CW-MD_LMW) sub-fractions, with weight-average M_w of ∼150–350 × 10³ and <10 × 10³, respectively, as confirmed by size-exclusion chromatography. The monosaccharide composition of the sub-fractions indicated a more extended enzymic degradation of β-glucans and glucomannans than arabinoxylans. Some differences in composition and molecular structure of the cell wall constituents among the three barley samples were related to their solubility and enzymic digestibility.     

Replicate and die for your own good: Endoreduplication and cell death in the cereal endosperm

Development of the cereal endosperm involves different stages that include cell proliferation, endoreduplication, the biosynthesis and accumulation of storage compounds, and programmed cell death (PCD). During endoreduplication, repeated genome duplications without mitosis or cytokinesis result in large polyploidy nuclei. Endoreduplication is affected by genetic, epigenetic, hormonal and environmental factors, as well as by several cell cycle-controlling genes. Although endoreduplication is strongly correlated with an increase in cell size, the accumulation of storage reserves, and high metabolic rates, its exact role(s) in endosperm development has not been demonstrated.     

Isolation of the wheat aleurone layer for 2D electrophoresis and proteomics analysis

Whereas the endosperm of bread wheat has been studied for many years for obvious ecoomic reasons, studies of the aleurone layer of the seed only started recently after the discovery of its nutritional and health benefits. In this paper, we describe two different techniques to isolate either the peripheral layers including the aleurone layer or only the aleurone layer (AL) which can be used for 2D electrophoresis and proteomic analysis. The two techniques provided similar 2D electrophoresis profiles although the time needed for dissection of the kernel and isolation of the cell layer was different. The two 2D protein profiles shared more than 80% identity and enabled us to observe approximately 700 spots in the aleurone layer. Two bread wheat cultivars, Chinese Spring and Recital, were used and the two techniques revealed that their AL shared at least 70% of common spots. Several spots not present in AL and coming from peripheral layers were identified using mass spectrometry and database interrogation. These dissection techniques will enable proteomic analysis of AL which can be used for genetic analysis of its components, for investigating the AL response to fungi attack and helpful for improving nutritional and health value of wheat.     

Compressive strength of Super Soft wheat endosperm

Wheat endosperm texture (hardness) largely determines end-product suitability. Since its development 25 years ago, the single kernel classification system (SKCS, a mechanical instrument that measures, among other properties, the force imparted on a kernel during crushing) has been used in breeding programs to differentiate soft wheats from hard wheats. Nominally, these have a soft to hard SKCS hardness index (HI) range of 25–75 (dimensionless units). However, in recent years, breeders have developed extremely soft (‘Super Soft’) lines having SKCS HI < 0. Until now, these very low SKCS HIs have not been corroborated with traditional methodologies that characterize mechanical strength. Herein, we report on the relationships between SKCS HI and three compressive strength properties (maximum stress, Young's modulus, and work) in Super Soft wheat. With respective correlation coefficients of 0.76, 0.66, and 0.75, we have found that the relationships between SKCS HI and compressive strength agree with prior research involving ordinary soft and hard wheats.     

Spectroscopic analysis of diversity in the spatial distribution of arabinoxylan structures in endosperm cell walls of cereal species in the HEALTHGRAIN diversity collection

Two varieties each of spelt (Triticum aestivum var. spelta), durum wheat (Triticum turgidum var. durum), rye (Secale cereale), barley (Hordeum vulgare), oats (Avena sativa), einkorn (Triticum monococcum var. monococcum) and emmer (Triticum turgidum var. dicoccum) (all members of the Pooideae sub-family of grasses) were selected according to variation in their contents of soluble and/or total arabinoxylan (AX) determined during the HEALTHGRAIN diversity screen, together with one genotype of the related “model” grass species Brachypodium distachyon. The spatial distribution of low substituted (LS-AX) and highly substituted arabinoxylan (HS-AX) was determined using FT-IR spectroscopic mapping of transverse thin cross-sections consisting of cell walls only. Variation in cell wall AX composition was observed between the cereals, and compared with that observed for wheat (Triticum aestivum var. aestivum). One line of each cereal type was analysed in more detail using ¹H NMR spectroscopy. The results of the two analyses were consistent, showing variation in the composition and structure of the endosperm cell wall AX that is consistent with the genetic relationships of the cereals studied.     

Protein bodies ontogeny and localization of prolamin components in the developing endosperm of wheat caryopses

During caryopsis development, prolamins are initially stored in individual protein bodies, then generate a protein matrix in the ripe caryopsis. The ontogeny of the protein bodies was analyzed by fluorescence and electron microscopy from 7 to 43 days after anthesis (dAA), a period of time from the cellularization of endosperm to its desiccation. A series of antibodies specific to each prolamin type (α/β-, γ-, ω-gliadins, low-molecular weight and high-molecular weight glutenin subunits) made it possible to localize and co-localize the different prolamins in organelles of endosperm cells at different developmental stages. Protein bodies containing prolamins were observed as early as 7 dAA. At the early developmental stages, protein bodies were spherical with diameters around 1–2 μm. Later, around 15 dAA, the PBs enlarged, and aggregation and/or coalescence were prominent at 21 dAA. From 33 dAA, individual PBs were no longer visible, but a protein matrix was confined in the space between starch granules. All prolamins were found in the same protein bodies, without any segregation according to their types. Immunochemical labelling of prolamins failed to reveal in TEM analyses any particular internal organization in protein bodies. Glutenin subunits and gliadins were observed in the Golgi apparatus at the early stages of endosperm development.     

Pulsed laser ablation: A new approach to reveal wheat outer layer properties

A new methodology based on pulsed lasers has been developed in order to estimate wheat outer layer mechanical properties without sample preparation. Laser experiments were carried out with an Argon Fluoride (λ = 193 nm) excimer laser source delivering pulses of 15 ns duration. Wheat grains from two cultivars were irradiated by single laser pulses with a quasi-uniform irradiation and two fluences (2.5 and 5 J cm⁻²). The ablation flux was characterized by environmental scanning electron microscopy before measuring the removed material on cross-sections observed by confocal scanning laser microscopy. Specific image treatment was carried out to obtain the ablation flux (amount of removed matter per pulse). Pericarp, seed coat and aleurone layer were gradually ablated under the laser conditions used in this work. Their ablation thresholds were different and could be related to tissue cohesion. Specific behaviour of seed coat layer (8 μm) could be emphasised with this technique. Pulsed laser ablation could be a potential methodology to reveal indirectly wheat grain layer cohesion.     

不同类型专用小麦品种胚乳发育的比较研究

为揭示不同类型专用小麦品种胚乳发育的差异,以强筋小麦皖麦38和弱筋小麦扬麦9号为材料,比较了两者胚乳细胞游离核分裂状况、细胞数目的增殖变化、细胞体积的变化、糊粉层发育、淀粉体和蛋白质体发育等内容,主要结果如下：（1）皖麦38游离核有丝分裂所占比例较大,扬麦9号无丝分裂所占比例较大,且受温度影响较大;（2）胚乳细胞增殖均呈"S"型曲线变化,皖麦38胚乳细胞增殖较快,胚乳体积较大,最终粒重较高;（3）皖麦38胚乳细胞中大淀粉体数目多于扬麦9号,而小淀粉体数目表现却相反;（4）皖麦38与扬麦9号相比较,糊粉层出现时间较晚,而且糊粉层细胞壁较厚,被甲苯胺蓝染色更浓;（5）成熟籽粒中淀粉粒形状有饼形、椭圆形和近圆球形三种。皖麦38胚乳中饼形和椭圆形淀粉粒较多,且相互结合较紧密;扬麦9号饼形和近球形淀粉粒较多,胚乳结构疏松。     

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.     

Effect of glutathione on gelatinization and retrogradation of wheat flour and starch

Reduced glutathione (GSH) commonly exists in wheat flour and has remarkable influence on gluten properties. In this study, effect of GSH on the gelatinization and retrogradation of wheat flour and wheat starch were investigated to better understand the GSH-gluten-starch interactions in wheat flour. Compared with wheat starch, wheat flour showed significant decreases in peak and final viscosity, and gelatinization onset temperature with increasing GSH concentration. GSH depolymerized gluten and thereby broke down the protein barrier around starch granules to make the starch easily gelatinized. However, the interaction between GSH and wheat starch restrained starch swelling. GSH addition resulted in weakened structure with higher water mobility in freshly gelatinized wheat flour dispersions but decreased water mobility in wheat starch dispersions. After storage at 4 °C for 7 d, GSH increased elasticity and retrogradation degree in wheat flour dispersions but retarded retrogradation in wheat starch dispersions. The results indicated that GSH promoted retrogradation of wheat flour, which mainly attributed to the depolymerized gluten embedding in the leached starch chains, and inhibiting the re-association of amylose, and subsequently promoted the starch intermolecular associations and starch retrogradation. This study could provide valuable information for the control of the quality of wheat flour-based products.     

Fractionation of wheat and wheat flour into starch and gluten: overview of the main processes and the factors involved

The starch and gluten components of wheat flour or whole wheat kernels can be separated by a number of industrial processes. This review provides a summary of these processes from both starting materials. The wheat constituents of importance in the fractionation processes are briefly introduced, and the different fractionation processes described with emphasis on the parameters affecting the separation, such as flour composition, mixing and washing water, processing aids (with an emphasis on enzymes) and kernel pre-treatment (pearling) in the case of flour fractionation and steeping conditions and processing aids in the case of whole wheat. Although fractionation of flour is the basis for the current industrial processes, starch yields are impaired by starch damage as a result of milling and loss of starch to milling streams. On the other hand fractionation of whole kernels often leads to impaired gluten production as a result of harsh process conditions which ‘devitalise’ the gluten.     

Impact of re-grinding on hydration properties and surface composition of wheat flour

The combination of two analytical methodologies (water vapor sorption isotherm by using the DVS and chemical surface composition by using the XPS) has been used to enhance the understanding of the impact of re-grinding on the wheat flour hydration mechanism. A controlled atmosphere microbalance was used to construct water sorption isotherms at 25 °C of different samples of wheat flours obtained by successive re-grinding of native wheat flour.     

Effect of medium hydrostatic pressure on the properties of wheat flour main biopolymers

In our work, the effect of medium hydrostatic pressure on the properties of wheat flour's main ingredients, starch and proteins, is presented and discussed. The symbiotic effect of the different constituents via size exclusion chromatography, water binding, gelatinization experiments as well as atomic force microscopy measurements and X-ray diffraction is elucidated. From results of size exclusion chromatography and protein content analysis, prolamin fractions seemed to be most sensitive to pressure. Hydrostatic pressure, however, had a significant influence on the amount of bound water and gelatinization enthalpy, especially at moderate pressures and higher temperatures. In this case, an optimal interaction between macromolecules and water occurs. This was also confirmed by atomic force microscopy images and X-ray diffraction patterns. Amorphous and crystalline regions of starch granules were modified, depending on pressure and temperature. At medium pressure (200 MPa), water was pressed into the starch cavities remaining there, whereas higher pressure (600 MPa) led to complete flattening of the surface. With X-ray diffraction, it was shown that medium pressure had nearly no effect on molecular structure, whereas higher pressure caused thermal-like molecular modifications.     

Heat induced formation of free radicals in wheat flour

Oxidative Stability of bread is increasingly being recognized as important for long shelf-life. Formation of free radicals in whole wheat flour and white flour during heating was compared using Electron Spin Resonance spectroscopy in order to identify the primary oxidative events. Heating lead to a higher content of free radicals in wholemeal wheat flour than in white flour, since components in the husk seem to make a major contribution. Two different pathways are suggested for the formation of free radicals in wheat flour. The activation energy for the radical formation in the flours was estimated by Arrhenius plot as 34 kJ mol⁻¹ up to 453 K, and above 473 K higher activation energies were observed. The change in activation energies indicates a change in reaction mechanisms for oxidation probably involving species with different mobility, i.e. different molecular weight. The accumulated concentration of free radicals in heated flour increased during one month storage and it is slightly affected by the water activity in the storage container.     

Analysis of the milling reduction of bread wheat farina: Physical and biochemical characterisation

The reduction of coarse farina during milling from five representative hard and soft French bread wheat cultivars was followed step by step. Particle size distribution of the milled products and energy required to dissociate wheat farina revealed large differences between cultivars. A grinding index (required energy to produce 1 kg of flour), K′, corresponding to the energy necessary to obtain a given amount of flour, was found to be related with both grain hardness and vitreousness. Analysis of particle size distribution suggested a major influence of hardness in the production of fine particles <50 μm during the reduction process. On the other hand, vitreousness appeared to impact on the extent of coarse particle size reduction. Differences in farina reduction behaviour of the wheats analysed were related to their endosperm mechanical properties as determined by compression tests and structural characteristics as measured by Hg intrusion porosity. Vitreousness appeared to affect the endosperm extensibility and degree of porosity. The profile of the different lipid and protein classes in the reduction products of two cultivars showing opposite behaviour as well as different PINA/PINB ratio were determined. The results suggest that the most resistant parts of the grain are enriched in glycolipids, whereas phospholipids appear associated with the most friable parts, at least in the soft wheat grains tested.     

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.     

Influence of sulphur fertilisation on quantities and proportions of gluten protein types in wheat flour

Although different supplies of sulphur (S) during wheat growth are known to influence the quantitative composition of gluten proteins in flour, an effect on the amount and on the proportions of single protein types has yet not been determined. Therefore, wholemeal flours of the spring wheat ‘Star’ grown on two different soils and at four different levels of S fertilisation (0, 40, 80, 160 mg S per container) were analysed in detail using an extraction/HPLC procedure. The results demonstrated that the amount of total gluten proteins as well as of the crude protein content of flour was little influenced, whereas amounts and proportions of single protein types were strongly affected by the different S fertilisation. The changes were clearly dependent on the Cys and Met content of each protein type. The amount of S-free ω-gliadins increased drastically, and that of S-poor high-molecular-weight (HMW) glutenin subunits increased moderately in the case of S deficiency. In contrast, the amounts of S-rich γ-gliadins and low-molecular-weight (LMW) glutenin subunits decreased significantly, whereas the amount of α-gliadins was reduced only slightly. S deficiency resulted in a remarkable shift of protein proportions. The gliadin/glutenin ratio increased distinctly; ω-gliadins became major components, and γ-gliadins minor components, whereas the ratio of HMW to LMW glutenin subunits was well-balanced.     

高水分蒸煮挤压小麦混合粉的研究

食品蒸煮挤压技术是多学科交叉所产生的一门高新技术,蒸煮挤压作为一种高温短时的加工方法,能够将输送、压缩、混合、蒸煮、杀菌、成型等多种操作单元同时完成。本实验优化了双螺杆机的加工工艺参数,DS56-Ⅲ型双螺杆挤压膨化机挤压腔内限流区的温度在125℃．含水率为40％、螺杆转速为215rpm,该条件下得到的最好产品膨化度为2．51,经过蒸煮挤压后的食品含水率为35％～37％。