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.     

Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition

Hand dissection of mature grains from two common wheats (Triticum aestivum L., cv. Caphorn and cv. Crousty) were performed to quantitatively assess their tissue composition and to obtain homogeneous samples of embryonic axis, scutellum, starchy endosperm, aleurone layer, hyaline layer, outer pericarp and a composite layer made up of testa+hyaline layer+inner pericarp. Polymeric neutral sugar and phenolic acid contents of the samples were determined and used to identify specific composition patterns in each tissue irrespective of the cultivar. The scutellum and embryonic axis showed the lowest amount of carbohydrates with similar relative amounts of arabinose and xylose (Ara+Xyl), but the scutellum differed from the embryonic axis in its high phenolic acid, in particular ferulate dehydrodimer, content. The peripheral layers of the grains were mainly composed of cell wall polysaccharides, chiefly arabinoxylans but with differing structures. The hyaline layer was mostly composed of arabinoxylan with extremely low Ara/Xyl ratio (0.1), with high amounts of ferulic acid monomers and hence very weakly crosslinked. The aleurone layer differed from the outer pericarp by its much lower Ara/Xyl ratio and lower amounts of ferulic acid dimers and trimers. High proportions of ether-linked phenolic acids (released by alkali at 170 °C) were detected specifically in the seed coat and tissues in the crease region. The possible application of biochemical markers found in the various tissues to monitor wheat grain fractionation processes is discussed.     

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.     

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.     

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.     

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.     

Field emission scanning electron and atomic force microscopy,and Raman and X-ray photoelectron spectroscopy characterization of near-isogenic soft and hard wheat kernels and corresponding flours

Secondary field emission scanning electron microscopy (FE SEM), atomic force microscopy (AFM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to investigate native near-isogenic soft and hard wheat kernels and their roller milled flours. FE SEM images of flat-polished interior endosperm indicated distinct differences between soft and hard wheats with less internal continuity in the soft wheat, whereas individual starch granules were much less evident in the hard kernel due to a more continuous matrix. AFM images revealed two different microstructures. The interior of the hard kernel had a granular texture with distinct individual spheroid features of 10–50 nm while the images obtained for the soft kernel revealed less distinct small grains and more larger features, possibly micro-structural features of starch granules. Raman spectra resolved identical distinct frequencies for both kernel types with slightly different intensities between types. Finally, the chemical surface compositions of flour for these two types of kernels obtained by XPS provided subtle insight into the differences between soft and hard wheat kernels. These combined advanced microscopic and spectroscopic analyses provide additional insight into the differences between the soft and hard wheat kernels.     

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.     

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.     

Variability in dehydrodiferulic acid composition of durum wheat (Triticum durum Desf.) and distribution in milling fractions

The dehydrodiferulic acid content of different common and durum wheat grains and milling fractions was determined by an HPLC procedure. The 8-O-4′, 5–8′ benzofuran, 5–8′ and 5-5′ dehydrodimers were identified in all samples studied and occurred in decreasing relative amounts, respectively. Durum wheats were twice as concentrated in dimers as common wheats. An important genetic variation for dehydrodiferulic acid content was shown within durum wheat grains, whereas the agronomic conditions had no effect. There was 10 to 20 times more dehydrodiferulic acids in external layers (aleurone, bran) than in the starchy endosperm of durum wheat grains. The content and composition in dimers of the inner endosperm did not vary according to genotypes and cultivation conditions. The ratio of dehydrodimers to monomers of ferulic acid which represented an index of dimerisation, was 1·6 times higher in the external layers of the grain than in the endosperm. No relation was found, however, between the degree of ferulic acid dimerisation and the milling behaviour of durum wheat grains.     

Endosperm and aleurone cell structure in barley and wheat as studied by optical and Raman microscopy

Grain ultrastructure is of utmost importance when designing grain processing procedures in the food industry. In this study, wheat and barley grain components were localised using optical and Raman microscopy. The optical microscopic analyses were performed using several selective stains to localise β-glucan, protein and starch or autofluorescence to image the ferulic acid and other fluorescing substances. Alternatively, Raman microscopy was applied to localise the grain components without any need for preceding staining or other sample pretreatment. Both methods provided consistent information on the grain structures, illustrating the distribution of polysaccharides, aromatics and protein in endosperm and aleurone layers. In aleurone layers of both barley and wheat, a distinct difference between the anticlinal and periclinal cell walls was observed. The anticlinal cell walls were enriched with aromatic substances which were present in remarkably lower concentrations in the inner periclinal cell walls but for barley, an even higher concentration in the outer periclinal cell wall was observed. In addition, Raman spectroscopy illustrated the detailed distribution of substances across the aleurone cell walls: β-glucan was adjacent to proteins and it was deficient in the middle lamella whereas arabinoxylan was enriched in the outer cell wall layers and middle lamella.     

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.     

Rheological Behaviour of Wheat Endosperm—Proposal for Classification Basedon the Rheological Characteristics of EndospermTest Samples

A simple, rapid method for the preparation of parallelepiped-shaped samples from a grain is used in the proposal of a study of the rheological behaviour of wheat endosperm. Compression rupture, creep and relaxation tests are used. A series of compression tests on mealy and vitreous endosperm of different wheat varieties (soft, hard and durum) shows that the rheological properties are influenced by both the genetic origin and grain vitreousness. The main mechanical characteristics—Young's modulus, elastic and rupture stresses, rupture energy and rupture strain—were determined at moisture contents of 12 to 17%. The influence of the moisture content on rheological behaviour is demonstrated. The vitreous endosperm of some wheat varieties displays considerable ductility before rupture. The nature of this plasticity was analysed by creep and relaxation tests on hard and soft wheats. Comparison of the different endosperm rheology values clarifies the notions of vitreousness and hardness. Wheat classification based on endosperm mechanical characteristics is proposed. It seems that Young's modulus characterises hardness whereas rupture energy is related to the vitreousness of the different varieties studied. Entering the results in a Young's modulus–rupture energy system leads to a classification of wheats according to two essential factors: hardness of varietal origin and vitreousness of cultural origin.     

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.     

巴西橡胶树果皮及种皮的发育

描述巴西橡胶树果皮和种皮在各个不同发育时期的解剖结构。（１）授粉后约１２０ｄ,果皮和种皮发育成熟,（２）果皮由于子房壁形成,授粉３０ｄ后,果皮可区分为外果皮,中果皮和内果皮三层结构,外果皮由表皮和表皮下７－８层绿色扁平细胞构成,中果皮由等径薄壁细胞构成,其上分布维管束及密集成群的初生乳管,内果皮由三层走向不同的长形细胞组成,授粉后６０～７０ｄ,内果皮细胞开始木质化果实成熟时,外中果皮革质而干燥,内     

Localization of alkylresorcinols in wheat,rye and barley kernels

Cereal alkylresorcinols (AR), a group of phenolic lipids mainly found in the outer parts of wheat and rye kernels, are currently being studied for the possibility to use them as biomarkers for the intake of whole grain wheat and rye foods. In this work, AR were localised in grains by using light microscopy and gas chromatographic analysis of hand-dissected botanical and pearling fractions. GC-analysis of hand-dissected fractions showed that more than 99% of the total AR content was located in an intermediate layer of the caryopsis, including the hyaline layer, testa and inner pericarp. Microscopic examination showed that the outer cuticle of testa/inner cuticle of pericarp was the exact location, and that no AR were found in the endosperm or in the germ, suggesting that AR could be used as a selective marker of testa.     

Structure,chemical composition and enzymatic activities of pearlings and bran obtained from pearled wheat (Triticum aestivum L.) by roller milling

While abrasive pearling (also referred to as debranning) of wheat kernels prior to milling increases the quality of the resultant flour for producing bread, the potential applications of the co-products of pearling is largely unknown. We studied the impact of different degrees of pearling (0, 3, 6, 9 and 12% by weight) on the composition of pearlings and bran obtained when subsequently roller milling pearled wheat kernels. Pearling does not remove the kernel outer tissues homogeneously as abrasion affects especially the accessible parts of the kernels. Nevertheless, the first 3% removed consisted of mainly pericarp. With 6% or more removed, a significant amount of starchy endosperm ended up in the pearlings. The starting bran material and bran obtained by subsequent roller milling of pearled wheat kernels had similar compositions but the latter had a lower average particle size. Moreover, removal of the outermost kernel layers substantially decreased the enzyme activity levels in the bran.     

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.     

鲜食糯玉米的果皮性状研究

以果皮脆性、厚度和柔嫩性不同的糯玉米自交系为材料,对鲜样和熟样的果皮厚度、拉力和硬度指标进行研究。结果表明,果皮厚度、拉力和硬度指标,鲜、熟样的不同品系间均存在明显差异,熟样均比鲜样降低;拉力和硬度的差值,鲜、熟样之间差异较大,且熟样使品系间的差值幅度拉大,变异系数增加;果皮厚度,鲜样与熟样之间呈极显著正相关;果皮硬度、拉力,鲜样与熟样之间呈高度正相关;果皮拉力、硬度与厚度之间均无相关关系。研究发现,熟样可使样品间的果皮厚度、拉力和硬度值差异放大,即熟样能更容易进行品种间比较。果皮适口性的好坏不仅与果皮厚度,还与其组织结构有关。     

Effects of Popping on the Endosperm Cell Walls of Sorghum and Maize

The structure of the vitreous endosperm of raw and popped grains of popcorn maize and sorghum has been examined by light and scanning electron microscopy. In both cereals, popping produces everted grains consisting of expanded endosperm foam attached to the pericarp and embryo tissue. As previously reported, each bubble of the foam is formed from an individual starch granule inflated by internal steam pressure. Large fissures may contribute significantly to the expansion of the endosperm foam. The cell walls of the vitreous endosperm of both cereals are shattered into small fragments, which separate slightly as the cell contents expand during popping. Despite this, the endosperm cells retain their polygonal outline. Intact cell walls of raw endosperm, wall fragments from popped endosperm foam, and fragments isolated after treatment of the foam withalpha -amylase, were visualised through the autofluorescence of their ferulic acid content. The in vitro digestibility of popped sorghum was unchanged compared to raw sorghum, whereas that of wet-cooked sorghum was greatly reduced. It is suggested that popping-induced wall fragmentation improves the accessibility of the protein and starch reserves of the endosperm to digestive enzymes.