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.     

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.     

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.     

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.     

Mechanical properties of outer layers from near-isogenic lines of common wheat differing in hardness

The mechanical properties of the combined outer layers from near-isogenic wheats differing by hardness were determined. Results from traction tests showed significant differences between the isogenic lines, outer layers from grains of the soft type showing higher extensibility. Determination of the mechanical properties of the corresponding component tissues revealed significant differences between the isolated tissues from soft or hard wheat grains. It also allowed analysis of their respective contribution to the properties of the combined peripheral tissues using a simulation of their rupture as unseparated tissues. According to the results, if the component layers displayed similar maximum lineic force to rupture, the rupture of combined outer layers occurs when the least extensible individual tissue breaks. The major cell wall biochemical components of the combined outer layers and of their component tissues were analysed. The phenolic acid composition of soft wheat pericarp contained more ferulic acid in either monomeric or polymeric forms than the pericarp from hard wheat. Arabinoxylans in walls of the soft wheat pericarp appeared 1.6 times more cross-linked by ferulic acid dehydrodimers than walls of hard wheat. These differences in arabinoxylan cross-linking may explain the observed differences in pericarp mechanical properties.     

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.     

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.     

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.     

Genetics and chemistry of pigments in wheat grain – A review

Cereal grains contain many phytochemicals, some of which significantly influence the grain colour. Anthocyanins are accumulated in the aleurone or pericarp layer and give blue, purple or combination of these colours. Flavonoids, such as yellow C-glycosides of flavones, flavonols, flavanonols, proanthocyanidins and reddish-coloured phlobaphenes are mainly present in the outer layer of grains while carotenoids that are responsible for yellow grain are in the endosperm. Therefore, accumulation of these pigments in the grain can represent an important target in breeding programmes aimed at increasing the concentrations of bioactive components in grain and products. This review therefore summarises our current knowledge of anthocyanin and carotenoid pigments, their genetic control and variation in levels in different wheat lines.     

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.     

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.     

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.     

Structural and Histochemical Characterization of Developing Rice Caryopsis

The development of pericarp, seed coat, starchy endosperm and aleurone of the rice caryopsis was investigated, histochemically and structurally, from the time of flowering to maturity. The results showed that during its growth, the maximum length of the caryopsis was attained first, followed by width and then thickness. Histochemical examination of the caryopsis showed that starch was mainly accumulated in the endosperm, but the endosperm showed no metabolic activity, while embryo and pericarp contained a few starch grains, and embryo and aleurone were strongly active. Aleuronic cells contained many aleurone grains and spherosomes, and aleurone in the dorsal region developed earlier and contained more layers of cells. Amyloplasts in endosperm contained many starch granules and were spherical at early stages but polyhedric at late stages. The protein bodies appeared later than amyloplasts, and the number of protein bodies in subaleurone was greater than those in the starchy endosperm. The white-belly portion of endosperm might be relative to the status of amyloplast development.     

四个不同粒重水稻品种颖果发育的比较

 以粒重差异较大的4个水稻品种为供试材料,采用树脂切片、酶解胚乳细胞和显微观察等方法,比较研究了品种间在颖果生长、胚乳细胞增殖、果皮和胚乳结构等方面的差异,探讨了影响颖果生长的因素。 大粒品种颖果发育时间较小粒品种长,其胚乳细胞数、胚乳干质量及单个胚乳细胞平均干质量均高于小粒品种。在粒重相近的情况下,籼稻颖果发育和淀粉积累快于粳稻。与小粒品种相比,大粒品种子房壁细胞中淀粉粒多,子房壁细胞生长的持续时间长,果皮及背部维管束衰亡迟。 小粒品种胚乳外层细胞在花后7 d已转化成糊粉层细胞,大粒品种胚乳外层细胞要在花后10 d才转化成糊粉层细胞。 大粒品种的库容大和生理活性期长是其颖果能显著增大的生理原因。     

The use of neutron tomography for the structural analysis of corn kernels

Neutron tomography was studied as a technique for non-destructively analyzing the internal structure of dried corn kernels. The study had two goals: first, to determine if the analysis could identify well-known anatomical features of the kernels; and second, to determine if it could distinguish between different types and treatments of kernels. Specifically, kernels which were infected vs. uninfected with the aflatoxin-producing fungus Aspergillus flavus were analyzed. Two different varieties of corn were used: VA35 (susceptible to A. flavus infection) and GT-MAS:gk (resistant). It was found that many anatomical features of the kernels could be identified using neutron tomography, including the scutellum, endosperm, aleurone, pericarp, pedicel, coleorhizae, radical, plumule, and coleoptile. Furthermore, differences were detected between susceptible kernels that had been inoculated and those that had not. Infected kernels were found to have lower neutron attenuation in the scutellum and embryo regions, possibly caused by lower hydrogen concentrations due to fungal degradation. No systematic structural differences were detected between resistant inoculated and resistant uninoculated kernels, as expected. This study indicated that neutron tomography could be a useful technique for the structural analysis of corn, and possibly other grains or small biological objects.     

Antioxidant and anti-inflammatory capacity of bioaccessible compounds from wheat fractions after gastrointestinal digestion

Wholegrain consumption is associated with several health benefits, in contrast to the consumption of refined grains. This can partly be related to the antioxidant compounds in the outer parts of the grain kernel. The bioaccessibility of these antioxidant compounds from the wholegrain matrix during gastrointestinal digestion is crucial for their absorption and bioavailability. In the current study, the bioaccessible compounds from aleurone, bran and flour were obtained from a dynamic in vitro model of the upper gastrointestinal tract. They were collected at 1 h time intervals to assess their antioxidant capacity (TEAC assay) and also their anti-inflammatory effect (TNF-α reduction in U937 macrophages stimulated with LPS). The bioaccessible compounds from aleurone had the highest antioxidant capacity and provided a prolonged anti-inflammatory effect, shown by the TNF-α reduction of a relatively late time-interval (3–4 h after start of digestion). The contribution of ferulic acid to those effects was minor due to its low bioaccessibility. Aleurone seems a promising wheat fraction for cereal products with a healthy added value.     

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.     

Structural Features of (Glucurono)Arabinoxylans Extracted from Wheat Bran by Barium Hydroxide

A (glucurono)arabinoxylan extract obtained from water-unextractable cell wall material of industrial wheat bran was fractionated by means of anion-exchange chromatography and graded ethanol precipitation. Methylation analysis and enzymic degradability of various fractions were used to elucidate their structures. Approximately one third of the extracted (glucurono)arabinoxylan was very lowly substituted (Ara/Xyl≈0·2). Substitution occurred predominantly by arabinose at theO-3 position of xylose residues. Enzymic degradation showed a random distribution of the substituents, which are probably interrupted by stretches of 6 or more contiguous unsubstituted xylose residues. More than half the extracted (glucurono)arabinoxylan was highly substituted (Ara/Xyl≥1). The complexity of the structure was shown by poor enzymic degradability and the presence of considerable amounts of branched arabinose and terminal xylose. Substitution of xylose occurred not only throughO-3 mono-,O-2 andO-3 disubstitution by terminal arabinose andO-2 monosubstitution by (4-O-methyl)glucuronic acid, but also through dimeric arabinose, xylose and possibly galactose containing branches as well as through 2,3-linked arabinose. The remainder of the extracted (glucurono)arabinoxylan (15%) was either intermediately substituted (Ara/Xyl≈0·5) or very highly substituted (Ara-Xyl≈1·2) and associated with protein and lignin.     

Chalcone synthase expression and pigments deposition in wheat with purple and blue colored caryopsis

Red and white caryopses are typical in common wheat. Genotypes with purple and blue caryopses are also described. This coloring is caused by anthocyanins which deposit in the pericarp (purple) or aleurone layer (blue). The anthocyanins biosynthetic pathway is well described. The key enzyme is chalcone synthase (CHS). It catalyzes the first step. We observed the deposition of anthocyanins in the pericarp and aleurone layer, the expression of a chalcone synthase gene and the amount of two anthocyanins - cyanidin-3-glycoside (pericarp of purple caryopses), and delphinidin-3-glycoside (aleurone layer of blue caryopses) during caryopsis development. Purple pigment deposition was not homogeneous and/or uniform. At first, small isolated spots of purple color were formed and thereafter they expanded. In blue caryopses, however, the coloring process was more homogeneous. The expression of chalcone synthase mRNA occurred five days before pigment deposition and finished earlier than expected. Amounts of cyanidin-3-glycoside and delphinidin-3-glycoside increased in a similar manner. Amounts of these fell at the end of caryopses development probably due to formation of more complex substances.