Inactive fluorescently labeled xylanase as a novel probe for microscopic analysis of arabinoxylan containing cereal cell walls

A new technique to visualize cereal cell walls by fluorescence microscopy was developed. The novel staining technique is based on an inactive fluorescently labeled xylanase binding to arabinoxylan (AX), an important polysaccharide in grain cell walls in terms of the technological and physiological functionalities of grain. The xylanase probe could stain AX in the seed coat, nucellar epidermis, aleurone layer, and starchy endosperm, but not the highly substituted AX of the pericarp layer. The advantage of this new staining technique over the existing immunolabeling techniques is that the staining procedure is clearly faster and less laborious, and uses a smaller probe that can easily be produced by marking a well characterized enzyme with a fluorescent label. In the future, the here proposed technology can be used to develop probes having specificity also for cell wall components other than AX and thus to study plant cell walls further through fluorescence microscopy.     

Deposition of cell wall polysaccharides in wheat endosperm during grain development: Fourier transform-infrared microspectroscopy study

The time course and pattern deposition of the cell wall polysaccharides in the starchy endosperm of wheat (Triticum aestivum cv. Recital) during grain development was studied using Fourier transform infrared microspectroscopy (micro-FTIR). Three stages of grain development identified as key stages for cell wall construction were retained as follows: the end cellularization, the beginning of cell differentiation, and the beginning of maturation. Micro-FTIR revealed that beta-(1-->3),(1-->4) glucans and arabinoglactan proteins are the main cell wall components of endosperm at the end of the cellularization stage, whereas arabinoxylans (AX) appeared only at the cell differentiation stage. Past the differentiation stage, FTIR spectra were dominated by AX features. Cell walls at the beginning of cell differentiation and at endosperm maturation could be distinguished by spectral features that were ascribed to AX substitution. AX appeared more substituted at the beginning of cell differentiation. Moreover, a difference in the degree of AX substitution was found between peripheral and central parts of the grain at the cell differentiation stage; AX in central cells was less substituted. Thus, dramatic changes in endosperm cell wall composition were detected during wheat grain development with respect to both the relative occurrence of individual constituents and the fine structure of the AX.     

Spectroscopic analysis of diversity of Arabinoxylan structures in endosperm cell walls of wheat cultivars (Triticum aestivum) in the HEALTHGRAIN diversity collection

Fifty bread wheat (Triticum aestivum L.) cultivars were selected from the HEALTHGRAIN germplasm collection based on variation in their contents of total and water-extractable arabinoxylan. FT-IR spectroscopic mapping of thin transverse sections of grain showed variation in cell wall arabinoxylan composition between the cultivars, from consisting almost entirely of low-substituted arabinoxylan (e.g., T.aestivum 'Claire') to almost entirely of highly substituted arabinoxylan (e.g., T.aestivum 'Manital') and a mixture of the two forms (e.g., T.aestivum 'Hereward'). Complementary data were obtained using endoxylanase digestion of flour followed by HP-AEC analysis of the arabinoxylan oligosaccharides. This allowed the selection of six cultivars for more detailed analysis using FT-IR and (1)H NMR spectroscopy to determine the proportions of mono-, di-, and unsubstituted xylose residues. The results of the two analyses were consistent, showing that variation in the composition and structure of the endosperm cell wall arabinoxylan is present between bread wheat cultivars. The heterogeneity and spatial distribution of the arabinoxylan in endosperm cell walls may be exploited in wheat processing as it may allow the production of mill streams enriched in various arabinoxylan fractions which have beneficial effects on health.     

Comparative Studies on Composition and Distribution of Phenolic Acids in Cereal Grain Botanical Fractions

The phenolic acid composition and concentration of four manually separated fractions (pericarp, aleurone layer, germ, and endosperm fractions) as well as whole grains of yellow corn, wheat, barley, and oats were analyzed by HPLC‐MS/MS following microwave‐assisted alkaline aqueous extraction. Phenolic acid compositions in whole grains and their fractions were similar, with minor differences among the grain fractions. Significant differences (P < 0.05), however, were observed in phenolic acid concentrations among cereal types, within cereal varieties, and among grain fractions, with yellow corn exhibiting the highest values. The concentrations of p‐coumaric and syringic acid in the pericarp were 10‐ to 15‐fold and 6‐ to 10‐fold higher, respectively, in yellow corn than in wheat, barley, and oats. In the aleurone layer, sinapic and vanillic acids in yellow corn were about 8‐ and 30‐fold more than in wheat. The germ fraction of wheat had 1.8 times more syringic acid than yellow corn germ. Grain fractions, excluding endosperm, had enhanced levels of phenolic acids compared with whole grain. Sinapic acid was more concentrated in the pericarp and germ of wheat, whereas isoferulic acid was concentrated in the germ of purple barley. Syringic and vanillic acids were concentrated in the pericarp and sinapic acid in the aleurone layer of yellow corn. These findings are important in understanding the composition and distribution of phenolic acids, and they act as a guide in identification of grain fractions for use as food ingredients. In addition, yellow corn fractions (aleurone and pericarp) may be potential alternative phenolic‐rich functional food ingredients in grain‐based food products.     

Genotype and Environment Variation for Arabinoxylans in Hard Winter and Spring Wheats of the U.S. Pacific Northwest

The development of high‐quality wheat (Triticum aestivum L.) cultivars depends on a thorough understanding of the constituents of grain and their variation due to genetics and environment. Arabinoxylans (pentosans) are key constituents of wheat grain and have broad and far‐reaching influences on milling and baking quality. However, variation in arabinoxylans due to genotype and environment are not fully understood. In this study, 25 hard winter and 25 hard spring wheat commercial cultivars and advanced breeding lines developed from eight public and private breeding programs in the U.S. Pacific Northwest were analyzed for water‐extractable and total arabinoxylan contents (WE‐AX and total AX), and the proportion of total AX that was water‐extractable. Winter and spring genotypes were grown in three environments each. The results indicated that there were significant differences among both sets of hard wheat genotypes for WE‐AX, total AX, and proportion of total AX that was WE‐AX. The WE‐AX and total AX mean content ranges for the winter cultivars were 0.390–0.808 and 3.09–4.04%, respectively; and for the spring cultivars 0.476–0.919 and 3.94–4.70%, respectively. WE‐AX as a percentage of total AX was similar between the two genotype sets, 11.7–23.0%. Arabinoxylan fractions were generally not correlated with grain protein, test weight, and kernel hardness. The two highest correlations for winter wheats were between protein and total AX (r = –0.40) and test weight and percentage of total AX that were water‐extractable (r = 0.37) for winter wheats. Among spring wheats, single‐kernel characterization system hardness was negatively correlated with WE‐AX and proportion of total AX that was WE‐AX (r = –0.46 and –0.51, respectively). Although often significant, arabinoxylan fractions were usually not highly intercorrelated, indicating some independence of traits. Notable genotypes, being especially high or low for one or more arabinoxylan fraction and, thus, candidates for further genetic study and cross‐breeding, included Juniper, Eddy, and ORN980995 winter wheats, and Hollis, Alta Blanca, and WQL9HDALP spring wheats. Although the results indicate that arabinoxylan fractions of wheat grain can be highly influenced by environment, there is clear support for the existence of genetic differences, especially for WE‐AX and the proportion of total AX that is water‐extractable. As such, the manipulation of arabinoxylan content of wheat grain seems to be a reasonable breeding objective.     

Differences in the Aleurone Layer Fate Between Hard and Soft Common Wheats at Grain Milling

In the milling process, efficient separation between the starchy endosperm and the other grain tissues is a key parameter estimated by ash measurement. Because this separation occurs near the aleurone layer interface, better understanding of this tissue fractionation is critical for a better analysis of the wheat milling behavior. Samples from hard and soft common wheat cultivars that had the same protein content were processed on a pilot mill, and whole grain meals or flour streams were analyzed for ash content. The para‐coumaric acid (p‐CA) and phytic acid flour contents were compared with ash measurement and used as markers of the aleurone cell walls or aleurone cell content, respectively. A greater amount of phytic acid in hard wheat flour compared with soft wheat flour was found and reveals a distinct milling behavior between those wheat classes, mainly at the breaking step. Therefore simple ash content measurement is not sufficient to analyze flour purity. At the reduction stage, quantity of phytic acid increases with the other markers and may result from the overall mechanical resistance of the aleurone tissue. As a consequence, wheat hardness not only determines grain milling behavior but also affects flour composition.     

Structure, chemical composition, and xylanase degradation of external layers isolated from developing wheat grain

The external layers of wheat grain were investigated during maturation with respect to chemical and structural features and xylanase degradability. Cytochemical changes were observed in the isolated peripheral tissues of the wheat grain at four defined stages following anthesis. Marked chemical changes were highlighted at 11 days after anthesis, for which protein and lipid contents varied weakly. The profile of esterified ferulic acid showed large variation in the maturing peripheral layers of grain in contrast to the deposition of ferulate dimers, p-coumaric and sinapic acids. Lignin was monitored at the latest stages of ripening, which corresponds to the cessation of reserve accumulation in the grain. Arabinoxylans (AX) reached a maximum at 20 days and did not display any significant change in arabinosyl substitution proportion until ripeness. When submitted to xylanase, all outer layers were similarly altered in the proportion of soluble AX except for the peripheral tissues of the 11-day-aged wheat grain that had very little AX. Aleurone and nucellar layers were mostly degraded, whereas pericarp stayed intact at all stages of maturation. This degradation pattern was connected with the preferential immunolocalization of xylanase in aleurone and nucellar layers irrespective of the developmental stages. Further chemical examination of the enzyme-digested peripheral tissues of the grain supports the facts that ferulic ester is not a limiting factor in enzyme efficiency. Arabinose branching, ferulic dimers, and ether-linked monomers that are deposited early in the external layers would have more relevance to the in situ degradability of AX.     

Formation of phenolic microbial metabolites and short-chain Fatty acids from rye, wheat, and oat bran and their fractions in the metabolical in vitro colon model

Rye bran and aleurone, wheat bran and aleurone, and oat bran and cell wall concentrate were compared in their in vitro gut fermentation patterns of individual phenolic acids and short-chain fatty acids, preceded by enzymatic in vitro digestion mimicking small intestinal events. The formation of phenolic metabolites was the most pronounced from the wheat aleurone fraction. Phenylpropionic acids, presumably derived from ferulic acid (FA), were the major phenyl metabolites formed from all bran preparations. The processed rye, wheat, and oat bran fractions contained more water-extractable dietary fiber (DF) and had smaller particle sizes and were thus more easily fermentable than the corresponding brans. Rye aleurone and bran had the highest fermentation rate and extent probably due to high fructan and water-extractable arabinoxylan content. Oat samples also had a high content of water-extractable DF, β-glucan, but their fermentation rate was lower. Enzymatic digestion prior to in vitro colon fermentation changed the structure of oat cell walls as visualized by microscopy and increased the particle size, which is suggested to have retarded the fermentability of oat samples. Wheat bran was the most slowly fermentable among the studied samples, presumably due to the high proportion of water-unextractable DF. The in vitro digestion reduced the fructan content of wheat samples, thus also decreasing their fermentability. Among the studied short-chain fatty acids, acetate dominated the profiles. The highest and lowest production of propionate was from the oat and wheat samples, respectively. Interestingly, wheat aleurone generated similar amounts of butyrate as the rye fractions even without rapid gas production.     

Influence of Cultivar and Environment on Water‐Soluble and Water‐Insoluble Arabinoxylans in Soft Wheat

Arabinoxylans are hydrophilic nonstarch polysaccharides found in wheat grain as minor constituents. Arabinoxylans can associate with large amounts of water through hydrogen bonding and can form oxidative gels. These properties are important factors in end‐use quality of wheat. The objective of this study was to delineate the influence of wheat cultivar and growing environment on variation in water‐soluble (WS‐AX), waterinsoluble (WI‐AX), and total (TO‐AX) arabinoxylan contents of flour and whole grain meal. This study included seven spring and 20 winter soft white wheat cultivars grown in 10 and 12 environments, respectively (each evenly split over two crop years). Univariate analysis of variance (ANOVA) and multivariate analysis of variance with canonical analysis (MANOVA) was used to evaluate sources of variation. Variation in arabinoxylan contents and absolute amounts (xylose equivalents) among the two cultivar sample sets (spring and winter) was similar, and both cultivar and environment were significant sources of variation. The cultivar‐by‐environment interaction was relatively unimportant. Results indicate that the variation in arabinoxylan content is primarily influenced by cultivar and secondarily influenced by environment. Within arabinoxylan fractions, WS‐AX content is primarily influenced by genotype, while WI‐AX content is more greatly influenced by the environment.     

Impact of Wheat Fiber on Frozen Dough Shelf Life and Bread Quality

Freezing and prolonged frozen storage of dough results in constant deterioration in the overall quality of the final product. In this study the effect of wheat bran and wheat aleurone as sources of arabinoxylan (AX) on the quality of bread baked from yeasted frozen dough was investigated. Wheat fiber sources were milled to pass through a 0.5 mm screen, prehydrated for 15 min, and incorporated into refined wheat flour at 15% replacement level. Dough products were prepared from refined flour (control A), whole wheat flour (control B), aleurone composite flour (composite flour A), and bran composite flour (composite flour B) and stored at –18°C for 28 weeks. Dough samples were evaluated for breadmaking quality at zero time, 14 weeks, and 28 weeks of storage. Quality parameters evaluated were loaf weight, loaf specific volume, and crumb firmness. Composite flour bread samples showed the most resistance to freeze damage (less reduction in the overall product quality), indicating a possible role of some fiber components (e.g., AX) in minimizing water redistribution in the dough system and therefore lessening adverse modifications to the gluten structure. The data suggest that the shelf life of frozen dough and quality of obtained bread can be improved with the addition of an AX source.     

In‐Depth Study of Durum Wheat Grain Tissue Distribution at Milling

The starchy endosperm proportion in durum wheat grain and its ability to be isolated from the peripheral tissues appear as main intrinsic characteristics potentially related to the milling value but still difficult to assess. In this study, several durum wheat samples displaying distinct grading characteristics were analyzed and processed through a pilot mill. The histological composition of grains and milling fractions was monitored by using identified biochemical markers of each wheat grain tissue. Contrasted milling yields of semolina and flour were observed between samples, despite displaying a similar starchy endosperm proportion determined by hand dissection. These yields were related both to differences in the starchy endosperm extraction and to the presence of the aleurone layer, particularly its cellular content. Furthermore, two distinct types of fractionation behavior of the aleurone layer were distinguished depending on the wheat grain sample. Extraction of the envelopes and embryonic axis into semolina and flours were found negligible in comparison with the other tissues.     

Distribution of Protein Bodies and Phytate‐Rich Inclusions in Grain Tissues of Low and High Iron Rice Genotypes

The present study aims to understand whether genotypic differences in grain iron (Fe) concentration in four rice genotypes are related to its association with protein bodies containing phytate‐rich inclusions. Rice genotypes with high and low grain Fe concentrations in unpolished brown rice were grown in a greenhouse at Chiang Mai, Thailand, and grains were harvested at maturity. The presence of protein bodies and phytate‐rich inclusions in rice grain tissues were examined by means of light and transmission electron microscopy (TEM). The composition of mineral elements in different grain tissues was examined using energy dispersive X‐ray microanalysis (EDX) and chemical analysis. The relative distribution pattern of protein bodies in the tissues was similar among the four rice genotypes, which resembled the pattern of grain N concentrations in these tissues. The high grain Fe genotypes (based on brown rice Fe concentration) had more protein bodies containing phytate‐rich inclusions in the embryo and aleurone layer tissues than the low Fe genotypes. Phytate‐rich inclusions were not detected in the endosperm tissues in all genotypes. In conclusion, the presence of protein bodies with phytate‐rich inclusions predominantly in the embryo and aleurone regions of the grain is an important parameter contributing to the variation in brown rice Fe concentration among the genotypes, but not in the white rice (the endosperm). Iron associated with the phytate‐rich inclusions present in the embryo and aleurone layer tissues are largely lost during the polishing process to produce white rice.     

Use of Two Endoxylanases with Different Substrate Selectivity for Understanding Arabinoxylan Functionality in Wheat Flour Breadmaking

A Bacillus subtilis endoxylanase (XBS) with a strong selectivity for hydrolysis of water‐unextractable arabinoxylan (WU‐AX) and an Aspergillus aculeatus endoxylanase (XAA) with a strong selectivity for hydrolysis of water‐extractable arabinoxylan (WE‐AX) were used in straight‐dough breadmaking with two European wheat flours. Dough, fermented dough, and bread characteristics with different levels of enzyme addition were evaluated with a strong emphasis on the arabinoxylan (AX) population. The WU‐AX solubilized by XBS during breadmaking were mainly released during mixing and had higher molecular weight, in contrast to their counterparts solubilized by XAA, which were mainly released during fermentation and had lower molecular weight. This coincided with increased loaf volume with XBS and a negative to positive loaf volume response with XAA. Bread firmness and dough extract viscosity also were affected by endoxylanase addition. Results confirmed that WU‐AX are detrimental for breadmaking, while WE‐AX and solubilized AX with medium to high molecular weight have a positive impact on loaf volume.     

Influence of Structural Characteristics of Aleurone Layer on Milling Behavior of Durum Wheat (Triticum durum Desf.)

The structure of the aleurone layer was considered for many years as a potential factor influencing wheat milling efficiency. Eight durum wheat samples of different milling values, including distinct cultivars and harvesting conditions, were employed to investigate the structural characteristics of the aleurone layer through image analysis of kernel sections. Particular attention was paid to tissue thickness and structural irregularity of its interface with the starchy endosperm. Wheat cultivar, agricultural conditions, and location of measurement within the grain had an influence similar to both thickness and irregularity of the aleurone layer. Conversely, grain weight and morphology showed no effect on these parameters. Statistical investigation demonstrated no correlation between structural characteristics and wheat milling behavior. However, the negative correlation between the extraction rate of semolina and starch content in the bran fraction, which was used as an indicator of the endosperm‐aleurone dissociation extent, demonstrated the relevance of the tissue adhesion on milling efficiency.     

Microstructure and Composition of Digitaria exilis Stapf (acha): A Potential Crop

Microstructure of the mature caryopsis of Digitaria exilis Stapf was studied by light and scanning electron microscopy and compared to chemical composition. The general structure of the caryopsis was similar to that of other grains, notably the millets. Thin bracts (the palea and lemma) and two glumes encased the caryopsis which consists of the thin, compressed layers of pericarp, testa, and cuticle surrounding the endosperm and embryonic tissues. The endosperm consisted of a single layer of aleurone cells and the starchy endosperm. The aleurone layer was thin over most of the starchy endosperm and thicker at the junction of the embryo and starchy endosperm. Aleurone cells contained lipid droplets and protein bodies. The cell contents of the starchy endosperm consisted of simple, polyhedral starch granules, lipid droplets, and protein bodies. Protein bodies were more abundant toward the periphery, and diminished toward the central portion of the starchy endosperm. Cells in certain regions of the embryo contained few, small, spherical starch granules and an abundance of protein bodies. Protein bodies containing phytic acid inclusions were located in the scutellum of the embryo. Compositional analyses revealed that the grain contained 8.2% protein, 2.1% fat, 0.48% fiber, and 1.4% ash.     

Comparison of Swiss red wheat grain and fractions for their antioxidant properties

Swiss red wheat grain, bran, aleurone, and micronized aleurone were examined and compared for their free radical scavenging properties against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH*), radical cation ABTS*+ and peroxide radical anion O(2)*-, oxygen radical absorbance capacity (ORAC), chelating capacity, total phenolic content (TPC), and phenolic acid composition. The results showed that micronized aleurone, aleurone, bran, and grain may significantly differ in their antioxidant properties, TPC, and phenolic acid composition. Micronized aleurone had the greatest antioxidant activities, TPC, and concentrations of all identified phenolic acids, suggesting the potential of postharvesting treatment on antioxidant activities and availability of TPC and phenolic acids. Ferulic acid was the predominant phenolic acid in Swiss red wheat and accounted for approximately 57-77% of total phenolic acids on a weight basis. Ferulic acid concentration was well correlated with scavenging activities against radical cation and superoxide anion, TPC, and other phenolic acid concentrations, suggesting the potential use of ferulic acid as a marker of wheat antioxidants. In addition, 50% acetone and ethanol were compared for their effects on wheat ORAC values. The ORAC value of 50% acetone extracts was 3-20-fold greater than that of the ethanol extracts, indicating that 50% acetone may be a better solvent system for monitoring antioxidant properties of wheat. These data suggest the possibility to improve the antioxidant release from wheat-based food ingredients through postharvesting treatment or processing.     

Ferulic acid from aleurone determines the antioxidant potency of wheat grain (Triticum aestivum L.)

Grain is an important source of phytochemicals, which have potent antioxidant capacity. They have been implicated in the beneficial health effect of whole grains in reducing cardiovascular disease and type 2 diabetes. The aim of the present study was to identify the most important antioxidant fractions of wheat grain. It was found that the aleurone content of these fractions was highly correlated with the antioxidant capacity of the fractions (r = 0.96, p < 0.0001). Ferulic acid appeared to be the major contributor to the antioxidant capacity in fractions with higher antioxidant capacity. The contribution of protein was rather limited. It was concluded that the antioxidant potency of wheat grain fractions is predominantly determined by aleurone content, which can be attributed to the presence of relatively large amounts of phenolic compounds, primarily ferulic acid.     

Structural features of arabinoxylans extracted with water at different temperatures from two rye flours of diverse breadmaking quality

The water extractable (WE) arabinoxylans from two rye flours differing in baking quality were studied following sequential extraction with water at 4, 40, and 100 degrees C. Ammonium sulfate fractionation of the resulting WE fractions and subsequent analysis revealed substantial differences in the structure of the isolated materials. Furthermore, it allowed us to identify the factors contributing to arabinoxylan water extractability. Our results provide compelling evidence for the existence of separate polymers in rye WE arabinoxylans with different substitution degrees, ranging from quantitatively dominating, lowly substituted populations (arabinose to xylose ratio, Ara/Xyl approximately 0.5) to comparatively less abundant, highly substituted analogues (Ara/Xyl approximately 1.3). Generally, arabinoxylan water extractability was governed by the relative proportion of lowly and highly branched structures. A gradually increasing proportion of highly substituted populations was observed from cold to hot WE fractions. This was associated with the lower proportion of monosubstituted xylopyranosyl residues in the backbone, the higher proportion of disubstituted xylopyranosyl residues, and the higher level of substitution with feruloyl residues. Notable differences in the ratio of phenolic compounds to arabinose residues were observed between corresponding polymers isolated from rye flours of high and low baking quality, whereas the differences in their molecular weights were much less pronounced.     

FT‐Raman Spectra of Unsoaked and NaOH‐Soaked Wheat Kernels,Bran, and Ferulic Acid

The sodium hydroxide (NaOH) test for determining wheat color class depends on the observation that on soaking in NaOH, red wheat turns a darker red and white wheat turns straw yellow. To understand the mechanism of this test, Raman spectra of wheat bran, wheat starch, ferulic acid, and whole kernels of wheat, before and after NaOH soak, were studied. The major observable components in the whole kernel were that of starch, protein, and ferulic acid, perhaps esterified to arabinoxylan and sterols. When kernels are soaked in NaOH, spectral bands due to ferulic acid shift to lower energy and show a slightly reduced intensity that is consistent with deprotonation of the phenolic group and extraction of a portion of the ferulic acid into solution. Other phenolic acids, alkyl resorcinols, and flavonoids observed in the NaOH extracts of wheat by HPLC were not observed in the Raman spectra. Wheat bran accounts for most of the ferulic acid in the whole kernel, as indicated by the increased intensity of the doublet at 1,631 and 1,600 cm^‐1 in the bran. The intense starch band at 480 cm^‐1 in whole kernel wheat was nearly absent in the wheat bran.     

Esterases in Barley and Malt

Several esterases from barley and malt have been separated on polyacrylamide gels. The slowest moving bands appear to represent a single enzyme displaying a spread of migration owing to differences in surface charge. During malting, this enzyme, which is located in the starchy endosperm, shifts to a more migratory form. Two other main esterase groups are identified through gel electrophoresis, notably a highly anionic, highly labile enzyme, MW 62,000, located in the aleurone. The slowest and fastest moving bands have been partially purified using salt fractionation and ion‐exchange chromatography. The former, MW 47,000, has strong capability for hydrolyzing acetylxylan and it is speculated that its role in the starchy endosperm is as part of the enzyme system that hydrolyzes the cell walls.