In vitro protein digestibility of RuBisCO-enriched wheat dough: a comparative study with pea and gluten proteins.

Growing demand for sustainable, plant-based protein sources has stimulated interest in new ingredients for food enrichment. This study investigates the nutritional and digestive implications of enriching wheat dough with RuBisCO, in comparison to pea protein-enriched and gluten-enriched doughs. The protein quality and digestibility of these enriched doughs were analysed through dough characterization, in vitro digestion experiments and biochemical analysis of digesta. Our findings indicate that an enrichment at 10% of RuBisCO or pea proteins improves the chemical score and the in vitro PDCAAS (IV-PDCAAS) score of wheat dough as compared to the control dough. Digestibility assays suggest that RuBisCO introduction modifies the protein hydrolysis kinetics: the nitrogen release is lower during gastric digestion but larger during intestinal digestion than other samples. The analysis of the protein composition of the soluble and insoluble parts of digesta, using size-exclusion chromatography, reveals that the protein network in RuBisCO-enriched dough is more resistant to gastric hydrolysis than the ones of other doughs. Indeed, non-covalently bound peptides and disulfide-bound protein aggregates partly composed of RuBisCO subunits remain insoluble at the end of the gastric phase. The digestion of these protein structures is then mostly performed during the intestinal phase. These results are also discussed in relation to the digestive enzymatic cleavage sites, the presence of potential enzyme inhibitors, the protein aggregation state and the secondary structures of the protein network in each dough type.

Maize Internode Autofluorescence at the Macroscopic Scale: Image Representation and Principal Component Analysis of a Series of Large Multispectral Images.

A quantitative histology of maize stems is needed to study the role of tissue and of their chemical composition in plant development and in their end-use quality. In the present work, a new methodology is proposed to show and quantify the spatial variability of tissue composition in plant organs and to statistically compare different samples accounting for biological variability. Multispectral UV/visible autofluorescence imaging was used to acquire a macroscale image series based on the fluorescence of phenolic compounds in the cell wall. A series of 40 multispectral large images of a whole internode section taken from four maize inbred lines were compared. The series consisted of more than 1 billion pixels and 11 autofluorescence channels. Principal Component Analysis was adapted and named large PCA and score image montages at different scales were built. Large PCA score distributions were proposed as quantitative features to compare the inbred lines. Variations in the tissue fluorescence were clearly displayed in the score images. General intensity variations were identified. Rind vascular bundles were differentiated from other tissues due to their lignin fluorescence after visible excitation, while variations within the pith parenchyma were shown via UV fluorescence. They depended on the inbred line, as revealed by the first four large PCA score distributions. Autofluorescence macroscopy combined with an adapted analysis of a series of large images is promising for the investigation of the spatial heterogeneity of tissue composition between and within organ sections. The method is easy to implement and can be easily extended to other multi-hyperspectral imaging techniques. The score distributions enable a global comparison of the images and an analysis of the inbred lines' effect. The interpretation of the tissue autofluorescence needs to be further investigated by using complementary spatially resolved techniques.

Darkfield and Fluorescence Macrovision of a Series of Large Images to Assess Anatomical and Chemical Tissue Variability in Whole Cross-Sections of Maize Stems.

The proportion and composition of plant tissues in maize stems vary with genotype and agroclimatic factors and may impact the final biomass use. In this manuscript, we propose a quantitative histology approach without any section labelling to estimate the proportion of different tissues in maize stem sections as well as their chemical characteristics. Macroscopic imaging was chosen to observe the entire section of a stem. Darkfield illumination was retained to visualise the whole stem cellular structure. Multispectral autofluorescence images were acquired to detect cell wall phenolic compounds after UV and visible excitations. Image analysis was implemented to extract morphological features and autofluorescence pseudospectra. By assimilating the internode to a cylinder, the relative proportions of tissues in the internode were estimated from their relative areas in the sections. The approach was applied to study a series of 14 maize inbred lines. Considerable variability was revealed among the 14 inbred lines for both anatomical and chemical traits. The most discriminant morphological descriptors were the relative amount of rind and parenchyma tissues together with the density and size of the individual bundles, the area of stem and the parenchyma cell diameter. The rind, as the most lignified tissue, showed strong visible-induced fluorescence which was line-dependant. The relative amount of para-coumaric acid was associated with the UV-induced fluorescence intensity in the rind and in the parenchyma near the rind, while ferulic acid amount was significantly correlated mainly with the parenchyma near the rind. The correlation between lignin and the tissue pseudospectra showed that a global higher amount of lignin resulted in a higher level of lignin fluorescence whatever the tissues. We demonstrated here the potential of darkfield and autofluorescence imaging coupled with image analysis to quantify histology of maize stem and highlight variability between different lines.

Preserving the Cellular Tissue Structure of Maize Pith Though Dry Fractionation Processes: A Key Point to Use as Insulating Agro-Materials.

Plant biomass has various compositions and structures at different scales (from the component organs to their constitutive tissues) to support its functional properties. Recovering each part of the plant without damaging its structure poses a challenge to preserving its original properties for differential dedicated end uses, and considerably increases its added value. In this work, an original combination of grinding based on shearing stress and separation based on particle size and density was successfully used to sort rind (65% w/w) and pith (35% w/w) from maize stem internodes. More than 97% of the rind was isolated. The pith alveolar structure was well preserved in coarse particles, making them suitable for insulation bio-based composite materials, a promising alternative to conventional nonbiodegradable insulation panels. Boards produced from the dry fractionated pith exhibited thermal conductivities like those produced from hand dissected pith, with values equal to 0.037 W·mK-1 and 0.039 W·mK-1, respectively. In the finest fraction (particle size <1 mm), the pith vascular bundles (around 300-400 µm in diameter) were dissociated from parenchyma cells and successfully isolated using a cutting-edge electrostatic separator. Their structures, which provide the plant structural support, make them potentially valuable for reinforcement in composite materials.

Enzymatic degradation of maize shoots: monitoring of chemical and physical changes reveals different saccharification behaviors.

BACKGROUND: The recalcitrance of lignocellulosics to enzymatic saccharification has been related to many factors, including the tissue and molecular heterogeneity of the plant particles. The role of tissue heterogeneity generally assessed from plant sections is not easy to study on a large scale. In the present work, dry fractionation of ground maize shoot was performed to obtain particle fractions enriched in a specific tissue. The degradation profiles of the fractions were compared considering physical changes in addition to chemical conversion. RESULTS: Coarse, medium and fine fractions were produced using a dry process followed by an electrostatic separation. The physical and chemical characteristics of the fractions varied, suggesting enrichment in tissue from leaves, pith or rind. The fractions were subjected to enzymatic hydrolysis in a torus reactor designed for real-time monitoring of the number and size of the particles. Saccharification efficiency was monitored by analyzing the sugar release at different times. The lowest and highest saccharification yields were measured in the coarse and fine fractions, respectively, and these yields paralleled the reduction in the size and number of particles. The behavior of the positively- and negatively-charged particles of medium-size fractions was contrasted. Although the amount of sugar release was similar, the changes in particle size and number differed during enzymatic degradation. The reduction in the number of particles proceeded faster than that of particle size, suggesting that degradable particles were degraded to the point of disappearance with no significant erosion or fragmentation. Considering all fractions, the saccharification yield was positively correlated with the amount of water associated with [5-15 nm] pore size range at 67% moisture content while the reduction in the number of particles was inversely correlated with the amount of lignin. CONCLUSION: Real-time monitoring of sugar release and changes in the number and size of the particles clearly evidenced different degradation patterns for fractions of maize shoot that could be related to tissue heterogeneity in the plant. The biorefinery process could benefit from the addition of a sorting stage to optimise the flow of biomass materials and take better advantage of the heterogeneity of the biomass.

Arabinoxylan content and grain tissue distribution are good predictors of the dietary fibre content and their nutritional properties in wheat products.

Wheat millstreams and wheat-based foods (pasta, biscuits and bread) enriched or not in dietary fibre with fractions extracted from wheat grains, have been characterized either for their total dietary fibre content (TDF) and their arabinoxylan (AX) content. A strong correlation (r2 = 0.98) is observed between the AX and TDF contents indicating that AX can be used to estimate TDF content in wheat products. Moreover, by adding a previous step including enzymatic hydrolysis with a xylanase, a functional evaluation of DF is proposed based on the amount of AX released by the enzyme. Xylanase hydrolysable AX are likely also released by microbiota's enzymes in the gut and therefore an indicator for the proportion of fermentable DF in grain fractions and wheat-based foods (pasta, biscuits and bread). This assay opens the door for simple characterization of qualitative attribute of cereal DF.

Ferulate and lignin cross-links increase in cell walls of wheat grain outer layers during late development.

Important biological, nutritional and technological roles are attributed to cell wall polymers from cereal grains. The composition of cell walls in dry wheat grain has been well studied, however less is known about cell wall deposition and modification in the grain outer layers during grain development. In this study, the composition of cell walls in the outer layers of the wheat grain (Triticum aestivum Recital cultivar) was investigated during grain development, with a focus on cell wall phenolics. We discovered that lignification of outer layers begins earlier than previously reported and long before the grain reaches its final size. Cell wall feruloylation increased in development. However, in the late stages, the amount of ferulate releasable by mild alkaline hydrolysis was reduced as well as the yield of lignin-derived thioacidolysis monomers. These reductions indicate that new ferulate-mediated cross-linkages of cell wall polymers appeared as well as new resistant interunit bonds in lignins. The formation of these additional linkages more specifically occurred in the outer pericarp. Our results raised the possibility that stiffening of cell walls occur at late development stages in the outer pericarp and might contribute to the restriction of the grain radial growth.

Structural, Culinary, Nutritional and Anti-Nutritional Properties of High Protein, Gluten Free, 100% Legume Pasta.

Wheat pasta has a compact structure built by a gluten network entrapping starch granules resulting in a low glycemic index, but is nevertheless unsuitable for gluten-intolerant people. High protein gluten-free legume flours, rich in fibers, resistant starch and minerals are thus a good alternative for gluten-free pasta production. In this study, gluten-free pasta was produced exclusively from faba, lentil or black-gram flours. The relationship between their structure, their cooking and Rheological properties and their in-vitro starch digestion was analyzed and compared to cereal gluten-free commercial pasta. Trypsin inhibitory activity, phytic acid and α-galactosides were determined in flours and in cooked pasta. All legume pasta were rich in protein, resistant starch and fibers. They had a thick but weak protein network, which is built during the pasta cooking step. This particular structure altered pasta springiness and increased cooking losses. Black-gram pasta, which is especially rich in soluble fibers, differed from faba and lentil pasta, with high springiness (0.85 vs. 0.75) and less loss during cooking. In comparison to a commercial cereal gluten-free pasta, all the legume pasta lost less material during cooking but was less cohesive and springy. Interestingly, due to their particular composition and structure, lentil and faba pasta released their starch more slowly than the commercial gluten-free pasta during the in-vitro digestion process. Anti-nutritional factors in legumes, such as trypsin inhibitory activity and α-galactosides were reduced by up to 82% and 73%, respectively, by pasta processing and cooking. However, these processing steps had a minor effect on phytic acid. This study demonstrates the advantages of using legumes for the production of gluten-free pasta with a low glycemic index and high nutritional quality.

Protein enriched pasta: structure and digestibility of its protein network.

Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher ß-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest.

Multiscale characterization of arabinoxylan and ß-glucan composite films.

Composite films made with Arabinoxylans (AXs) (with high, middle and low level of substitution by arabinose) and (1 → 3)(1 → 4)-ß-D-glucans (BGs) extracted from cereal cell walls have been prepared and analyzed using microscopy (SEM and LSCFM), DSC, mechanical tests and TD-NMR spectroscopy. The objectives were to correlate molecular and physico-chemical properties of films with mechanical and hydration properties of wheat cell walls. A phase separation phenomenon was observed for films made with highly substituted AXs and BGs at a ratio AX/BG of 60/40. This phase separation was correlated with lower dipolar interactions between polysaccharide chains and a decrease of ultimate strain and stress of films. Highly substituted AX and BG composite films exhibited very weak mechanical properties in agreement with weaker interactions between the polymer chains. This effect was supported by NMR results showing that interactions between AXs and BGs decreased with increased substitution of AXs in composite films. Lower dipolar interactions between polysaccharides favored the water mobility in relation with a higher specific surface area of polysaccharides in films but also higher distances between polysaccharide chains so larger nanopores in composite films made within highly substituted AXs. These multiscale characterizations agreed with the structural changes observed in wheat grain during its development.

Hydration and mechanical properties of arabinoxylans and ß-D-glucans films.

Arabinoxylans (AX) and (1→3)(1→4)-ß-d-glucans (BG) are the main components of the cell walls in the endosperm of wheat grain. The relative occurrence of these two polysaccharides and the fine structure of the AX are highly variable within the endosperm. Films of AX and BG were used as models of the cell wall to study the impact of polymer structure on the hydration and mechanical properties of the cell walls. Effective moisture diffusivities (Deff) of AX and BG films were determined from 0 to 95% relative humidity (RH) at 20°C. Deff was influenced by the water content, and the structure of polysaccharides. Higher Deff was obtained for films made with highly substituted AX compared to values obtained for films made with BG or lowly substituted AX. Proton dipolar second moments M2 and water T2 relaxation times measured by TD-NMR, indicated that the highly branched AX films exhibited a higher nano-porosity, favoring water motions within films. Results from traction tests showed significant different mechanical properties between the AX and BG films. BG films exhibited much higher extensibility than AX films. Strength and extensibility of AX films decreased with increasing arabinose to xylose ratio. Our results show that the water motions and the mechanical properties of AX and BG films can be linked to the polysaccharide chains interactions that modulate the nanostructure of films.

The peroxidase/H2O2 system as a free radical-generating agent for gelling maize bran arabinoxylans: rheological and structural properties.

The oxidative gelation of maize bran arabinoxylans (MBAX) using a peroxidase/H(2)O(2) system as a free radical-generating agent was investigated. The peroxidase/H(2)O(2) system led to the formation of dimers and trimer of ferulic acid as covalent cross-link structures in the MBAX network. MBAX gels at 4% (w/v) presented a storage modulus of 180 Pa. The structural parameters of MBAX gels were calculated from swelling experiments. MBAX gels presented a molecular weight between two cross-links (Mc), a cross-linking density (ρ(c)) and a mesh size (x) of 49 × 103 g/mol, 30 × 10-6 mol/cm3 and 193 nm, respectively.

Prediction of relative tissue proportions in wheat mill streams by fourier transform mid-infrared spectroscopy.

Fourier-transform mid-infrared (FTIR) spectroscopy was investigated as a method to quantify the relative wheat grain tissue proportion in milling fractions. Spectra were acquired with a FTIR spectrometer equipped with an attenuated total reflectance device on ground samples, and the relative tissue proportion was determined according to the biochemical marker methodology as the reference method. Partial least-squares models were developed independently to predict the amount of outer pericarp, aleurone layer, starchy endosperm, and an intermediate layer (made up of inner pericarp plus seed coat plus nucellar epidermis). Good quality of prediction was obtained regardless of the target tissue. The standard errors of prediction obtained for the outer pericarp, intermediate layer, aleurone layer, and starchy endosperm quantification were, respectively, 3.4, 1.3, 3.4, and 4.6%.

Water mobility within arabinoxylan and ß-glucan films studied by NMR and dynamic vapour sorption.

BACKGROUND: The main purpose of this research was to determine the impact of the structure and organisation of polysaccharides on the hydration properties of the cell walls of cereal grains. In order to remodel the lamellar assembly of arabinoxylan (AX) and (1 → 3)(1 → 4)-ß-D-glucan (BG) within the endosperm cell walls, films were prepared and analysed using dynamic vapour sorption and time domain nuclear magnetic resonance spectroscopy. RESULTS: The water diffusivities within the AX and BG films were measured at 20 °C by observing the water sorption kinetics within a mathematical model based on Fick's second law. The evolution of spin-spin relaxation times of water protons measured by increasing the temperature is explained by the additional contributions of motion of the protons of polysaccharides and/or rapid chemical exchanges of protons between water and hydroxyl groups of polysaccharides. CONCLUSION: The difference between patterns of water behaviour within the AX and BG films can be related to the difference in their nanostructures. The smaller nanopores of the BG films cause their nanostructure to be more compact.

Brachypodium distachyon grain: characterization of endosperm cell walls.

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ∼52% (w/w) of the endosperm in comparison with 2-7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1-3) (1-4)-ß-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1-3) (1-4)-ß-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell-cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains.

Laser-induced breakdown spectroscopy and chemometrics: a novel potential method to analyze wheat grains.

Laser-induced breakdown spectroscopy (LIBS) has been widely used to evaluate the elemental composition (e.g., minerals or metal accumulation) on vegetal tissues. The main objective of this work was to differentiate wheat outer tissues during the grain ablation using LIBS and univariate/multivariate analysis. A high resolution spectrometer and a Nd:YAG laser (532 nm, 5 ns) was first used in order to easily identify atomic wheat emission lines. Then a pulsed excimer laser ArF (193 nm, 15 ns) and a compact fiber optic spectrometer was used to acquire LIBS spectral data from each pulse. Univariate and multivariate analyses (MW2D, PLS-DA) were carried out to provide more in depth information from the LIBS experiment. The number of pulses needed to ablate wheat tissues was successfully predicted by the supervised pattern recognition procedure. LIBS used in conjunction with multivariate analysis could be an interesting technique for rapid structural analysis of vegetal material.

Characterization using Raman microspectroscopy of arabinoxylans in the walls of different cell types during the development of wheat endosperm.

The time course and pattern of arabinoxylan deposition in the wheat (Triticum aestivum) endosperm during grain development were studied using Raman spectroscopy. The presence of arabinoxylans (AX) is detected at the beginning of grain filling. At this stage, AX appear more substituted than at the later stages. Feruloylation of AX increases during the grain-filling stage, especially in the case of the aleurone layer. Whatever the stage of grain development, four populations of cells could be defined according to Raman arabinoxylan signatures. In the walls of the aleurone cells, AX appeared to be little substituted and highly esterified with phenolic acids. In the walls of prismatic cells, AX were found to be highly substituted and poorly esterified. Apart from aleurone and prismatic cells, the substitution degree of AX in endosperm was in the same range. Cells in the crease region were distinguished from cells in the starchy endosperm by their lower amount of esterified phenolic compounds.

Structural heterogeneity of wheat arabinoxylans revealed by Raman spectroscopy.

A set of arabinoxylan samples differing in their arabinose composition and various samples of arabino-xylo-oligosaccharide samples were analysed by Raman spectroscopy. Specific signatures for arabinose substitution were found in several spectral regions, that is, 400-600, 800-950 and 1030-1100 cm(-1). A linear relationship was observed between the peak ratio 855/895 cm(-1) of the second derivative spectra and the A/X ratio determined by chemical analysis. Moreover, spectral changes were observed in the 400-600 cm(-1) region assigned to the coupled vibrations mode in the skeleton: while the intensity of the band at 570 cm(-1) increased with the degree of substitution, that at 494 cm(-1) decreased. Similarly, a linear relationship was observed between the peak intensity ratio 570/494 cm(-1) calculated on the second derivative spectra and the composition data. Analysis of Raman spectra of arabino-xylo-oligosaccharides allowed to identify specific spectral features of disubstitution.

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.

FT-IR investigation of cell wall polysaccharides from cereal grains. Arabinoxylan infrared assignment.

The FT-IR fingerprint of wheat endosperm arabinoxylan (AX) was investigated using a set of polysaccharides exhibiting variation of their degree of substitution and xylo-oligosaccharides comprising xylose units mono- or disubstituted by arabinose residues. Substitution of the xylose backbone by arabinose side units was more particularly studied in the 1000-800 cm(-1) spectral region, by taking advantage of second-derivative enhancement. The 920-1020 cm(-1) spectral region revealed two absorption bands at 984 and 958 cm(-1), the intensities of which varied according to the degree of substitution. Whereas the intensity of the band at 958 cm(-1) increased with the degree of substitution, that at 984 cm(-1) decreased. The second-derivative spectral data of xylo-oligosaccharides indicated that these changes could be attributed to substitution of the xylan backbone by arabinose residues, and the band at 958 cm(-1) was ascribed to the presence of disubstituted xylose residues. Principal component analysis of FT-IR spectra of model mixtures of AX, beta-glucans, and arabinogalactans suggested that it is possible to evaluate the relative proportions of the polymers and degree of substitution of AX in complex mixtures such as the cell wall of cereal grains.