Molecular clustering,interrelationships and carbohydrate conformation in hull and seeds among barley cultivars

The objective of this study was to use molecular spectral analyses with the diffuse reflectance Fourier transform infrared spectroscopy (DRIFT) bioanlytical technique to study carbohydrate conformation features, molecular clustering and interrelationships in hull and seed among six barley cultivars (AC Metcalfe, CDC Dolly, McLeod, CDC Helgason, CDC Trey, CDC Cowboy), which had different degradation kinetics in rumen. The molecular structure spectral analyses in both hull and seed involved the fingerprint regions of ca. 1536–1484 cm⁻¹ (attributed mainly to aromatic lignin semicircle ring stretch), ca. 1293–1212 cm⁻¹ (attributed mainly to cellulosic compounds in the hull), ca. 1269–1217 cm⁻¹ (attributed mainly to cellulosic compound in the seeds), and ca. 1180–800 cm⁻¹ (attributed mainly to total CHO C–O stretching vibrations) together with an agglomerative hierarchical cluster (AHCA) and principal component spectral analyses (PCA). The results showed that the DRIFT technique plus AHCA and PCA molecular analyses were able to reveal carbohydrate conformation features and identify carbohydrate molecular structure differences in both hull and seeds among the barley varieties. The carbohydrate molecular spectral analyses at the region of ca. 1185–800 cm⁻¹ together with the AHCA and PCA were able to show that the barley seed inherent structures exhibited distinguishable differences among the barley varieties. CDC Helgason had differences from AC Metcalfe, MeLeod, CDC Cowboy and CDC Dolly in carbohydrate conformation in the seed. Clear molecular cluster classes could be distinguished and identified in AHCA analysis and the separate ellipses could be grouped in PCA analysis. But CDC Helgason had no distinguished differences from CDC Trey in carbohydrate conformation. These carbohydrate conformation/structure difference could partially explain why the varieties were different in digestive behaviors in animals. The molecular spectroscopy technique used in this study could also be used for other plant-based feed and food structure studies.     

Characterizing the molecular structure features of newly developed hulless barley cultivars with altered carbohydrate traits (Hordeum vulgare L.) by globar-sourced infrared spectroscopy in relation to nutrient utilization and availability

Four newly developed huless barley cultivars with altered carbohydrate traits were developed at the Crop Development Centre (CDC), University of Saskatchewan which varied for amylose (1–40% DM), and β-glucan (5–10% DM) content. The four hulless barley cultivars include zero-amylose waxy, CDC Fibar; 5%-amylose waxy, CDC Rattan; normal-amylose, CDC McGwire and high-amylose, HB08302. CDC Copeland was also included as a hulled barley control. Our previous study revealed the altered carbohydrate traits in barley affected nutrition availability and rumen degradation kinetics. These biological differences may be caused by differences in their molecular structure features. The objectives of this study were to (1) reveal molecular structure features in the four hulless barley cultivars with altered carbohydrate traits, and (2) quantify the molecular structural features in relation to rumen degradation kinetics, intestinal nutrient digestion and predicted protein supply to dairy cattle. Conventional Molecular Infrared-vibration Spectroscopy was applied to detect biochemical characteristics of functional group bands including protein, non-starch carbohydrate (β-glucan and cellulosic compounds) and total carbohydrate. Spectral data were further analyzed using univariate analysis of recording absorption peak parameters (baseline, region, relative height and area) and also multivariate analysis with agglomerative hierarchical cluster analysis and principal components analysis. Although correlation results showed weak correlation (P < 0.05) between identified functional group bands and ruminal degradation kinetics and estimated protein supply from both models, molecular structure differences of hulless barley cultivars can still be detected by the Molecular Infrared-vibration Spectroscopy technique and potential truly protein supply (MP) was significantly affected (P < 0.05) by protein molecular structure characteristics in hulless barley cultivars.     

Mass spectrometry imaging of BARLEYmax fructooligosaccharides

BARLEYmax is a novel hull-less barley cultivar lacking activity of a key enzyme required for starch synthesis (SSIIa), resulting in a grain with reduced total starch, higher dietary fiber, and higher fructan content. In this study, we examined the quantitative differences in fructan among 2 hull-less barley lines (BARLEYmax and Mannenboshi) and 2 peeled barley lines (Glutinous barley rice and Hindmarsh). We found that BARLEYmax had a higher fructan content, particularly 2 types of fructooligosaccharides (FOS), namely kestose and nystose, the important components of fructan that have some beneficial effects for human health. We established a mass spectrometry imaging (MSI) method to further investigate the content and localization of FOS in the barley lines. We found that kestose, nystose, and fructosyl-nystose showed increased molecular ion intensities in BARLEYmax compared with those in other barley lines. These results confirmed that the loss of SSIIa activity in BARLEYmax leads to a decrease in starch in the cytoplasm and an increase in fructan in the vacuole as an energy store. Overall, these findings demonstrate that MSI analysis is helpful for understanding FOS localization in cereal grains and for visual comparison among matured grains.     

Soluble arabinoxylans extracted from soft and hard wheat show a differential prebiotic effect in vitro and in vivo

Arabinoxylans (AX) are part of dietary fiber. They are currently under study due to their potential prebiotic effect. Wheat whole grain flours contain all the grain layers and, therefore, present a higher arabinoxylan content than white flour. It is known that the chemical structure of these compounds varies with the type of wheat cultivar and the tissue from which they are extracted. In this work, water soluble extractable arabinoxylans (WE-AX) from two types of wheat whole flours (hard and soft) were extracted. We characterized the molecular size distribution and the potential prebiotic effect of those extracts. The prebiotic effect was evaluated in vitro and confirmed in vivo. Bacterial group abundance (Lactobacillus, Bifidobacterium, Clostridium, Enterococcus, Bacteriodes and total bacteria) was determined by quantitative RT-PCR. The molecular size of AX from hard wheats was significantly higher than AX from soft wheats. Both extracts showed potential prebiotic activity by increasing the growth of beneficial bacteria in vitro and in vivo, decreasing the pathogens in the profile of intestinal microorganisms and increasing the amount of short chain fatty acids in the intestine. WE-AX from hard wheat showed a higher prebiotic activity. Prebiotic effect assessed in vitro and in vivo assays showed a significant correlation between both types of analysis. This finding suggests that the in vitro indices performed allow predicting the potential prebiotic effect in vivo.     

Application of an orcinol-ferric chloride colorimetric assay in barley and wheat accessions for water-extractable and total arabinoxylan

Arabinoxylan is an important hemicellulose potentially affecting wheat baking qualities, barley malt quality, and may impart prebiotic benefits. Water-extractable (WEAX) and total arabinoxylan (TOAX) were characterized in 204 wheat and barley accessions, respectively, using an orcinol-ferric chloride assay. WEAX measurement accuracy was optimum when glucose concentration was greater than 12–13 times pentose concentration. The modified method removed enough excess glucose spectral absorbance to make the corrected and uncorrected lines different, but still significantly correlated (p = 0.009, R = 0.94). Mean WEAX (expressed as percentage WEAX of TOAX) for the wheat accessions was 15.90%, ranging from 8.82% to 24.87%, and for barley accessions WEAX the mean was 7.10%, and ranged from 2.98% to 13.86%. Conclusions are 1) the assay is useful for a breeding program because of its semi-high throughput design for the simultaneous analysis of 16 (WEAX) to 40 (TOAX) barley lines or 24 (WEAX) to 40 (TOAX) wheat lines, 2) the trichromatic measurement reduces the impact of glucose, and 3) the broad range of WEAX measured showed that barley and wheat accessions vary in their extractable and unextractable components.     

The agronomic performance and nutritional content of oat and barley varieties grown in a northern maritime environment depends on variety and growing conditions

Warmer temperatures and increasing interest in high provenance food and drink products are creating new opportunities for cereal growing in northern Europe. Nevertheless, cultivation of oats and barley in these areas for malting and milling remains a challenge, primarily because of the weather, and there are few reports of their nutritional content from this region. In this study, trials in Orkney compared agronomic characteristics and nutritional content of recommended UK oat and barley varieties with Scandinavian varieties over three years. For a subset of varieties, nutritional content was compared with samples cultivated in more southerly sites. For Orkney, barley was considered a more suitable crop than oats because varieties matured earlier. In both crops, Scandinavian varieties matured earlier than UK varieties and some produced comparable yields. The range of values for macronutrients and minerals in oats and barley in Orkney were similar to those reported previously for other locations, but there were some significant differences attributable to variety and year. Compared with grain samples from more southerly locations, oats in Orkney had a significantly lower β-glucan and higher sodium content. The lower β-glucan may have resulted from higher rainfall and lower temperatures during the months of grain filling and maturation.     

Effect of altered carbohydrate traits in hulless barley (Hordeum vulgare L.) on nutrient profiles and availability and nitrogen to energy synchronization

Four hulless barley cultivars (zero-amylose waxy, CDC Fibar; 5%-amylose waxy, CDC Rattan; normal-amylose, CDC McGwire and high-amylose, HB08302) were developed at the Crop Development Centre, University of Saskatchewan, with differences in carbohydrates traits on the basis of amylose (1–20% DM) and β-glucan (5–10% DM) content. The objectives of this research were to determine: 1) the effect of the alteration of these carbohydrate traits in hulless barley on nutrient availability in ruminants, and 2) carbohydrate structure conformation on hourly effective rumen degradation ratio, in comparison with hulled barley-CDC Copeland. Among the hulless barley lines, HB08302 was greater (P < 0.05) in rumen bypass crude protein (BCP: 52.1% CP), effective degradability (ED) of neutral detergent fibre (EDNDF: 74 g/kg DM) than these in CDC Fibar, whereas CDC Fibar showed greater (P < 0.05) effective degradable crude protein (EDCP: 90 g/kg DM) than the other hulless barley lines. Compared with hulless barley, the hulled CDC Copeland showed relatively greater (P < 0.05) BNDF (62.5% NDF or 98 g/kg DM), starch degradation rate (K_d: 17.4%/h) and EDST (75.9% ST) but reduced (P < 0.05) rumen undegradable protein (RUP: 49 g/kg DM). With the respect to hourly effective degradation (ED) ratios, hulless barley cultivars exhibited relatively optimal hourly ED ratios, ranging from 23 to 34 g N/kg CHO, higher than the hulled barley. Among hulless barley cultivars, CDC Fibar and Rattan had the highest (P < 0.05) ratios (34 g N/kg CHO), while the ratio for CDC McGwire (23 g N/kg CHO) was the lowest (P > 0.05). In conclusion, hulless barley lines with altered carbohydrate traits have the potential to increase rumen nutrient availability to ruminants. The altered carbohydrate conformation of hulless barley affected hourly ED ratios, thus affecting rumen nitrogen to energy synchronization.     

Extractability and chromatographic separation of rye (Secale cereale L.) flour proteins

A size exclusion – high performance liquid chromatography (SE-HPLC) method originally developed for separating wheat, barley or rice proteins was applied to study the extractability and molecular weight (MW) distribution of rye flour proteins. These were extracted with 50 mmol/l sodium phosphate buffer (pH 6.8) containing 2.0% (w/v) sodium dodecyl sulfate (SDS) and, optionally, 1.0% (w/v) dithiothreitol (DTT). About 95% of the proteins were extracted in buffer containing 2.0% SDS. Addition of 1.0% DTT to such buffer increased the protein extractability to 100%, indicating that rye flour contains some proteins cross-linked by disulfide (SS) bonds. The SE-HPLC profiles revealed that rye flour contains SS-linked HMW-secalins and 75 k γ-secalins which elute in specific peaks. Upon reduction, these SS-linked protein aggregates dissociate and some entrapped albumins, globulins and/or ω-secalins are released. Rye flour albumins and globulins elute over the entire SE-HPLC profile. In contrast, the monomeric ω-secalins and 40 k γ-secalins are detected in specific well resolved SE-HPLC peaks. The applied fast and reproducible method can be used to characterise and quantify rye flour proteins and to determine changes as a result of processing.     

Detect unique molecular structure associated with physiochemical properties in CDC varieties of oat grain with unique nutrient traits [Feed Type vs. Milling Type] in comparison with barley grain using advanced molecular spectroscopy as a non-destructive biological tool

The objective of this study was to determinate grain unique protein inherent molecular structure that are related physiochemical and nutrient profiles in CDC developed oat varieties [CDC Nasser (Feed Type) and CDC Seabiscuit (Milling Type)] grown in cool climate condition in western Canada in comparison with conventional barley variety of CDC Meredith as a control using advanced molecular spectroscopy. Multivariate analyses, including an agglomerative hierarchical cluster analysis (CLA) and principal component analysis (PCA), were performed to identify protein molecular structural differences among the grains. The results revealed that CDC Seabiscuit contained greater (P < 0.05) protein structural Amide I and II than CDC Nasser and CDC Meredith, while the greater (P < 0.005) structural Amide I to II area and height ratios was detected in CDC Meredith. New oat grains had greater (P < 0.05) β-sheet height than barley grains, however, there was no difference in α-helix to β-sheet ratio values among the varieties. In conclusion, CDC Nasser and CDC Meredith had no difference in protein molecular structural features, while CDC Seabiscuit contains different protein structural characteristics as compared to CDC Meredith grain. The molecular structure features are highly associated with physiochemical and nutrient profiles in grains, which indicate that it also affect nutrient utilization and availability.     

Spatial distribution of functional components in the starchy endosperm of wheat grains

The starchy endosperm of the mature wheat grain comprises three major cell types, namely sub-aleurone cells, prismatic cells and central cells, which differ in their contents of functional components: gluten proteins, starch, cell wall polysaccharides (dietary fibre) and lipids. Gradients are established during grain development but may be modified during grain maturation and are affected by plant nutrition, particularly nitrogen application, and environmental factors. Although the molecular controls of their formation are unknown, the high content of protein and low content of starch of sub-aleurone cells, compared to the other starchy endosperm cells types, may result from differences in developmental programming related to the cells having a separate origin (from anticlinal division of the aleurone cells). The gradients within the grain may be reflected in differences in the compositions of mill streams, particularly those streams enriched in the central and outer cells of the starchy endosperm, respectively, allowing the production of specialist flours for specific end uses.     

Polymerisation of gluten proteins in developing wheat grain as affected by desiccation

The breadmaking quality of wheat is affected by the composition of gluten proteins and the polymerisation of subunits that are synthesised and accumulated in developing wheat grain. The biological mechanisms and time course of these events during grain development are documented, but not widely confirmed. Therefore, the aim of this study was to monitor the accumulation of gluten protein subunits and the size distribution of protein aggregates during grain development. The effect of desiccation on the polymerisation of gluten proteins and the functional properties of gluten were also studied. The results showed that the size of glutenin polymers remained consistently low until yellow ripeness (YR), while it increased during grain desiccation after YR. Hence, this polymerisation process was presumed to be initiated by desiccation. A similar polymerisation event was also observed when premature grains were dried artificially. The composition of gluten proteins, the ratios of glutenin to gliadin and high molecular weight-glutenin subunits to low molecular weight-glutenin subunits, in premature grain after artificial desiccation showed close association with the size of glutenin polymers in artificially dried grain. Functional properties of gluten in these samples were also associated with polymer size after artificial desiccation.     

Pilot scale production of a non-immunogenic soluble gluten by wheat flour transamidation with applications in food processing for celiac-susceptible people

Celiac disease (CD) is an immune-mediated disease triggered by wheat gluten and related prolamins. A lifelong gluten-free (GF) diet is mandatory to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase of gluten was effective in suppressing the gliadin-specific inflammatory response in CD patients without influencing the main technological properties of wheat flour or semolina. In this study, we produced on a pilot scale a soluble form of transamidated gluten (soluble protein fraction, spf), characterised by a high protein content (88 mg/ml), while native gluten was dramatically reduced (32 ± 2 ppm; R5-ELISA). Using HLA-DQ8 transgenic mice as a CD model, we found suppression of interferon-γ secretion in gliadin-specific CD4⁺ T cells challenged with spf-primed dendritic cells. In terms of functional properties, spf showed both solubility and emulsifying activity values within the range of commercial soluble glutens. Notably, dough prepared by mixing rice flour with spf could leaven. After baking, blended rice bread had a higher specific volume (2.9 ± 0.1) than control rice bread (2.0 ± 0.1) and acquired wheat-like sensory features. Taken together, our results highlighted the technological value of transamidated soluble gluten to improve both nutritional and sensory parameters of GF food.     

Genotypic variations in zinc accumulation and bioaccessibility among wheat (Triticum aestivum L.) genotypes under two different field conditions

Zinc (Zn) is an essential micronutrient for human health. Breeding zinc-rich crop genotypes is considered as potential solution to Zn deficiency. In this study, variation of Zn uptake, accumulation, distribution and the estimated bioaccessibility among 30 wheat genotypes across two locations were investigated with field experiments. A significant difference in grain Zn concentrations occurred across the two locations, with the corresponding values of 55.24 and 57.14 mg kg⁻¹. Grain Zn concentration was significantly and positively correlated with grain Mn concentration (0.698**, 0.617** for two locations). The estimated grain Zn bioaccessibility also showed a significant difference, a trend similar to grain Zn concentrations but with lower values (13.87 and 13.49 mg Zn d⁻¹ for two locations). These results indicate that the interaction of locations * genotypes may play an important role in grain Zn concentrations and Zn bioaccessibility.     

Combined mutations in five wheat STARCH BRANCHING ENZYME II genes improve resistant starch but affect grain yield and bread-making quality

Increases in the proportion of amylose in the starch of wheat grains result in higher levels of resistant starch, a fermentable dietary fiber associated with human health benefits. The objective of this study was to assess the effect of combined mutations in five STARCH BRANCHING ENZYME II (SBEII) genes on starch composition, grain yield and bread-making quality in two hexaploid wheat varieties. Significantly higher amylose (∼60%) and resistant starch content (10-fold) was detected in the SBEII mutants than in the wild-type controls. Mutant lines showed a significant decrease in total starch (6%), kernel weight (3%) and total grain yield (6%). Effects of the mutations in bread-making quality included increases in grain hardness, starch damage, water absorption and flour protein content; and reductions in flour extraction, farinograph development and stability times, starch viscosity, and loaf volume. Several traits showed significant interactions between genotypes, varieties, and environments, suggesting that some of the negative impacts of the combined SBEII mutations can be ameliorated by adequate selection of genetic background and growing location. The deployment of wheat varieties with increased resistant starch will likely require economic incentives to compensate growers and millers for the significant reductions detected in grain and flour yields.     

Responses of glutamine synthetase activity and gene expression to nitrogen levels in winter wheat cultivars with different grain protein content

Glutamine synthetase (GS) plays a central role in plant nitrogen (N) metabolism, which improves crops grain protein content. A pot experiment in field condition was carried out to evaluate GS expression and activity, and grain protein content in high (Wanmai16) and low grain protein (Loumai24) wheat cultivars under two N levels (0.05 and 0.15 g N kg⁻¹ soil). High nitrogen (HN) resulted in significant increases in GS1 and GS2 expression at 10 days after anthesis (DAA), and higher GS activity during the entire grain filling stage. HN also significantly increased yield, grain protein content and protein fraction (except for glutenin of Luomai24) in two wheat cultivars, which indicated that it increased grain yield and protein content by improving nitrogen metabolism. Wanmai16 showed higher grain protein content, gliadin and glutenin content, and had higher expression level of GS2 both in flag leaves and grains at early grain filling stage. However, Luomai24 had greater yield and higher expression level of GS1. The difference expression of GS2 and GS1 genes indicates they had various contributions to the accumulation of protein and starch in wheat grains, respectively. The results suggest that GS2 would be serving as a potential breeding target for improving wheat quality.     

Modification of physicochemical properties and in vitro digestibility of wheat flour through superheated steam processing

Native and moistened wheat flours (moisture contents were 13.5 and 27.0%, respectively) were treated with superheated steam (SS) at different temperatures (140 and 170 °C) and times (1, 2 and 4 min). Their physicochemical and digestive properties were analyzed. For native flour, SS treatment altered the starch molecular structure and behavior slightly. While for moistened flour, crystalline degree, gelatinization enthalpy, amylose leaching (AML) and falling number significantly decreased, but thermal transition temperatures increased with the rise of treating severity. Clumping of starch granules, aggregation of proteins and formation of amylose-lipid complexes occurred in both native and moistened flours. Broader pasting temperature ranges and higher viscosities were found on SS-modified flours. Additionally, SS treatment on moistened flours increased resistant and slowly digestible starch contents. In general, SS treatment induced changes in starch molecular structure and reactions among flour components leading to more stable structures, thus affecting their pasting behavior, thermal properties and in vitro digestion.     

Factors that affect high voltage atmospheric cold plasma treatment efficacy on wet distillers’ grains: Shelf-life and nutrient composition

The increase in ethanol production continues to drive the availability of distillers' grains, which are used as an animal feed for cattle, swine and poultry. In its wet state this co-product of the ethanol production process must be fed or treated before spoiling and reaching its shelf-life, only 3–5 days after production. Novel treatment technologies, such as high voltage atmospheric cold plasma (HVACP), continue to show promise as a method to increase the shelf-life of many products such as contaminated animal feed. This study investigated the parameters grain depth, amount of sample and exposure time, for effective treatment of distillers’ grains to increase shelf-life using HVACP. This work evaluates the different combinations of treatments to determine, which parameters are critical for reduction in microbial growth and increase shelf-life. The efficacy of the reactive gas species (RGS) produced by HVACP, at 70 kV for up to 360 s, moving through the grain mass of distillers wet grains (DWG) was evaluated by measuring microbial load (log reduction), moisture content and pH after 0, 14 and 28 days of storage at 25 °C at grain depths of 1, 2.5 and 4.5 cm. Results showed a higher log reduction at the 1 cm depth than at 2.5 and 4.5 cm for measurements after the initial treatment. Stored samples continued to show reductions, however the greater depths had higher log reductions than the surface. Nutrient composition and amino acid profiles were also analyzed, showing no significant difference between treated and untreated samples over the storage period.     

Feasibility analysis of NIR for detecting sweet corn seeds vigor

This paper explores the feasibility of particle-based detection and grading of seed vigor based on a self-built seed single-granulation device using near infrared spectroscopy (NIRS). Sweet corn with uniform kernel size was used for this study. The seed samples were divided into three types, they were normal seeds, artificially aged seeds and heat-damaged seeds. A 2-part spectral acquisition of each seed were performed, one for the collection of seeds that fall into the detection zone within the separation pipe, another was on the static platform, whose collection was performed on 5 faces of each seed. Partial least squares discriminant analysis (PLS-DA) was used to classify the original data of the seeds. In the 2 parts, the discriminant results of the unprocessed normal seeds and the artificial accelerated aging seeds, the untreated normal seeds and the heat-damaged seeds showed that classification accuracy was higher than 98%. The research indicates that the spectral data of different positions of seeds can reflect their activity information, and it is feasible to detect and classify seeds in real time in the detection area of the separation pipeline.