Quantitative LC-MS proteoform profiling of intact wheat glutenin subunits

Methodology for the quantitative analysis of intact glutenin proteins by ESI-LC-MS was developed with 1 Da mass resolution, constituting the first published application of proteoform profiling to plant biology. Two parent lines and 28 F5 crosses were analyzed in two blocks, 14 months apart. Control sample data were used to align retention times and normalize abundance between sample sets. A total of 4622 observations of 347 distinct proteoforms between 17 899 and 88 744 Da were observed. Proteoform abundances spanned a 1000-fold range and were linear (r² > 0.990) with dilution. A novel method for the objective elimination of low intensity, noise-dominated data using replicate variability within the dataset is presented. Two abundant PTMs were detected; one known but uncharacterized Bx and Dy high-molecular-weight glutenin subunit (HMWGS) PTM and the other in 24 low molecular weight proteoform pairs. Finally, 16 abundant proteoforms were detected in progeny but not in either parent. This application should increase the statistical power of correlations between gluten complement and functional data and drive the detection of novel PTMs that may indicate differential regulation of the cellular processes related to quality.     

Variation in physicochemical properties and nutritional quality in chalky mutants derived from an indica rice

The physicochemical and nutritional quality of four chalky mutants (JM1-4) rice and their parent 93-11 were investigated. JM1 and JM4 are white-belly mutants, and JM2 is white-core mutant, while JM3 is floury mutant. JM2 is also a high amylose mutant with AAC of 29%. JM3 had the lipid content nearly three times of the parent, so it is also a high lipid mutant. JM1 had similar pasting viscosities with the parent, while JM2 and JM3 had smaller breakdown and setback than 93-11. JM2 had lower enthalpy of gelatinization, but higher enthalpy of retrogradation, so it had highest retrogradation percentage. JM3 had the highest accumulation of amino acids, fatty acids, and some major mineral elements such as P, K, Mg and Ca. The amino acids and fatty acids had negative correlation with PV, HPV and CPV. The pleiotropic effects caused by the mutations on starch and lipid metabolism deserve further investigation.     

GlutoPeak parameters of whole wheat flours for gluten quality evaluation in soft wheat breeding programs

In soft wheat breeding programs, the gluten strength of flours from specific genotypes is determined by various chemical and rheological tests. Based on such tests, the experimental wheat lines with very weak flour gluten are typically selected for the production of soft-dough biscuits, while the lines with medium gluten strength and extensibility are reserved for hard-dough biscuits. Often, the genotypes having high gluten strength are removed from such breeding programs. In the present study, the usability of the GlutoPeak tester on whole wheat flour samples was investigated for assessing the gluten strength of soft wheat breeding materials. In the study, 25 soft wheat genotypes, grown in seven locations for three years, were categorized by commonly used gluten-quality-related parameters. Based on the results of the study GlutoPeak whole wheat flour PMT values ranging from 30.0 to 50.0 s and AM values from 15.0 to 20.0 GPU were found to be suitable for soft-dough biscuit products, whereas the values between 40.0 and 60.0 s and 20.0 and 23.0 GPU were appropriate for hard-dough biscuit products. The genotypes exhibiting AM values > 24.0 GPU and PMT values > 60.0 s were judged to have too-strong gluten, and thus eliminated from the breeding program. The gluten aggregation energy (AGGEN), and the torque after the maximum torque (PM) values were only useful and applicable to flours for soft-dough products. The maximum torque (BEM) values were not effective in discriminating against the genotypes. The results of this study demonstrated that the GlutoPeak whole wheat PMT and AM parameters can be recommended as quick and accurate parameters especially for early generation screening with small-scale tests in soft wheat improvement programs.     

Effect of curdlan on the quality of frozen-cooked noodles during frozen storage

Frozen-cooked noodles (FCN) and its components undergo quality changes during frozen storage, such as reduced textural and cooking qualities, weakened gluten network, and damaged starch properties; thus, storage condition is a critical factor affecting the final quality of FCN. In this study, in view of the thermoirreversible high-strength gel property (at ≥ 80 °C) of curdlan, strong hydrophilicity, and freeze-thawed stability of high-strength gel prepared from curdlan powder, the effect of curdlan on the quality of FCN during frozen storage was first evaluated. The results showed that curdlan was effective in reducing cooking loss, enhancing water absorption, and improving textural properties of FCN; the improving effect presented a trend of first increasing and then decreasing with the amount of increasing curdlan (0.1%–0.9%); and the addition of 0.5% curdlan was most effective in improving the quality of FCN. Thermal gravimetric analysis indicated that curdlan enhanced thermal stability of FCN, implying curdlan could strengthen the gluten network. Meanwhile, structural observations revealed that, during frozen storage, FCN with added curdlan exhibited a more continuous and compact gluten network accompanied with more uniform and smaller ice crystals. Thus, curdlan is desirable to be used as a novel gum in FCN to provide specific functionality and minimize the negative effect of frozen storage. This study provides new insights into the quality improvement of FCN and further expands the application potential of curdlan in food industry.     

Genetic responses in milling,flour quality,and wheat sensitivity traits to grain yield improvement in U.S. hard winter wheat

A rising global population necessitates continued genetic improvement of wheat (Triticum spp.), but not without monitoring of unintended consequences to processors and consumers. Our objectives were to re-establish trends of genetic progress in agronomic and milling traits using a generational meter stick as the timeline rather than cultivar release date, and to measure correlated responses in flour quality and human wheat-sensitivity indicators. Grain yield and kernel size showed stepwise increases over cycles, whereas wheat protein content decreased by 1.1 g/100 g. Reduced protein content, however, did not result in lower dough strength pertinent to bread baking. A novel method of directly testing gluten elasticity via the compression-recovery test indicated a general increase in gluten strength, whereas the ratio of total polymeric to total monomeric proteins remained stable. Also showing no change with genetic progress in yield were flour levels of gluten epitopes within the key immunotoxic 33-mer peptide. The oligosaccharide fructan, present in milled and wholemeal flours, increased with increasing grain yield potential. While yield improvement in U.S. bread wheat was not accompanied by a decline in gluten strength or systematic shift in a key wheat sensitivity parameter, the unanticipated rise in total fructans does implicate potentially new dietary concerns.     

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.     

Impact of ethanol,succinic acid,and the combination thereof at levels produced during sponge fermentation on hard wheat,soft wheat,and durum wheat farinograph rheology

The sponge and dough mixing process is one of the most common in the world, yet the mechanistic understanding of this process has yet to be sufficiently explored. In this study, aqueous solutions of ethanol, succinic acid, and their combination were prepared at concentrations intended to replicate fermentation times of 3, 4 and 6 h. These solutions were added to a farinograph mixer to make dough using hard wheat, soft wheat, and durum wheat flour. The results indicate that these yeast metabolites (ethanol, succinic acid) impact the mixing resistance, peak mixing value, and dough mixing stability in each of the flour types, likely primarily affected by the ratios of gliadin to glutenin and LMW glutenin in each flour type. Results suggest a stabilizing non-covalent interaction imparted by gliadin at peak mixing time, a stabilizing effect of HMW glutenin during break down, and synergistic effects of ethanol and succinic acid that leads to a faster rate of breakdown in later stages of mixing. It also suggests an increase in mixing resistance when acidulants are added to durum wheat dough. Taken together, this study adds new insights on the sponge and dough mixing process in a way that has not previously been conducted.     

Characterization and comparison of predominant aroma compounds in microwave-treated wheat germ and evaluation of microwave radiation on stability

The present study was performed to evaluate the effects of microwave (MW) output power and treatment time on moisture content, lipase and lipoxygenase activities as well as colour changes of wheat germ (WG). In addition, the key aroma compounds in different MW-power-treated WG, which is of importance to the flavour of WG products, were also investigated. The obtained results showed that MW treatment maintained the inherent colour of WG and significantly reduced the moisture content (maximum reduction of 95%) and the activities of lipase and lipoxygenase (maximum reduction of 65% and 99%, respectively). In terms of aroma compounds, with the increase of the MW output power, the content of esters, alkanes, alcohols and acids decreased, while the content of heterocyclic compounds, nitrogen-containing compounds, aldehydes and ketones increased, providing more compounds with roasted flavour and less volatiles with grass-like flavour. Therefore, MW treatment was an effective stabilization method for WG utilization.     

Breeding and biotechnological efforts in Jatropha curcas L. for sustainable yields

Jatropha is a non-edible, important bioenergy plant, which can grow in marginalized land. The seeds possess about 36% oil and this would be converted into biodiesel or biojet-fuel. Jatropha provides an option for sustainable feed and fuel production due to its inherent qualities including hardy nature, drought tolerance and surviving with limited amount of water, tolerance to unfavorable conditions and excessive moisture. However, heterozygosity, low productivity and poor understanding of its genome are the major impediments to elite line development. Further, classical breeding and advanced technological investments remain limited owing to long juvenile phase and breeding cycles. Scientific technologies that lead to identification of elite genotypes and development of high yielding elite Jatropha lines and effective methods of detoxification of seeds needs immediate priority. Efficient tissue culture system, doubled haploids (DH) and genomic tools are increasingly made available to improve the seed yield and its oil quantity through the development of geminivirus disease resistant lines. The application of advanced, sequencing technologies has presented a repertoire of genomic information for this important yet orphan crop. In the present investigation, we highlight the achievements made in Jatropha towards development of high yielding, virus resistant elite lines and hybrids with yield potential ranging from 3 to 5 tons per hectare in a year, which is a first ever report in the world. We also developed potential biotechnological tools such as genetic transformation, genome editing and next generation genomics tools including linkage maps and QTLs for accelerating breeding efforts through marker assisted selection. Because of our concerted and continuous efforts during the past 15 years, we have overcome all the obstacles and developed high yielding, disease resistant hybrids/lines, advanced cultivation technologies with thorough knowledge on Agronomy of the Jatropha crop. The Jatropha cultivation technology developed for the first time in the world, could open up new avenues for higher yield productivity of commercial viability.     

Effects of wheat tempering and stone rotational speed on particle size,dough rheology and bread characteristics for a stone-milled weak flour

The poor technological performance of weak wheat flours means that they are usually considered difficult to be transformed into satisfactory bread. During milling, there are several settings that can affect flour characteristics. In this study, we tested two operative parameters that have the potential to affect flour quality – stone rotational speed and wheat tempering. Tempering moistures were set at 11%, 13%, 15%, and 17%, while stone rotational speeds were set at 173, 260, and 346 rpm. Both factors were found to affect operative milling parameters, notably flour yield, process productivity and specific energy consumption. Grain moisture had a significant effect on both dough rheology and bread characteristics (dough stability, tenacity, and extensibility). Dough stability was maximum at 13% moisture. Dough tenacity decreased as moisture increased, while extensibility increased as moisture increased. Bread specific volume and crumb specific volume were improved at 13% and 15% moisture. In conclusion, wheat tempering can be used to improve the potential of a weak flour and bread characteristics, while stone rotational speed affects operative parameters and white flour yield.     

Biofortification of sweet corn hybrids for provitamin-A,lysine and tryptophan using molecular breeding

Traditional sweet corn is poor in provitamin-A, lysine and tryptophan, deficiency of which causes serious health problems. Here, parental lines of two shrunken2 (sh2) -based sweet corn hybrids viz., ASKH-1 and ASKH-2 were targeted for introgression of crtRB1 and opaque2 (o2) genes through marker-assisted backcross breeding. Gene-based markers; umc1066 (SSR) and 3′TE-InDel were utilized for foreground selection of o2 and crtRB1, respectively in BC₁F₁, BC₂F₁ and BC₂F₂ generations. Background selection employing 102–113 polymorphic SSRs led to >90% recovery of recurrent parent genome. Reconstituted hybrids recorded high mean provitamin-A (18.98 μg/g) with a maximum of 7.7-fold increase over original hybrids (3.12 μg/g). High mean lysine (0.39%) and tryptophan (0.10%) with an average enhancement of 1.71- and 1.79-fold, respectively was recorded among reconstituted hybrids over original versions (lysine: 0.23%, tryptophan: 0.06%). Improved hybrids exhibited high phenotypic resemblance with their original hybrids. The average cob yield (11.82 t/ha) and brix (17.66%) of improved hybrids was at par with their original versions (cob yield: 11.27 t/ha, brix: 17.04%). These biofortified sweet corn hybrids rich in provitamin-A, lysine and tryptophan hold immense significance as multinutrient-rich balanced food. This is the first report to stack sh2, crtRB1 and o2 genes to improve nutritional quality in sweet corn.     

New angiotensin-converting enzyme inhibitory peptide from Coix prolamin and its influence on the gene expression of renin-angiotensin system in vein endothelial cells

Coix seed, which is a traditional Chinese medicine, has been used to treat hypertension for thousands of years. It has been shown that Coix prolamin peptides display high levels of angiotensin I converting enzyme (ACE) inhibitory activity. Hence, we purified the ACE inhibitory peptides from Coix prolamin hydrolysates and evaluated the influence of the most potent peptide on the renin-angiotensin system (RAS) genes expression in human umbilical vein endothelial cells (HUVECs). In this study, Coix prolamin peptides were sequentially separated by ultrafiltration, ion exchange chromatography, gel filtration chromatography and RP-HPLC, while the peptide structure was analyzed by mass spectrometry. Next, in silico proteolysis, pharmacophore and molecular docking were further applied to screen and optimize the structure of peptides. Finally, a novel ACE inhibitory peptide VDMF was obtained, in which its influence on the gene expression of RAS signaling pathway in AngⅡ-injury HUVECs was evaluated by quantitative real-time PCR. VDMF significantly down-regulated ACE, AngII type 1 receptor (AT1R) and ACE2 mRNA expression in comparation with model group, while up-regulating Mas gene expression. Hence, we obtained a novel antihypertensive candidate that was derived from the Coix peptides, which could involve a multi-modulation mechanism that regulates blood pressure.     

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.     

Camelina sativa,a short gestation oilseed crop with biofuel potential: Opportunities for Indian scenario

Camelina is an oilseed crop which is being commercially produced globally as feedstock for biodiesel. Being a non-edible oil bearing low input crop owing to its low fertilizer and water requirement, fits well for biofuel production. In India, targets for biofuel blending has been set by New Biofuel Policy-2018 and to meet these targets efforts are being made to harness the potential of available feedstock in the country. Among these feedstock, contribution of short gestation oilseed bearing crop has been very important. Camelina has been introduced in India during 2009–10 as experimental crop by DIBER, DRDO. Since then various efforts have made to standardize the production technology of this crop under various agro-climatic regions of the country, crop improvement, oil quality analysis and development of high energy by-products. Camelina has various advantages to offer for Indian biofuel sector. This paper reviews the potential of this crop for Indian Biofuel scenario.     

Enzymatic degradation of FODMAPS via application of β-fructofuranosidases and α-galactosidases- A fundamental study

Cereals and pulses often contribute to the intake of Fermentable Oligo-, Di-, Monosaccharides, and Polyols (FODMAPs) due to high amounts of fructans or galactooligosaccharides (GOS). FODMAPs can trigger symptoms of Irritable Bowel Syndrome (IBS) and therefore, the development of foods and beverages with a lower FODMAP-content are favourable for IBS patients. Enzyme technology is a promising tool to reduce the FODMAP-content in foods and to maintain product quality. This fundamental study investigates the efficiency of invertase, inulinase, and α-galactosidase as potential food additives to reduce the total FODMAP content of food ingredients. Extracts of high FODMAP ingredients, such as wheat and lentil, and standard solutions of various fructans and GOS were incubated with invertase, inulinase and α-galactosidase for 1 h and 2 h. Contents of oligosaccharides before and after treatment and related IBS-triggering reaction products were quantified using ion chromatography. Inulinase showed a high degradation yield (over 90% of degradation) for both GOS and fructans. For invertase only low degradation yields were measured. α-Galactosidase showed the highest efficiency in decomposing GOS (100% of degradation) and led to non-IBS triggering degradation products. This indicates a high potential for a combined inulinase/α-galactosidase treatment for products containing both fructans and GOS.     

Localization of amino acids in germinated rice grain: Gamma-aminobutyric acid and essential amino acids production approach

Germinated rice is popularly consumed for its high gamma-aminobutyric acid (GABA) and bioactive compounds. Supporting information on how to germinate rice with high GABA content and essential amino acids is lacking. White and colored rough rice were germinated for 0, 24, 48, 72, 96, 120 and 144 h. GABA, GABA substrates and essential amino acids were also investigated using GC-MS and MALDI-MSI. GABA was more concentrated after germination and shifted into the coleoptile. High correlation was recorded between germination time and GABA (0.79–0.83, p ≤ 0.01) and glutamic acid (0.88–0.89, p ≤ 0.01). Highest rates of GABA and glutamic production were observed within 48 h of germination in both rice varieties (26.12 and 34.28 mg/100g) and reached maximum value at 96 h (31.36 and 38.75 mg/100g). Colored rice germination showed higher GABA, GABA substrates and deficient amino acids than white rice. GABA and essential amino acids drastically increased after germination, supporting the consumption of germinated rice as a functional food.     

Benzoxazinoid and alkylresorcinol content,and their antioxidant potential,in a grain of spring and winter wheat cultivated under different production systems

The health-promoting properties of the grains of common wheat (Triticum aestivum L.) are associated with the presence of unique phytochemicals. This study determines the profile of alkylresorcinols (ARs) and benzoxazinoids (BXs) in T. aestivum spring and winter cultivars grown in Poland under the two different production systems: conventional and organic. Wheat grain extracts were subjected to qualitative and quantitative UPLC-UV-MS/MS analyses. The ARs profile consisted of five 5-n-alkylresorcinol derivatives, among which 5-n-heneicosylresorcinol (C21:0) and 5-n-nonadecanylresorcinol (C19:0) predominated; while six different BXs were determined in hydrothermally treated grains. Our research shows significant differences in the contents of ARs and BXs among wheat cultivars, as well as the two production systems used. Organically grown varieties had their total contents of ARs and BXs significantly higher than those grown conventionally. Another aim of the study was to determine the antioxidant capacity of alkylresorcinol extracts from tested wheat cultivars. The quantitative TLC-DPPH• method for determining the antiradical capacity of wheat ARs extracts was developed. We observed a positive relationship between the free radical scavenging activity of extracts and the total amount of ARs. The biological activity research is important for developing value-added wheat cultivars, having an improved profile and composition of nutritional substances.     

Characterization and bioactivities of young rice protein hydrolysates

Immature rice was reported to contain higher quantities of bioactive compounds than mature rice. Young rice protein is easy to digest and has hypoallergenic potential, with protein content of 7.2–11.5% compared to rice bran at 9.8%. Few studies have reported on bioactivities and characterization of young rice proteins and their hydrolysates. Bioactivities of native protein and protein hydrolysates of two rice varieties (white rice and colored rice) were characterized and investigated for four development stages (flowery, milky, dough, and mature). Degree of hydrolysis of young rice protein was considerably higher than at the mature stage. Highest DPPH and iron chelating activity were found in alcalase® protein hydrolysate during the flowery-to-milky stage. Iron chelating activity was constant in all development stages because of the low polar amino acid content in rice. The ACE activity of alcalase® protein hydrolysate was higher than native protein at the same development stage, as observed in the milky and dough stages. Inhibitory activity of young rice hydrolysate HepG2 cells was concentration-dependent and not correlated with protein molecular size.     

Variability and cluster analysis of arabinoxylan content and its molecular profile in crossed wheat lines

Non-starch polysaccharides (NSP) in cereals, after starch and gluten proteins, determine the technological and nutritional properties of flour, dough and end product. From NSPs, the arabinoxylans are the most important components studied in wheat nowadays. The novelty of our study is the investigation of the variability and clustering ability of the wheat lines based on different quantitative and qualitative traits of arabinoxylans. The quantitative properties, the total and the water-extractable arabinoxylan content of wheat flours were measured separately by gas chromatography. The qualitative property referring to the structure of the molecule was the molecular weight distribution measured by size exclusion liquid chromatography. According to the results the variability of arabinoxylan properties in the breeding lines varied between wider ranges then the values measured for the parental varieties. The correlation between the quantitative parameters, and a trend between quantitative and qualitative parameters were described. During the cluster analysis, the parents were well separated into different groups. The parameters that played an important role in the clustering were the ratio of the probability of occurrence of molecular size ranges along with the quantitative traits of AX. The selected samples can be used for further targeted breeding while the methodology was used in this work can be suitable for selecting lines/varieties for special food or other industrial applications.     

Variation in avenanthramide content in spring oat over multiple environments

Avenanthramides (AVNs), a group of phytochemicals which are unique to oats, provide health benefits through antioxidant activity and other bioactivities. In this study, we explored genotype-by-environment interactions and heritability for AVN concentrations in oats. Avenanthramide concentrations were quantified for 100 breeding lines and cultivars at three locations over two years. While year and environment had an influence on AVN concentrations, with the influence of year being more apparent than that of environment within a year, genotype had the largest impact on AVN concentration. All three major AVNs were found to be heritable. Two methods of calculating heritability on a line mean basis were used. The statistical method yielded heritability estimates of 0.34, 0.39, and 0.41 for AVN 2c, AVN 2p, and AVN 2f, respectively. By comparison the traditional plant breeding method yielded heritability estimates of 0.82, 0.88, and 0.89 for AVN 2c, AVN 2p, and AVN 2f, respectively, indicating that expected gains will be dependent on the scale of the breeding program, the number of target environments, and climate variability. The estimated heritabilities and the 11-fold range in AVN concentrations in the 100 genotypes studied provided evidence that variability for AVN concentration should allow breeding progress for higher AVN concentration.