Transcriptome and metabolome analyses reveal regulatory networks associated with nutrition synthesis in sorghum seeds.

Cereal seeds are vital for food, feed, and agricultural sustainability because they store and provide essential nutrients to human and animal food and feed systems. Unraveling molecular processes in seed development is crucial for enhancing cereal grain yield and quality. We analyze spatiotemporal transcriptome and metabolome profiles during sorghum seed development in the inbred line 'BTx623'. Morphological and molecular analyses identify the key stages of seed maturation, specifying starch biosynthesis onset at 5 days post-anthesis (dpa) and protein at 10 dpa. Transcriptome profiling from 1 to 25 dpa reveal dynamic gene expression pathways, shifting from cellular growth and embryo development (1-5 dpa) to cell division, fatty acid biosynthesis (5-25 dpa), and seed storage compounds synthesis in the endosperm (5-25 dpa). Network analysis identifies 361 and 207 hub genes linked to starch and protein synthesis in the endosperm, respectively, which will help breeders enhance sorghum grain quality. The availability of this data in the sorghum reference genome line establishes a baseline for future studies as new pangenomes emerge, which will consider copy number and presence-absence variation in functional food traits.

Dry fractionation process operations in the production of protein concentrates: A review.

The market for plant proteins is expanding rapidly as the negative impacts of animal agriculture on the environment and resources become more evident. Plant proteins offer competitive advantages in production costs, energy requirements, and sustainability. Conventional plant-protein extraction is water and chemical-intensive, posing environmental concerns. Dry fractionation is an energy-efficient and environmentally friendly process for protein separation, preserving protein's native functionality. Cereals and pulses are excellent sources of plant proteins as they are widely grown worldwide. This paper provides a comprehensive review of the dry fractionation process utilized for different seeds to obtain protein-rich fractions with high purity and functionality. Pretreatments, such as dehulling and defatting, are known to enhance the protein separation efficiency. Factors, such as milling speed, mill classifier speed, feed rate, seed type, and hardness, were crucial for obtaining parent flour of desired particle size distribution during milling. The air classification or electrostatic separation settings are crucial in determining the quality of the separated protein. The cut point in air classification is targeted based on the starch granule size of the seed material. Optimization of these operations, applied to different pulses and seeds, led to higher yields of proteins with higher purity. Dual techniques, such as air classification and electrostatic separation, enhance protein purity. The yield of the protein concentrates can be increased by recycling the coarse fractions. Further research is necessary to improve the quality, purity, and yield of protein concentrates to enable more efficient use of plant proteins to meet global protein demands.

Performance of a Handheld MicroNIR Instrument for Determining Protein Levels in Sorghum Grain Samples.

Near infrared (NIR) spectroscopy is widely used for evaluating quality traits of cereal grains. For evaluating protein content of intact sorghum grains, parallel NIR calibrations were developed using an established benchtop instrumentation (Perten DA-7250) as a baseline to test the efficacy of an adaptive handheld instrument (VIAVI MicroNIR OnSite-W). Spectra were collected from 59 grain samples using both instruments at the same time. Cross-validated calibration models were validated with 33 test samples. The selected calibration model for DA-7250 with a coefficient of determination (R2) = 0.98 and a root mean square error of cross validation (RMSECV) = 0.41% predicted the protein content of a test set with R2 = 0.94, root mean square error of prediction (RMSEP) = 0.52% with a ratio of performance to deviation (RPD) of 4.13. The selected model for the MicroNIR with R2 = 0.95 and RMSECV = 0.62% predicted the protein content of the test set with R2 = 0.87, RMSEP = 0.76% with an RPD of 2.74. In comparison, the performance of the DA-7250 was better than the MicroNIR, however, the performance of the MicroNIR was also acceptable for screening intact sorghum grain protein levels. Therefore, the MicroNIR instrument may be used as a potential tool for screening sorghum samples where benchtop instruments are not appropriate such as for screening samples in the field or as a less expensive option compared with benchtop instruments.

Variations in Total Protein and Amino Acids in the Sequenced Sorghum Mutant Library.

Sorghum (Sorghum bicolor) is the fifth most important cereal crop worldwide; however, its utilization in food products can be limited due to reduced nutritional quality related to amino acid composition and protein digestibility in cooked products. Low essential amino acid levels and digestibility are influenced by the composition of the sorghum seed storage proteins, kafirins. In this study, we report a core collection of 206 sorghum mutant lines with altered seed storage proteins. Wet lab chemistry analysis was conducted to evaluate the total protein content and 23 amino acids, including 19 protein-bound and 4 non-protein amino acids. We identified mutant lines with diverse compositions of essential and non-essential amino acids. The highest total protein content in these lines was almost double that of the wild-type (BTx623). The mutants identified in this study can be used as a genetic resource to improve the sorghum grain quality and determine the molecular mechanisms underlying the biosynthesis of storage protein and starch in sorghum seeds.

Effect of Flour Particle Size on the Glycemic Index of Muffins Made from Whole Sorghum, Whole Corn, Brown Rice, Whole Wheat, or Refined Wheat Flours.

The unique properties of sorghum are increasingly being studied for potential health benefits, with one area of emphasis being the impact of sorghum consumption on mitigating type 2 diabetes. The glycemic index (GI) of muffins made from whole grain sorghum flour ground to three different particle sizes (fine, intermediate, coarse) was tested on eight healthy volunteers (ages 18-40) and compared to the glycemic index of whole grain corn, wheat, and rice flours produced using a similar product formula. Sorghum flour ground through a 0.5 mm screen ("fine") had an overall similar particle size to that of the brown rice flour ground using a 0.5 mm screen. The range of GI values was 32 to 56, with only the GI of intermediate milled sorghum flour being lower than that of corn, rice, or wheat (p < 0.05). The lowest glycemic index (32 +/- 17) was found when using sorghum flour with an intermediate particle size (167 +/- 4 µm). Muffins made using brown rice had the next lowest glycemic index at 37 +/- 17. All GI values calculated had large standard deviations, which is common for these types of studies. These results can assist in the product development process to advance the quality of healthy, gluten-free sorghum-based foods for consumers. Further research should investigate if these results can be duplicated and the possible reason for the lower GI of intermediate particle size sorghum flour.

Sorghum Flour Application in Bread: Technological Challenges and Opportunities.

Sorghum has a long history of use in the production of different types of bread. This review paper discusses different types of bread and factors that affect the physicochemical, technological, rheological, sensorial, and nutritional properties of different types of sorghum bread. The main types of bread are unleavened (roti and tortilla), flatbread with a pre-ferment (injera and kisra), gluten-free and sorghum bread with wheat. The quality of sorghum flour, dough, and bread can be improved by the addition of different ingredients and using novel and traditional methods. Furthermore, extrusion, high-pressure treatment, heat treatment, and ozonation, in combination with techniques such as fermentation, have been reported for increasing sorghum functionality.

Chemical Composition, Fatty Acid and Mineral Content of Food-Grade White, Red and Black Sorghum Varieties Grown in the Mediterranean Environment.

Grain sorghum (Sorghum bicolor) is a gluten-free cereal grown around the world and is a food staple in semi-arid and subtropical regions. Sorghum is a diverse crop with a range of pericarp colour including white, various shades of red, and black, all of which show health-promoting properties as they are rich sources of antioxidants such as polyphenols, carotenoids, as well as micro- and macro-nutrients. This work examined the grain composition of three sorghum varieties possessing a range of pericarp colours (white, red, and black) grown in the Mediterranean region. To determine the nutritional quality independent of the contributions of phenolics, mineral and fatty acid content and composition were measured. Minor differences in both protein and carbohydrate were observed among varieties, and a higher fibre content was found in both the red and black varieties. A higher amount of total saturated fats was found in the white variety, while the black variety had a lower amount of total unsaturated and polyunsaturated fats than either the white or red varieties. Oleic, linoleic, and palmitic were the primary fatty acids in all three analysed sorghum varieties. Significant differences in mineral content were found among the samples with a greater amount of Mg, K, Al, Mn, Fe, Ni, Zn, Pb and U in both red and black than the white sorghum variety. The results show that sorghum whole grain flour made from grain with varying pericarp colours contains unique nutritional properties.

Genetic control of source-sink relationships in grain sorghum.

MAIN CONCLUSION: QTL hotspots identified for selected source-sink-related traits provide the opportunity for pyramiding favorable alleles for improving sorghum productivity under diverse environments. A sorghum bi-parental mapping population was evaluated under six different environments at Hays and Manhattan, Kansas, USA, in 2016 and 2017, to identify genomic regions controlling source-sink relationships. The population consisted of 210 recombinant inbred lines developed from US elite post-flowering drought susceptible (RTx430) and a known post-flowering drought tolerant cultivar (SC35). Selected physiological traits related to source (effective quantum yield of photosystem II and chlorophyll index), sink (grain yield per panicle) and panicle neck diameter were recorded during grain filling. The results showed strong phenotypic and genotypic association between panicle neck diameter and grain yield per panicle during mid-grain filling and at maturity. Multiple QTL model revealed 5-12 including 2-5 major QTL for each trait. Among them 3, 7 and 8 QTL for quantum yield, panicle neck diameter and chlorophyll index, respectively, have not been identified previously in sorghum. Phenotypic variation explained by QTL identified across target traits ranged between 5.5 and 25.4%. Panicle neck diameter and grain yield per panicle were positively associated, indicating the possibility of targeting common co-localized QTL to improve both traits simultaneously through marker-assisted selection. Three major QTL hotspots, controlling multiple traits were identified on chromosome 1 (52.23-61.18 Mb), 2 (2.52-11.43 Mb) and 3 (1.32-3.95 Mb). The identified genomic regions and underlying candidate genes can be utilized in pyramiding favorable alleles for improving source-sink relationships in sorghum under diverse environments.

Modification of zein dough functionality using kafirin as a coprotein.

Kafirin, sorghum prolamin, was investigated as a coprotein for zein as visco-elastic masses and in starch-based model doughs. Regular kafirin and kafirins from waxy and high protein digestibility (HD) sorghum crosses were studied. HPLC revealed that waxy-HD kafirin was of smaller molecular size and low in ß-kafirin. It also had greater surface hydrophobicity. Kafirin addition to zein increased visco-elastic mass elasticity up to ≈50% stress-recovery, similar to wheat gluten. Waxy-HD kafirin gave the highest elasticity, possibly due to its hydrophobicity. Kafirin inclusion at 2:8 parts zein increased the tensile strength of model doughs. Maximum strength was, however, only 60% that of gluten-based dough. Kafirin from regular sorghum gave the highest strength, possibly because of greater disulphide-bonded polymerisation. Confocal laser scanning microscopy showed that zein-kafirin copolymers formed fairly linear fibrils in stretched doughs, indicating excellent compatibility between the proteins. Future research should establish how kafirin-zein copolymer performs in non-wheat flour products.

Effect of Tempering Conditions on White Sorghum Milling, Flour, and Bread Properties.

The effects of room temperature water, hot water, and steam tempering methods were investigated on sorghum kernel physical properties, milling, flour, and bread-making properties. Overall tempering condition and tempering moisture content were found to have a significant effect on the physical properties. Milling properties were evaluated using a laboratory-scale roller milling flowsheet consisting of four break rolls and eight reduction rolls. Room temperature tempering (18% moisture for 24 h) led to better separation of bran and endosperm without negatively impacting flour quality characteristics i.e., particle size distribution, flour yield, protein, ash, damaged starch, and moisture content. Bread produced from the flour obtained from milling sorghum kernels tempered with room temperature water (18% m.c for 24 h) and hot water (16% m.c at 60 °C for 18 h) displayed better bread-making properties i.e., high firmness, resilience, volume index, higher number of cells, and thinner cell walls when compared to other tempering conditions. Room temperature water tempering treatment (18% m.c for 24 h) could be a better pretreatment process for milling white sorghum kernels without negatively impacting the flour and bread-making quality characteristics.

Analysis of corn and sorghum flour mixtures using laser-induced breakdown spectroscopy.

BACKGROUND: In a world constantly challenged by climate change, corn and sorghum are two important grains because of their high productivity and adaptability, and their multifunctional use for different purposes such as human food, animal feed, and feedstock for many industrial products and biofuels. Corn and sorghum can be utilized interchangeably in certain applications; one grain may be preferred over the other for several reasons. The determination of the composition corn and sorghum flour mixtures may be necessary for economic, regulatory, environmental, functional, or nutritional reasons. RESULTS: Laser-induced breakdown spectroscopy (LIBS) in combination with chemometrics, was used for the classification of flour samples based on the LIBS spectra of flour types and mixtures using partial least squares discriminant analysis (PLS-DA) and the determination of the sorghum ratio in sorghum / corn flour mixture based on their elemental composition using partial least squares (PLS) regression. Laser-induced breakdown spectroscopy with PLS-DA successfully identified the samples as either pure corn, pure sorghum, or corn-sorghum mixtures. Moreover, the addition of various levels of sorghum flour to mixtures of corn-sorghum flour were used for PLS analysis. The coefficient of determination values of calibration and validation PLS models are 0.979 and 0.965, respectively. The limit of detection of the PLS models is 4.36%. CONCLUSION: This study offers a rapid method for the determination of the sorghum level in corn-sorghum flour mixtures and the classification of flour samples with high accuracy, a short analysis time, and no requirement for time-consuming sample preparation procedures. © 2020 Society of Chemical Industry.

Advancing provitamin A biofortification in sorghum: Genome-wide association studies of grain carotenoids in global germplasm.

Vitamin A deficiency is one of the most prevalent nutritional deficiencies worldwide. Sorghum [Sorghum bicolor L. (Moench)] is a major cereal crop consumed by millions of people in regions with high vitamin A deficiency. We quantified carotenoid concentrations in a diverse sorghum panel using high-performance liquid chromatography and conducted a genome-wide association study (GWAS) of grain carotenoids to identify genes underlying carotenoid variation. There was moderate variation for ß-carotene (00.8 µg g-1 ), lutein (0.3-9.4 µg g-1 ), and zeaxanthin (0.2-9.1 µg g-1 ), but ß-cryptoxanthin and α-carotene were nearly undetectable. Genotype had the largest effect size, at 81% for zeaxanthin, 62% for ß-carotene, and 53% for lutein. Using multiple models, GWAS identified several significant associations between carotenoids and single nucleotide polymorphisms (SNPs), some of which colocalized with known carotenoid genes that have not been previously implicated in carotenoid variation. Several of the candidate genes identified have also been identified in maize (Zea mays L.) and Arabidopsis (Arabidopsis thaliana) carotenoid GWAS studies. Notably, an SNP inside the putative ortholog of maize zeaxanthin epoxidase (ZEP) had the most significant association with zeaxanthin and with the ratio between lutein and zeaxanthin, suggesting that ZEP is a major gene controlling sorghum carotenoid variation. Overall findings suggest there is oligogenic inheritance for sorghum carotenoids and suitable variation for marker-assisted selection. The high carotenoid germplasm and significant associations identified in this study can be used in biofortification efforts to improve the nutritional quality of sorghum.

Allelochemicals targeted to balance competing selections in African agroecosystems.

Among major cereals domesticated as staple food, only sorghum has a high proportion of cultivars with condensed tannins in grain, which can trigger bitter taste perception in animals by binding to type 2 taste receptors (TAS2Rs). Here, we report the completion of uncovering of a pair of duplicate recessive genes (Tannin1 and Tannin2) underlying tannin presence. Three loss-of-function alleles from each gene were identified in non-tannin sorghum desired as palatable food. Condensed tannins effectively prevented sparrows from consuming sorghum grain. Parallel geographic distributions between tannin sorghum and Quelea quelea supported the role of tannins in fighting against this major herbivore threat. Association between geographic distributions of human TAS2R variants and tannin sorghum across Africa suggested that different causes had probably driven this bidirectional selection according to varied local herbivore threats and human taste sensitivity. Our investigation uncovered coevolution among humans, plants and environments linked by allelochemicals.

Factors Influencing Zein-Whole Sorghum Flour Dough Formation and Bread Quality.

Zein is known to able to form viscoelastic dough with wheat-like properties under certain conditions. Several studies have been conducted to explain the mechanism behind this ability and to improve the functionality and end-use quality of zein-based dough systems. However, most of this research has been conducted using zein in combination with isolated starches or high-starch flours. To investigate the production of additional zein-whole sorghum flour breads, experiments were conducted to determine factors impacting zein-whole sorghum flour dough and bread quality. Optimizing water levels, using defatted zein and/or sorghum flour, and increasing zein content in dough formulas were investigated as initial formulation steps. Of these factors, increasing zein content from 20% to 30% (flour weight basis) had the greatest impact, resulting in stronger zein-based dough and improved bread quality. Additives and zein treatments shown to impact zein functionality were then investigated for their effect of zein-whole sorghum flour breads. Mixing zein and whole sorghum flour with 0.5% hydrogen peroxide, 5% ethanol, or 3% hydroxypropyl methylcellulose resulted in improved dough strength and bread quality. Breads made from whole white sorghum flour had improved quality compared to zein-based breads made with black or high-tannin whole sorghum flour. PRACTICAL APPLICATION: Zein is known to be able to form wheat-like dough when mixed under the right conditions. Most of the research on zein-based dough and food products has used high-starch flours. This project investigated optimizing the production of zein-whole sorghum flour dough and bread as an alternative. Increasing the zein content in the formula and using additives including ethanol and HPMC produced breads from zein-whole sorghum flour that were like those made with zein and pure starch.

The effect of genotype and traditional food processing methods on in-vitro protein digestibility and micronutrient profile of sorghum cooked products.

Sorghum (Sorghum bicolor (L.) Moench) is one of the principal staple for millions of people in sub-Saharan Africa serving as the main sources of protein. However, protein digestibility is low in sorghum and this may be affected by processing methods. In this study 15 sorghum cultivars and one variety each of maize (Zea maize) and tef (Eragrostis tef) all of Ethiopian origin were investigated for in-vitro protein digestibility (IVPD), activity and concentration of anti-nutritional factors and micro nutrient profile in raw flour and various cooked food samples. Kafirin composition content and composition was also determined from raw flour samples of the sorghum cultivars. IVPD was significantly different between genotypes with both maize and tef superior to sorghum both in cooked and uncooked state except for the high lysine genotype Wetet Be-gunchie. Cooking significantly reduced IVPD in all crops but had only minor effect in maize. Results revealed a highly significant interaction between genotype and food processing methods where, occasionally, genotypes with highest IVPD under one processing method ended up to be the lowest under another. Trypsin inhibitor levels had a significant and negative correlation with IVPD (r2 = 0.1), while changes in phytic acid concentration and intrinsic phytase levels during processing followed opposite trends to each other. Processing increased mineral levels by 20-44% for iron and 4-29% for zinc perhaps due to degradation of phytic acid. Results demonstrated that protein digestibility and the concentration of anti- nutritional factors varied widely depending on the food type. Identification of specific genotypes for a specific food product may help improve the nutritional quality of sorghum based foods.

Interaction mechanisms of condensed tannins (proanthocyanidins) with wheat gluten proteins.

Proanthocyanidins (PA) crosslink wheat gluten, increasing its polymer size and strength. However, mechanisms behind these interactions are unknown. This study used PA of different MW profiles (mean degree of polymerization 8.3 and 19.5) to investigate how PA polymerize gluten. The higher MW PA had greater binding affinity for both glutenins and gliadins than lower MW PA, whereas both PA precipitated glutenins more efficiently than gliadins. The PA preferentially bound the largest of the protein fractions available: high MW glutenin subunits (HMW-GS) over low MW-GS, and ω-gliadins over α- and γ-gliadins. Furthermore, within the HMW-GS, PA bound more of the larger x-type than the smaller y-type. Proanthocyanidins reduced gluten solubility in urea and decreased surface hydrophobicity of glutenins, but not gliadins. The PA appear to preferentially crosslink HMW-GS via hydrophobic interactions and hydrogen bonding, whereas their interaction with gliadins is dominated by hydrogen bonding and is relatively weaker.

Effect of Salt and Ethanol Addition on Zein-Starch Dough and Bread Quality.

Development of viscoelastic doughs from non-wheat proteins allows for a wider range of gluten-free products. Little work has been completed to describe mechanisms of zein functionality in food systems. To identify factors responsible for dough development in zein-starch mixtures and their influence on zein bread quality, a mixture of 20% zein-80% maize starch was mixed with water and various reagents. Salts, NaSCN, NaCl, and Na2 SO4 were evaluated at concentrations from 0 to 2M for their influence on the properties of zein-starch dough systems. NaSCN at low concentrations produced softer dough. Ethanol treatments produced softer more workable dough in the absence of salts. Increasing concentrations of NaCl and Na2 SO4 resulted in coalescing of the proteins and no dough formation. The addition of ß-ME had minimal softening effects on zein-starch dough. Specific volumes of zein-starch bread increased with decreasing NaCl addition in bread formulations. Likewise, including 5% ethanol (v/v) in the bread formula increased bread quality.

The Effects of Egg and Diacetyl Tartaric Acid Esters of Monoglycerides Addition on Storage Stability, Texture, and Sensory Properties of Gluten-Free Sorghum Bread.

The impact of whole egg addition (as is) at 20%, 25%, or 30% (flour basis) on sorghum bread quality was evaluated. The use of the antistaling agent diacetyl tartaric acid esters of monoglycerides (DATEM) at 0.5% (flour basis) at each of the egg addition levels was also studied. Evaluated quality factors included color, specific volume, and crumb structure. Texture analysis was performed to evaluate the rate of quality loss based on changes in crumb hardness values over time. A trained sensory panel evaluated bread quality attributes by descriptive analysis. Sorghum breads with egg had larger specific volumes than the control, while DATEM had a negative effect on the volume of sorghum gluten-free bread. Inclusion of egg in the bread formula improved cell structure and produced darker crust (P < 0.05). The addition of egg reduced bread hardness and slowed the rate of quality loss over a 12-d storage period. Descriptive analysis confirmed the findings of texture analysis. Control breads were significantly harder (P < 0.05) than egg-containing bread at days 0 and 4. This research demonstrates that addition of eggs to a gluten-free sorghum bread formulation results in improved storage stability and better overall quality and acceptability of the product.

Effect of Condensed Tannin Profile on Wheat Flour Dough Rheology.

Proanthocyanidins (PA) cross-link proteins and could expand wheat gluten functionality; however, how the PA MW or gluten profile affect these interactions is unknown. Effect of PA MW profile (sorghum versus grape seed PA) on dough rheology of high versus low insoluble polymeric protein (IPP) wheat flour was evaluated using mixograph, large (TA.XT2i) and small (HAAKE Rheostress 6000) deformation rheometry. Sorghum PA (93% polymeric) more effectively (p < 0.05) strengthened both glutens than grape seed PA (45% polymeric), without reducing gluten extensibility. These effects were higher in low IPP (weak gluten) flour, e.g., sorghum PA doubled IPP, increased mix time by 75%, dough elasticity by 82%, and peak angle by 17° versus control. Grape seed PA increased IPP by 75% and elasticity by 36%, but reduced peak angle by 15°, indicating reduced mixing tolerance. Sorghum PA, but not grape seed PA, increased (p < 0.05) all above parameters in high IPP dough. Polymeric PA more effectively strengthened gluten than oligomeric PA, likely via more efficient protein cross-linking to overcome strong antioxidant effect of PA. High MW PA may be useful natural gluten strengtheners for diverse applications.

Grain sorghum proteomics: integrated approach toward characterization of endosperm storage proteins in kafirin allelic variants.

Grain protein composition determines quality traits, such as value for food, feedstock, and biomaterials uses. The major storage proteins in sorghum are the prolamins, known as kafirins. Located primarily on the periphery of the protein bodies surrounding starch, cysteine-rich ß- and γ-kafirins may limit enzymatic access to internally positioned α-kafirins and starch. An integrated approach was used to characterize sorghum with allelic variation at the kafirin loci to determine the effects of this genetic diversity on protein expression. Reversed-phase high performance liquid chromatography and lab-on-a-chip analysis showed reductions in alcohol-soluble protein in ß-kafirin null lines. Gel-based separation and liquid chromatography-tandem mass spectrometry identified a range of redox active proteins affecting storage protein biochemistry. Thioredoxin, involved in the processing of proteins at germination, has reported impacts on grain digestibility and was differentially expressed across genotypes. Thus, redox states of endosperm proteins, of which kafirins are a subset, could affect quality traits in addition to the expression of proteins.