Increasing protein content and digestibility in sorghum grain with a synthetic biology approach

Despite great genetic diversity, sorghum grain consistently suffers from poor protein digestibility. The physicochemical packaging of protein bodies which consist of protease-resistant β- and γ-kafirin is considered a major obstacle. A synthetic β-kafirin gene, which shares the endosperm-specific promoter and signal peptide with the native β-kafirin gene (Sobic.009G001600.1), was transformed into sorghum inbred line Tx430. The gene was modified with ten additional proteolytic sites. These sites were designed to be amenable to cleavage by pepsin and/or chymotrypsin proteinases. Five independent transgenic lines were regenerated by microprojectile transformation. Notably, considerably more protein was observed in the peripheral endosperm of transgenic lines under scanning electron microscopy. Microscopy revealed invaginated or irregularly shaped protein bodies in the endosperm of transgenic lines. Grains of transgenic lines contained 11–37% more protein, which was 11–21% more pepsin digestible and 7–25% more chymotrypsin digestible than Tx430. Additionally, the abundant synthetic β-kafirin protein (5.6% of total protein) was detected by mass spectrometry data analysis in the transgenic line 9-1. Field-grown homozygous transgenics retained higher protein content, larger seed size and no reduction in grain number per plant. The results illustrated that plant synthetic biology could play an important role in improving sorghum nutritional value.     

Comparison of the effects induced by different processing methods on sorghum proteins

The effects induced by cooking, dry heating, popping, fermentation and germination on sorghum proteins were studied. Changes promoted by these processing methods were compared via sodium dodecyl sulfate polycrylamide gel electrophoresis (SDS-PAGE), in vitro protein digestibility (IVPD) assay and Fourier Transform Infrared (FT-IR).     

Effect of germination time and temperature on the functionality and protein solubility of sorghum flour

Sorghum was germinated for different time (12, 24, 36 and 48 h) at different temperatures (25, 30 and 35 °C) and the changes in their nutritional and functional properties of germinated sorghum flour were assessed and compared with native sorghum flour. Germination inversely affects the crude protein, fat, fibre and ash content. A decrease in water absorption and swelling power and increase in oil absorption capacity was observed due to enzymatic starch modification as the germination duration progressed. Germination of sorghum increased the gel consistency while paste clarity was decreased as compared to native flour. Proteins were modified by action of enzymes during higher germination time and temperature conditions, which results in significantly higher protein solubility of germinated sorghum flour, which also result in enhancing the foaming and emulsifying properties of the flour. Lowest % synersis value and least gelation concentrations were observed in native sorghum has, which increased during germination and were highest in sorghum germinated for 48 h at 35 °C. Germination in overall can be used as low cost natural bio-processing technique for the preparation of modified flour with enhanced function properties without chemical modification or genetic engineering.     

Functionality and characterization of kafirin-rich protein extracts from different whole and decorticated sorghum genotypes

The study of food-grade isolated kafirin proteins has increased recently due to the interest of their potential applications in gluten-free products. However, functionality of extracted proteins and fractions have been poorly characterized. In this investigation, kafirins were extracted from whole or decorticated sorghum genotypes (white-regular, white-waxy, red-regular and high-tannin) with 70% aqueous ethanol with metabisulfite. Chemical composition, color parameters, functionality and molecular characterization of kafirin extracts were determined. When kafirin extracts were obtained from decorticated sorghums, higher protein purities (81.5–93.3%), lower lipid contents (1.9–12.6%), better color parameters, and higher yields were obtained compared to counterparts extracted from whole caryopses. Functional properties of the extracted kafirins showed low water solubility, free amino nitrogen, and emulsifying activity, but adequate water holding and fat absorption capacities and in vitro protein digestibility. Differences in functionality were attributed to sorghum genotype. SDS-PAGE showed extraction of α₁-, α₂-, β-, and γ-kafirins with no contamination of other protein fractions. FTIR indicated a reduction in α-helix:β-sheet after extraction, especially in proteins obtained from decorticated sorghums. High correlations found from Pearson analysis could be used to optimize protein yield and the functionality (emulsifying activity and solubility) of the extracted proteins to be applied in gluten-free food systems.     

Effect of Japanese mint (Mentha arvensis) oil as fumigant on nutritional quality of stored sorghum

Japanese mint (Mentha arvensis) oil (JMO) can be used effectively as fumigant againstSitophilus oryzae in stored sorghum. The effect of JMO at a dose of 166 µl/l of space on nutrient composition and protein quality was studied in infested and uninfested sorghum grains stored for 3 months. The results revealed non significant effect of JMO on gran moisture, total ash, crude fibre, crude fat, crude protein and fat acidity in infested and uninfested grains at the end of 3 months storage. The JMO treatment had small but significant effect on reducing and nonreducing sugars. The values of Protein Efficiency Ratio (PER) for uninfested JMO treated grains, infested JMO treated grains and for untreated control stored for 3 months were 1.11, 1.07 and 1.09, respectively against control casein diet for which it was 2.15.     

Pericarp thickness of sorghum whole grain is accurately predicted by NIRS and can affect the prediction of other grain quality parameters

The thickness of grain pericarp, the outer layer of the kernel, is an important breeding criterion for sorghum. This cereal is mainly used through traditional processing in family-based food systems in many regions of the world. We investigated in this study how pericarp thickness could be predicted by Near Infrared Reflectance Spectroscopy (NIRS), a fast and non-destructive measurement method that is commonly used to measure physico-chemical parameters of sorghum grains, and how this trait also influences the prediction of those parameters. We showed that, using a classification approach, it was possible to discriminate thick from thin pericarp whole grain samples with a good accuracy and that the proportion of thin and thick grains in mixed samples could also be predicted. In addition, pericarp thickness had a significant effect on the calibration performance for other grain parameters indicating that the pericarp can distort spectral information of whole grain samples. As a practical consequence, we suggest to develop separate whole grain calibration models for thin and thick pericarp samples, combined with a two-steps prediction approach to improve the accuracy of whole grain NIRS calibrations for grain quality parameters in sorghum.     

Digestibility of protein and starch from sorghum (Sorghum bicolor) is linked to biochemical and structural features of grain endosperm

Although a principal source of energy and protein for millions of the world's poorest people, the nutritional value of sorghum (Sorghum bicolor L. Moench) is diminished because of low digestibility of grain protein and starch. To address this problem, we analyzed the properties of two sorghum lines that have a common pedigree but differ in digestibility. Consistent with results based on a ruminal fluid assay, the protein and starch of one line (KS48) was more thoroughly digested than that of the other (KS51) using in vitro assays based on pepsin and α-amylase. The indigestibility of KS51 relative to KS48 was shown to be due to (i) a greater abundance of disulfide-bonded proteins; (ii) presence in KS51 of non-waxy starch and the accompanying granule-bound starch synthase; and (iii) the differing nature of the protein matrix and its interaction with starch. The current findings suggest that each of these factors should be considered in efforts to enhance the nutritional value of sorghum grain.     

Effect of processing on the phenolic contents,antioxidant activity and volatile compounds of sorghum grain tea

Sorghum grain is rich in phenolic compounds and may be used to develop functional tea beverages. This work investigated the effect of processing techniques on the phenolic contents, antioxidant activity, and volatile compounds of a white colour sorghum (Liberty) grain tea. Significant (P ≤ 0.05) increase of total phenolic content, total flavonoid content and condensed tannin content were observed during the processing, whereas the antioxidant activity was not statistically enhanced. A total of 63 volatile compounds were detected including 5 alcohols, 13 alkanes, 2 aldehydes, 2 carboxylic acids, 15 esters, 4 ketones, 3 pyrazines and 1 phenylenediamine, which were affected by the processing techniques. The sorghum tea made from powder form infusion had more abundant volatile compounds compared to whole grain form infusion. The findings of this research have potential to expand human consumption of sorghum grain in the new form of grain tea.     

Effect of fermentation on sorghum protein fractions and in vitro protein digestibility

Changes in pH, titratable acidity, total soluble solids and proteins ofDabar sorghum (Sorghum bicolor (Linn) Moench.) during naturalfermentation at 37 ^°C for up to 36 h were monitored. The pH ofthe fermenting material decreased sharply with a concomitant increase in the titratable acidity. Total soluble solids increased with progressivefermentation time. The crude protein and non-protein nitrogen slightlyincreased during the last stages of fermentation. The in vitroprotein digestibility markedly increased as a result of fermentation.The globulin plus albumin fractions increased significantly (p 0.05)during the first 8 h of fermentation. Kaffirin fraction decreasedduring the first 8 h of fermentation but increased sharply as fermentationprogressed. Cross-linked kaffirins fluctuated during the fermentationprocess. Glutelin like protein, which was the minor fraction, trueglutelins, the second most abundant fraction, together with non-extractableproteins fluctuated during the fermentation process.     

Preferential binding of sorghum tannins with γ-kafirin and the influence of tannin binding on kafirin digestibility and biodegradation

Kafirins, sorghum prolamins bind with sorghum condensed tannins (CTs). The binding of different kafirin species with sorghum CTs was investigated. Analysis by chemical assay and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), reversed-phase high-performance liquid chromatography (RP-HPLC), and free zone capillary electrophoresis (FZCE), showed that γ-kafirin bound more CTs than the other kafirin species. SDS–PAGE suggested that the γ-kafirin-bound tannins were in the form of aggregates of molecular size >200k. RP-HPLC and FZCE revealed that sample preparation and drying the kafirins prior to the binding assays had a significant impact on γ-kafirin solubility. The effect of tannin binding on kafirin and kafirin film digestibility and film biodegradation was determined. Kafirins bound to tannins had lower digestibilities than unbound kafirins. Films made from tannin-bound kafirin had much lower digestibility and were less biodegradable than films made from unbound kafirin. The increase in kafirin film life by tannin modification appears to be due to a decrease in protein digestibility caused by kafirin–tannin binding. These findings suggest that γ-kafirin content in sorghum may be manipulated to either reduce or increase tannin binding in order to change the functionality of the kafirin in food, feed or film applications.     

Effect of accelerated aging on maize, sorghum, and sorghum meal

Accelerated aging at 50 °C significantly affected the physical and chemical properties of sorghum and maize. Aging caused associations between starch granules, protein matrix, and cell walls. During aging, floury areas of the endosperm became more corneous; as the endosperm hardened, strong associations between starch and protein developed, causing the endosperm to fracture through endosperm cells instead of along cell walls, which is common for non-aged maize. Aging significantly decreased the pasting viscosity of starch, molecular solubility at 85 °C and the molecular weight of solubilized starch. Solubility of albumins and globulins decreased while solubility of proteins extracted by a reducing agent and/or in alkaline pH increased during aging. Decreased solubility and functionality of starch and protein in aged grain appear to be due to protein oxidation.     

Effect of extrusion cooking on chemical structure,morphology, crystallinity and thermal properties of sorghum flour extrudates

The effect of feed moisture content (10, 14 and 18%) and die temperature (110 and 160 °C) on functional properties, specific mechanical energy (SME), morphology, thermal properties, X-ray diffraction pattern (XRD), Fourier transform infrared spectroscopy (FTIR) and amylose-lipid complex formation of extruded sorghum flour was investigated. Results showed that the extrusion cooking significantly changed the functional properties of extruded sorghum flour. Increasing feed moisture increased the peak gelatinization temperature (T_p), the degree of gelatinization (%) and starch crystallinity (%) while it decreased the gelatinization temperature ranges (T_c - T₀), starch gelatinization enthalpy (ΔH_G) and amylose-lipid complex (%) formation. With increasing die temperature, the degree of gelatinization and amylose-lipid complex formation increased and the starch T_p, T_c-T₀, ΔH_G and crystallinity decreased. The FTIR spectra also showed that the extrusion cooking did not create new functional groups or eliminate them in sorghum protein, whereas the sorghum extrudate protein had random coil conformation.     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

Cultivar and processing effects on the pasting characteristics,tannin content and protein quality and digestibility of cowpea (Vigna unguiculata)

Four popular West African local cultivars of cowpea (Vigna unguiculata), with distinctly different seed coat colors, were evaluated for their relative amylograph pasting characteristics, condensed tannin content, in vitro protein digestibility and Tetrahymena protein efficiency ratio (t-PER). The effects of roasting and dehulling on these properties were also determined. There were wide variations in the hot paste viscosity characteristics of the different cultivars studied. The raw cowpea flour samples exhibited maximum paste viscosities ranging between 260 Brabender Units (BU) for the Mottled cultivar and 460 BU for the cream-colored Blackeye cultivar. Cowpea cultivars with the greatest peak viscosities showed low stabilities to extended cooking. Roasting depressed paste viscosity properties of all the cowpea cultivars studied. Tannin concentrations were 0.3–6.9 and 7.2–116 mg CE/g flour from whole cowpea seeds and seed coats respectively, increasing with intensity of seed color. Although dehulling removed 98% of the tannin content of raw cowpeas, improvement in protein quality as a result of dehulling was observed for only the highly-pigmented Maroon-red variety. Roasting significantly improved digestibility and more than doubled the t-PER of all cowpea cultivars studied. Roasted cowpeas possess adequate nutritional and functional qualities as protein supplements in cereal-based weaning foods. However, it appears that dehulling is necessary to enhance the nutritional quality of the highly pigmented cultivars of cowpea.     

Prediction of total phenols,condensed tannins,and 3-deoxyanthocyanidins in sorghum grain using near-infrared (NIR) spectroscopy

The reported high phenolic levels in sorghum have led an interest from sorghum breeding programs in developing and identifying germplasms with high phenolic levels, which require screening a large number of samples to find those with the highest levels. Since wet chemistry screening methods are slow, expensive, and destructive, the use of NIR calibration curves could be an alternative. The objectives of this project were to determine the variation range in total phenols, condensed tannins, and 3-deoxyanthocyanidin levels in a diverse set of sorghum genotypes and to assess the predictive value of NIR curves to estimate these compounds in sorghum. A calibration curve to estimate each compound was developed and validated with an independent validation set. Calibration curve correlations for total phenols, condensed tannins, and 3-deoxyanthocyanidins were r = 0.98, 0.97, and 0.99, respectively. Correlations between NIR-predicted values and reference values in the validation set were significant for total phenols (r = 0.93), condensed tannins (r = 0.81) and 3-deoxyanthocyanidin (r = 0.82). These indicated that sufficient variation for these compounds existed within sorghum and that NIR calibration curves could be used to rapidly and non-destructively predict total phenols, condensed tannins, and 3-deoxyanthocyanidins concentrations in whole grain sorghum.     

Protein bodies ontogeny and localization of prolamin components in the developing endosperm of wheat caryopses

During caryopsis development, prolamins are initially stored in individual protein bodies, then generate a protein matrix in the ripe caryopsis. The ontogeny of the protein bodies was analyzed by fluorescence and electron microscopy from 7 to 43 days after anthesis (dAA), a period of time from the cellularization of endosperm to its desiccation. A series of antibodies specific to each prolamin type (α/β-, γ-, ω-gliadins, low-molecular weight and high-molecular weight glutenin subunits) made it possible to localize and co-localize the different prolamins in organelles of endosperm cells at different developmental stages. Protein bodies containing prolamins were observed as early as 7 dAA. At the early developmental stages, protein bodies were spherical with diameters around 1–2 μm. Later, around 15 dAA, the PBs enlarged, and aggregation and/or coalescence were prominent at 21 dAA. From 33 dAA, individual PBs were no longer visible, but a protein matrix was confined in the space between starch granules. All prolamins were found in the same protein bodies, without any segregation according to their types. Immunochemical labelling of prolamins failed to reveal in TEM analyses any particular internal organization in protein bodies. Glutenin subunits and gliadins were observed in the Golgi apparatus at the early stages of endosperm development.     

Influence of endosperm texture on milling,chemical composition and nutritive quality in maize

Three maize varieties namely Vijay, SO/SN composite and Shakti, which differ in their nutritional quality and endosperm texture were processed to prepare semolina (sooji) and process-flour. The nutritional quality of these products was determined and compared with whole kernel flour of the respective variety. Distinct differences in milling and chemical composition of three varieties were recorded. In quality evaluation tests lysine and tryptophan content, biological value and true digestibility were better in semolina and process-flour. Moreover, decrease in acid value in semolina and process-flour compared with whole maize flour recorded in all the varieties suggested improvement in shelf-life.     

Effects of endosperm texture and cooking conditions on the in vitro starch digestibility of sorghum and maize flours

The effects of endosperm vitreousness, cooking time and temperature on sorghum and maize starch digestion in vitro were studied using floury and vitreous endosperm flours. Starch digestion was significantly higher in floury sorghum endosperm than vitreous endosperm, but similar floury and vitreous endosperm of maize. Cooking with 2-mercaptoethanol increased starch digestion in both sorghum and maize, but more with sorghum, and more with vitreous endosperm flours. Increasing cooking time progressively reduced starch digestion in vitreous sorghum endosperm but improved digestibility in the other flours. Pressure-cooking increased starch digestion in all flours, but markedly more in vitreous sorghum flour; probably through physical disruption of the protein matrix enveloping the starch. Irrespective of vitreousness or cooking condition, the alpha-amylase kinetic constant (k) for both sorghum and maize flours remained similar, indicating that differences in their starch digestion were due to factors extrinsic to the starches. SDS-PAGE indicated that the higher proportion of disulphide bond-cross-linked prolamin proteins and more extensive polymerisation of the prolamins on cooking, resulting in polymers of M_r>100k, were responsible for the lower starch digestibility of the vitreous sorghum endosperm flour.     

Effect of soy-fortification method on the fermentation characteristics and nutritional quality of fermented maize meal

Studies were conducted to develop an appropriate household/small-scale enterprise level technique for the production of soy-fortified fermented maize dough (or meal) by comparing different treatments, processing methods and fortification levels. The effects of fortification method of the Ghanaian traditional fermented maize dough with raw or heat-treated whole soybeans and full-fat soyflour at 0%, 10% and 20% replacement levels, on the rate of fermentation and product quality were investigated. Sensory characteristics, trypsin inhibitor activity, amino acid pattern, proximate composition and hot paste viscosity were used as the indices of quality. Addition of whole soybeans to maize before milling and fermentation reduced the fermentation time by 60% while increasing the protein content by 24% and 70% respectively for 10% and 20% levels of fortification. A significant improvement was also achieved in the amino acids pattern of the fortified dough. However, raw whole soybeans imparted an undesirable color and beany flavor and an appreciable concentration of trypsin inhibitor activity (TIA) to the dough. Boiling soybeans for 20 min before incorporation into the maize for milling and fermentation was found necessary for desirable flavor and low levels of TIA. Little or no changes in the pasting viscosity characteristics occurred in samples containing boiled soybeans, while the usual method of fortifying maize meal with soy flour was found to severely depress the pasting viscosity characteristics and drastically reduced the acidity of the fermented dough. Based on the findings of the study, the most appropriate technique for the production of soy-fortified high protein fermented maize dough has been suggested to involve incorporation of boiled whole soybeans in soaked maize before milling and fermentation for improved sensory characteristics, enhanced nutritive value and optimal functional properties.