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.     

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.     

Effect of Grain Structure and Cooking on Sorghum and Maize in vitro Protein Digestibility

Uncooked and cooked sorghum showed improvement in in vitro protein digestibility as the structural complexity of the sample reduced from whole grain flour through endosperm flour to protein body-enriched samples. This was not the case for maize. Cooking reduced protein digestibility of sorghum but not maize. Treating cooked sorghum and maize whole grain and endosperm flours with alpha -amylase to reduce sample complexity before in vitro pepsin digestion slightly improved protein digestibility. The reduction in sorghum protein digestibility on cooking was not related to the total polyphenol content of samples. Pericarp components, germ, endosperm cell walls, and gelatinised starch were identified as possible factors limiting sorghum protein digestibility. Electrophoresis of uncooked and cooked protein-body-enriched samples of sorghum and maize, and prolamin fractions of sorghum under non-reducing conditions showed oligomeric proteins with molecular weights (M_r) 45, 66 and >66 kDa and monomeric kafirins and zeins. Protein-body-enriched samples of sorghum had more 45–50 kDa oligomers than those of maize. In cooked sorghum, some of these were resistant to reduction. Pepsin-indigestible residues from protein-body-enriched samples consisted mainly of α-zein (uncooked and cooked maize) or α-kafirin (uncooked sorghum), whilst cooked sorghum had in addition, β- and γ-kafirin and reduction-resistant 45–50 kDa oligomers. Cooking appears to lead to formation of disulphide-bonded oligomeric proteins that occurs to a greater extent in sorghum than in maize. This may explain the poorer protein digestibility of cooked 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.     

Grain of high digestible,high lysine (HDHL) sorghum contains kafirins which enhance the protein network of composite dough and bread

The aim of this study was to determine whether protein body-free kafirins in high digestibility, high-lysine (HDHL) sorghum flour can participate as viscoelastic proteins in sorghum-wheat composite dough and bread. Dough extensibility tests revealed that maximum resistance to extension (g) and time to dough breakage (sec) at 35 °C for HDHL sorghum-wheat composite doughs were substantially greater (p < 0.01) than for normal sorghum-wheat composite doughs at 30 and 60% substitution levels. Functional changes in HDHL kafirin occurred upon exceeding its T_g. Normal sorghum showed a clear decrease in strain hardening at 60% substitution, whereas HDHL sorghum maintained a level similar to wheat dough. Significantly higher loaf volumes resulted for HDHL sorghum-wheat composites compared to normal sorghum-wheat composites at substitution levels above 30% and up to 56%, with the largest difference at 42%. HDHL sorghum-wheat composite bread exhibited lower hardness values, lower compressibility and higher springiness than normal sorghum-wheat composite bread. Finally, HDHL sorghum flour mixed with 18% vital wheat gluten produced viscoelastic dough while normal sorghum did not. These results clearly show that kafirin in HDHL sorghum flour contributes to the formation of an improved protein network with viscoelastic properties that leads to better quality composite doughs and breads.     

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.     

In vitro protein digestibility of developing and mature sorghum grain in relation to α-, β-, and γ-kafirin disulfide crosslinking

Sorghum (P721N, 1992) was harvested at selected days after half-bloom (DAHB) and at maturity, and analyzed for protein and moisture contents, protein digestibility, α-, β-, and γ-kafirin contents, and unextractable disulfide-bound complexes. α-Kafirin synthesis began before 10 DAHB, and β- and γ-kafirin at 20 DAHB. All the kafirins were as abundant at 40 DAHB as at maturity. Protein digestibilities of uncooked flour were about 90%, and dropped to 73% at maturity. Digestibilities of cooked flour dropped markedly at 35 DAHB, 40 DAHB and maturity. A comparison of 1992 and 1987 data indicates that digestibility decreases with moisture content, not days of development. The amount of disulfide “crosslinked” β- and γ-kafirins was insignificant at the earlier stages, though increased as the grain matured. Since kafirin synthesis is complete by 40 DAHB, we suggest that the decrease in digestibility in maturing grain is due to the drying effect and formation of disulfide-bound complexes involving β- and γ-kafirins. Cooking the flour may further promote protein interactions, particularly after kafirin synthesis has ceased.     

Content and quality of protein in proso millet (Panicum miliaceum L.) varieties

Proso millet belongs to the oldest cereals that human is using. Eight varieties of proso millet were cultivated in Ceske Budejovice from 1998 to 2000 and Cerveny Dvur from 1999 to 2000. The crude protein content was determined according to Kjehladl method and amino acid content was determined chromatographically after acid and oxidative acid hydrolysis. Although the protein content of proso (11.6% of dry matter) was similar to wheat, the grain of proso was significant richer in essential amino acids (leucine, isoleucine, methionine) then wheat. Hence, the protein quality of proso (Essential Amino Acid Index) was higher (51%) compared to wheat. The proso grain contained about 3.3 g kg(-1) of the limiting amino acid-lysine. Significant differences in protein and its quality were found among the evaluated proso varieties. The varieties Toldanskoe and Lipetskoe were the most different from the others in protein and amino acid content and Amino Acid Score of individual acids. They had the lowest content and quality of protein. The seed coat of these varieties was red. The amino acid and protein content was significantly influenced by weather during the year. Dry conditions caused an increase of protein but its quality was decreased.     

大豆蛋白营养品质和生理功能研究进展

本文综述了大豆蛋白营养品质和生理功能研究进展。蛋白消化率正后的氨基酸得分（ＰＤＣＡＡＳ）是一种新的、更准确的评价蛋白质营养品质的方法的指标;大豆蛋白ＰＤＣＡＡＳ值为满分（１,０）,表明大豆蛋白是完全蛋白质,可满足２岁以上人体对各种必需氨基酸的需求;临床研究表明,大豆蛋白可显著降低ＬＤＬ胆固醇浓度,而对ＨＤＬ胆固醇浓度有一定程度的提高作用;与其他优质蛋白相比,大豆蛋白有利于钙的代谢;用大豆蛋白代替膳     

An improved method for extraction of sorghum polymeric protein complexes

A method for fractionating sorghum proteins using extraction solvents and techniques designed to obtain polymeric protein structures (especially disulfide linked) was developed. Extraction and separation conditions were optimized in terms of completeness of protein extraction, sample stability, and analytical resolution. After pre-extraction of albumins and globulins, a 3-step sequential procedure involving no reducing agents was applied to ground whole sorghum flour. The three fractions obtained represented proportionally different protein polymer contents and molecular weight distribution as evidenced by comparative size exclusion chromatography. Protein composition also varied among the extracts with differences in kafirin composition and non-kafirin proteins detected in the fractions by RP-HPLC and SDS-PAGE analysis. The ability to quantify and further characterize sorghum polymeric protein complexes will be useful for additional studies linking protein structures with functionality and digestibility and variations for these properties within diverse sorghum germplasm.     

Disease and pest resistance in grains of sorghum and millets

In this review available information on the mechanisms of resistance to insect pests and fungal pathogens in sorghum and millets is discussed. The primary source of resistance lies in the chemical and physical make up of the grain. Phenolic compounds such as ferulic acid and tannins present in some sorghums are potent inhibitors of pests and pathogens. Grain hardness is a major deterrent to infection and infestation in low tannin grains. The prolamins, the grain storage proteins of sorghum, are organized into protein bodies and provide a physical and a nutritional barrier since they are resistant to digestion by insect and fungal proteases. A plethora of proteins that belong to the ‘pathogenesis related protein’ group are distributed in various parts of the grain. Some of them are located in protein bodies. Notwithstanding, sorghum is still susceptible to insect pests and fungal pathogens. An understanding of the natural mechanisms of resistance in the grain is paramount for the development of durable resistance against pests and pathogens. The pyramiding of resistance genes and the development of transgenic lines based on this understanding are two sources of hope for the future protection of sorghum and millets.     

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.     

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.     

Compositional variation amongst sorghum hybrids: Effect of kafirin concentration on metabolizable energy

Kafirins are stored proteins that negatively affect the nutritional quality of sorghum grain. Kafirin concentration and other chemical characteristics were determined in 12 sorghum hybrids and varied significantly, from 58% (HB1) to 42% (HB12) as percent total protein. Kafirin concentration correlated negatively with crude protein (CP) (−0.49), with acid detergent fiber (−0.40), apparent metabolizable energy (−0.61), and true metabolizable energy corrected for N (−0.63). HB12 was the hybrid with the lowest content of kafirins, amylose and tannins, and the highest content of apparent metabolizable energy. No differences were observed in the concentration of starch, but differences were found in apparent metabolizable energy (3325–2944 kcal kg⁻¹) probably due to a greater availability of starch, related to differences in kafirin concentration.     

The influence of milling on the nutritive value of flour from cereal grains. 6. Sorghum

Low-tannin sorghum was milled into flour with extraction rates between 100 and 64%. The nutritive value of the flours was studied by chemical analyses and in balance experiments with growing rats. The effects of amino acid supplementation was also examined. In general, the content of essential nutrients decreased when the extraction rate was lowered, but the content of fibre, tannins and phytate was also significantly reduced by milling. The digestibility of energy as well as protein was high, but the protein quality was poor, especially in flours of low extraction. Amino acid supplementation improved protein utilization and weight gain considerably. Femur zinc concentration was significantly depressed in the rats fed the amino acid supplemented diets, and sorghum seems to be a poor source of available zinc, in particular when it is highly refined. The poor zinc availability may be attributed to the high content of phytate.     

Specific expression of a sesame storage protein in transgenic rice bran

Rice oil bodies enclosed by unique structural proteins, oleosins, are found in the embryo and the aleurone layer, but not the starchy endosperm where starch and storage proteins are accumulated. To examine oleosin promoter specificity, a sesame storage protein, 2S albumin, was expressed in transgenic rice seeds under the control of a rice oleosin promoter. In all transgenic rice seeds, the sesame 2S albumin was found exclusively in the bran fraction after milling. Immunological staining revealed that the sesame 2S albumin was also located in the embryo and the outermost cells of the starchy endosperm. Furthermore, immunogold labeling showed that the transgenic 2S albumin was deposited in both type-I and type-II protein bodies of the outermost cells of the endosperm as well as in the type-II protein bodies of the embryo. The methionine and cysteine contents in the bran from four homozygous transgenic lines were elevated by 24–38 and 50–62%, respectively, compared with those of wild-type plants. The results suggest that the rice oleosin promoter is bran-specific and could be used to add value to rice bran, an abundant by-product of rice polishing, by genetic engineering.     

Content and Quality of Protein in Proso Millet (Panicum miliaceum L.) Varieties

Proso millet belongs to the oldest cereals that human is using. Eight varieties of proso millet were cultivated in Ceske Budejovice from1998 to 2000 and Cerveny Dvur from 1999 to 2000. The crude protein content was determined according to Kjehladl method and amino acid content was determined chromatographically after acid and oxidative acid hydrolysis. Although the protein content of proso (11.6% of dry matter) was similar to wheat, the grain of proso was significant richer in essential amino acids (leucine, isoleucine, methionine) then wheat. Hence, the protein quality of proso (Essential Amino Acid Index) was higher (51%) compared to wheat. The proso grain contained about 3.3 g kg⁻¹ of the limiting amino acid-lysine. Significant differences in protein and its quality were found among the evaluated proso varieties. The varieties Toldanskoe and Lipetskoe were the most different from the others in protein and amino acid content and Amino Acid Score of individual acids. They had the lowest content and quality of protein. The seed coat of these varieties was red. The amino acid and protein content was significantly influenced by weather during the year. Dry conditions caused an increase of protein but its quality was decreased.     

Compositional variation amongst sorghum hybrids: Effect of kafirin concentration on metabolizable energy

Kafirins are stored proteins that negatively affect the nutritional quality of sorghum grain. Kafirin concentration and other chemical characteristics were determined in 12 sorghum hybrids and varied significantly, from 58% (HB1) to 42% (HB12) as percent total protein. Kafirin concentration correlated negatively with crude protein (CP) (−0.49), with acid detergent fiber (−0.40), apparent metabolizable energy (−0.61), and true metabolizable energy corrected for N (−0.63). HB12 was the hybrid with the lowest content of kafirins, amylose and tannins, and the highest content of apparent metabolizable energy. No differences were observed in the concentration of starch, but differences were found in apparent metabolizable energy (3325–2944 kcal kg⁻¹) probably due to a greater availability of starch, related to differences in kafirin concentration.     

Novel food and non-food uses for sorghum and millets

Sorghum and millets have considerable potential in foods and beverages. As they are gluten-free they are suitable for coeliacs. Sorghum is also a potentially important source of nutraceuticals such antioxidant phenolics and cholesterol-lowering waxes. Cakes, cookies, pasta, a parboiled rice-like product and snack foods have been successfully produced from sorghum and, in some cases, millets. Wheat-free sorghum or millet bread remains the main challenge. Additives such as native and pre-gelatinised starches, hydrocolloids, fat, egg and rye pentosans improve bread quality. However, specific volumes are lower than those for wheat bread or gluten-free breads based on pure starches, and in many cases, breads tend to stale faster. Lager and stout beers with sorghum are brewed commercially. Sorghum's high-starch gelatinisation temperature and low beta-amylase activity remain problems with regard to complete substitution of barley malt with sorghum malt . The role of the sorghum endosperm matrix protein and cell wall components in limiting extract is a research focus. Brewing with millets is still at an experimental stage. Sorghum could be important for bioethanol and other bio-industrial products. Bioethanol research has focused on improving the economics of the process through cultivar selection, method development for low-quality grain and pre-processing to recover valuable by-products. Potential by-products such as the kafirin prolamin proteins and the pericarp wax have potential as bioplastic films and coatings for foods, primarily due to their hydrophobicity.     

Protein and lysine digestibility and protein quality of cooked Filipino rice diets and milled rice in growing rats

The protein quality of typical rice-based menu of Filipino preschool child and adult and cooked milled rice was assessed for true digestibility (TD), biological value (BV) and net protein utilization (NPU) in growing rats. Lysine and energy digestibilities were also determined. For the preschool child diet, TD was 88.8%, BV 90.0%, and NPU 79.9%. For the adult diet, TD was 87.3%, BV 86.6%, and NPU 75.5%, whereas cooked rice had 90.0% TD, 82.5% BV and 74.3% NPU. Lysine digestibility was 95.4% for preschool child diet, 95.7% for adult diet, and 100.0% for rice. Digestible energy was 91.3% for preschool child diet, 93.0% for adult diet, and 95.3% in rice. Amino acid scores were 100.0% for preschooler diet, 92.1% for adult diet, and 62.2% for rice. Protein quality based on amino acid score corrected for TD was 88.8% for preschool child diet. 80.4% for adult diet, and 56.0% for rice. If based on lysine digestibility instead of TD, protein quality would be 7.1% higher.