Turbidity Assay for Rapid and Efficient Identification of High Protein Digestibility Sorghum Lines

Recently, our laboratory reported a protein digestibility assay based on SDS‐PAGE that distinguishes mutant high protein digestibility from wild‐type sorghum lines. Using that assay, high protein digestibility sorghum lines were identified both qualitatively (visual observation) and quantitatively by measuring the SDS‐PAGE band intensity of the undigested α‐kafirin protein. Here, we report on a new turbidity assay that can be used for an even quicker quantitation of the undigested proteins with much higher throughput for screening purposes. Proteins remaining after 1 hr of pepsin digestion were extracted with a buffer of SDS, 2‐mercaptoethanol, and borate and an aliquot of the extract was precipitated using 72% trichloroacetic acid (TCA). Absorbance of the resulting turbid solution was then read at 562 nm. Lower readings corresponded to more digestible lines. The turbidity of the suspensions developed quickly and reached a plateau at ≈5 min for high protein digestibility lines and 10 min for wild‐type lines. The turbid solutions remained stable for at least 1 hr. Two distinct groups, wild‐type and high protein digestibility sorghum lines, were obtained when the assay was compared with a standard pepsin digestibility procedure and to our recently developed SDS‐PAGE assay. A comparison with the bicinchoninic acid (BCA) assay of protein quantitation indicated that the turbidity assay is more efficient in differentiating between wild‐type and high protein digestibility sorghum lines. We have further refined the turbidity assay for microtiter plate analysis making it possible for a single operator to analyze ≈200 sorghum lines per day, compared to 60 lines when using the SDS‐PAGE assay.     

Impacts of Kafirin Allelic Diversity,Starch Content,and Protein Digestibility on Ethanol Conversion Efficiency in Grain Sorghum

Seed protein and starch composition determine the efficiency of the fermentation process in the production of grain‐based ethanol. Sorghum, a highly water‐ and nutrient‐efficient plant, provides an alternative to fuel crops with greater irrigation and fertilizer requirements, such as maize. However, sorghum grain is generally less digestible because of extensive disulfide cross‐linking among sulfur‐rich storage proteins in the protein– starch matrix. Thus, the fine structure and composition of the seed endosperm directly impact grain end use, including fermentation performance. To test the hypothesis that kafirin (prolamin) seed storage proteins specifically influence the efficiency of ethanol production from sorghum, 10 diverse genetic lines with allelic variation in the β‐, γ‐, and (δ‐kafirins, including three β‐kafirin null mutants, were tested for ethanol yield and fermentation efficiency. Our selected lines showed wide variation in grain biochemical features, including total protein (9.96–16.47%), starch (65.52–74.29%), and free amino nitrogen (FAN) (32.84–73.51 mg/L). Total ethanol yield (ranging from 384 to 426 L/metric ton), was positively correlated to starch content (R² = 0.74), and there was a slight positive correlation between protein digestibility and ethanol yield (R² = 0.52). Increases in FAN content enhanced fermentation efficiency (R² = 0.65). The highest ethanol producer was elite staygreen breeding line B923296, and the line with the highest fermentation efficiency at the 72 h time point was inbred BT×623. A large‐seeded genotype, KS115, carrying a novel γ‐kafirin allele, was rich in FAN and exhibited excellent short‐term fermentation efficiency at 85.68% at the 20 h time point. However, the overall ethanol yield from this line was comparatively low at 384 L/metric ton, because of insufficient starch, low digestibility, and high crude protein. Multivariate analysis indicated an association between the β‐kafirin allele and variation in grain digestibility (P = 0.042) and FAN (P = 0.036), with subsequent effects on ethanol yield. Reversed‐phase HPLC profiling of the alcohol‐soluble kafirin protein fraction revealed diversity in protein content and composition across the lines, with similarities in peak distribution profiles among β‐kafirin null mutants compared with normal lines.     

Discovery of Grain Sorghum Germ Plasm with High Uncooked and Cooked In Vitro Protein Digestibilities

Grain sorghum has been documented to have low protein digestibility relative to other cereal grains. Low protein digestibility of sorghum is most pronounced in cooked foods and is ranked slightly lower than corn as a feed grain. In this article, sorghum germ plasm is identified that has substantially higher uncooked and cooked flour in vitro protein digestibility than normal cultivars. Sorghum lines were found within a high-lysine opulation derived from the mutant P721Q that have ≈10–15% higher uncooked and ≈25% higher cooked protein digestibilities using a pepsin assay. Highly digestible sorghum grain showed little reduction in digestibility after cooking, compared to the large reduction that is typical of normal sorghum cultivars. Using the three-enzyme pH-stat method, we showed that the highly digestible lines had the same degree of peptide bond hydrolysis in ≈5 min, as was found in 60 min in the normal cultivar, P721N. Differences in protein digestibility were related to enyzme susceptibility of the major storage prolamin, α-kafirin, that comprises ≈50–60% of the total sorghum grain protein. Using the enzyme-linked immunosorbent assay (ELISA) technique to track the pepsin digestion of α-kafirin, the highly digestible lines had ≈90–95% α-kafirin digested in 60 min compared to 45–60% for two normal cultivars. γ-Kafirin, a minor structural prolamin found mainly at the periphery of protein bodies, was also somewhat more digestible in the highly digestible sorghums. Highly digestible grain was of a floury kernel type, though recently this trait has been found in a modified background. More digestible protein from sorghum grain, that additionally is high in lysine content and has a fairly hard endosperm, could be of important benefit to populations who lack adequate protein in their diets, and may, pending further studies, prove to increase the value of sorghum as a feed grain.     

Transgenic sorghum with altered kafirin synthesis: kafirin solubility, polymerization, and protein digestion

Transgenic sorghum (TG) lines with altered kafirin synthesis, particularly suppression of γ-kafirin synthesis, and improved protein quality have been developed. The proportion of kafirin extracted with 60% tert-butyl alcohol alone was greatly increased in the TG lines. However, the total amount of kafirin remained unchanged. Further, in the TG lines, the kafirin was much less polymerized by disulfide bonding. There was also evidence of compensatory synthesis of other kafirin proteins. Cooked protein digestibility was increased in the TG form, even after removal of interfering starch. The TG protein bodies were intermediate in appearance between the normal type and the invaginated high digestibility mutants. Hence, the increased protein digestibility of these TG lines is probably related to their lower levels of disulfide-bonded kafirin polymerization, allowing better access of proteases. This work appears to confirm that disulfide bond formation in kafirin is responsible for the reduced protein digestibility of cooked sorghum.     

A Novel Modified Endosperm Texture in a Mutant High‐Protein Digestibility/High‐Lysine Grain Sorghum (Sorghum bicolor (L.) Moench)

Development of high‐protein digestibility (HPD)/high‐lysine (hl) sorghum mutant germplasm with good grain quality (i.e., hard endosperm texture) has been a major research objective at Purdue University. Progress toward achieving this objective, however, has been slow due to challenges posed by a combination of genetic and environmental factors. In this article, we report on the identification of a sorghum grain phenotype with a unique modified endosperm texture that has near‐normal hardness and possesses superior nutritional quality traits of high digestibility and enhanced lysine content. These modified endosperm lines were identified among F₆ families developed from crosses between hard endosperm, normal nutritional quality sorghum lines, and improved HPD/hl sorghum mutant P721Q‐derived lines. A novel vitreous endosperm formation originated in the central portion of the kernel endosperm with opaque portions appearing both centrally and peripherally surrounding the vitreous portion. Kernels exhibiting modification showed a range of vitreous content from a slight interior section to one that filled out to the kernel periphery. Microstructure of the vitreous endosperm fraction was dramatically different from that of vitreous normal kernels in sorghum and in other cereals, in that polygonal starch granules were densely packed but without the typically associated continuous protein matrix. We speculate that, due to the lack of protein matrix, such vitreous endosperm may have more available starch for animal nutrition, and possibly have improved wet‐milling and dry‐grind ethanol processing properties. The new modified endosperm selections produce a range that approaches the density of the vitreous parent, and have lysine content and protein digestibility comparable to the HPD/hl opaque mutant parent.     

Sorghum Bran as a Potential Source of Kafirin

Sorghum bran, a coproduct of sorghum dry milling, could be a source of protein for industrial applications. Condensed tannin‐free red and white sorghum samples were decorticated by abrasion until ≈10 or 25% grain by weight was removed. Kafirin was then extracted from the milling fractions using an aqueous ethanol based solvent system. The brans were darker and considerably higher in protein and fat compared with the whole grain flours and decorticated grain flours, with the 25% bran having higher protein than the 10% bran. This is due to increased contamination of the bran with protein‐dense, corneous endosperm. The protein extracted from all the milling fractions, including the brans, was pure kafirin. However, the yield of kafirin from the brans (15.9–26.7% of total protein present) was somewhat lower than that from whole grain and decorticated grain flours (45.0–57.9% of total protein present), due to the fact that kafirin is located solely in the endosperm. Also, the kafirin from bran was more contaminated with fat, polyphenols, and other substances, and more highly colored, particularly the kafirin from red sorghum. Thus, sorghum bran could be used as a source of kafirin but further purification steps may be necessary.     

Genetic Analysis of Kafirins and Their Phenotypic Correlations with Feed Quality Traits,In Vitro Digestibility,and Seed Weight in Grain Sorghum

Twenty‐three entries of grain sorghum (Sorghum bicolor (L.) Moench), including eight inbred lines (five males and three females) and 15 hybrids, were evaluated to determine the proportion of γ, αII, and β‐αI‐kafirins and their association with contents of crude protein, fat, and starch; protein digestibility; in vitro dry matter disappearance; and seed weight. The male lines included three normal‐seeded lines (TX2737, TX435, and P954063) and two large‐seeded lines (Eastin1 and PL‐1). Female lines consisted of three common U.S. seed parent lines (Wheatland, Redlan, and SA3042). The lines and their hybrids were grown under dryland conditions at two locations in Kansas using a randomized complete block design. The effects of genotype, location, and males were significant for all kafirins. Wide variations in composition and general combining ability (GCA) for kafirin content were noted among parent lines and hybrids, with TX2737, Eastin1, and PL1 having the largest GCA values for γ (1.37), αII (1.99), and β‐αI (2.57), respectively. Correlations among kafirins ranged from ‐0.89 to 0, whereas those of kafirins with feed quality traits, digestibility, and seed weight ranged from ‐0.45 to 0.48.     

Use of SDS to Extract Sorghum and Maize Proteins for Free Zone Capillary Electrophoresis (FZCE) Analysis

Two different extraction methods for extracting sorghum (Sorghum bicolor L. Moench.) storage proteins for free zone capillary electrophoresis (FZCE) analysis were compared. A traditional solvent based on 60% t‐butanol was compared with a pH 10 borate buffer containing the anionic detergent SDS followed by precipitation of nonkafirins using 60% t‐butanol. FZCE analysis of both types of extracts showed identical patterns, despite the fact that the SDS should have given all proteins equal charge‐to‐mass ratios. This methodology was also successfully applied to maize proteins. The use of t‐butanol to precipitate nonkafirins, combined with electrophoresis at low pH, is thought to have removed the SDS from the storage proteins. The SDS extraction procedure produced more stable extracts for FZCE analysis. These extracts could also be used directly for SDS capillary electrophoresis (SDS‐CE) separations. Kafirins from 15 genotypes were extracted with this procedure and analyzed by FZCE and SDS‐CE. Resolution of the kafirins by FZCE was much higher than the SDS‐CE, demonstrating that the kafirin proteins possessed a high level of charge density variability within a relatively small molecular size distribution. Two distinct groups of α‐kafirins could be seen in the FZCE electropherograms.     

Extraction and Film Properties of Kafirin from Coarse Sorghum and Sorghum DDGS by Percolation

The high cost of kafirin and zein restricts their use for bioplastic and food applications. Effective, simple, and rapid kafirin/zein isolation processes are required. Here a percolation‐type aqueous ethanol solvent extraction process from coarse meals (grits) and coarse sorghum distillers dried grains and solubles (DDGS) for kafirin and zein isolation employing a low ratio of extractant to meal (2.5:1) was investigated, which is potentially applicable in the grain bioethanol industry. Postextraction filtration times were more than twice as fast using coarse meals compared with fine flours. Washing the meals prior to extraction to remove starch improved protein preparation purity to 73–85% compared with 68–72% for unwashed meals. Hence, no subsequent filtration or centrifugation step is required to clean up the kafirin/zein solution prior to solvent evaporation. With a single extraction step, kafirin/zein yields were 48% (protein basis) for DDGS and 53–70% for washed sorghum/maize meals. Cast films were used as a model bioplastic system to evaluate extracted kafirin/zein functional properties. DDGS kafirin films had rough surfaces but had the lowest water uptake and in vitro digestibility, owing to heat‐induced disulfide crosslinking during DDGS processing. Extraction by percolation using coarse meal/DDGS has potential to improve kafirin/zein viability.     

Low α-Amylase Starch Digestibility of Cooked Sorghum Flours and the Effect of Protein

The comparably low starch digestibility of cooked sorghum flours was studied with reference to normal maize. Four sorghum cultivars that represent different types of endosperm were used. Starch digestibilities of 4% cooked sorghum flour suspensions, measured as reducing sugars liberated following α-amylase digestion, were 15–25% lower than for cooked maize flour, but there were no differences among the cooked pure starches. After the flours were predigested with pepsin to remove some proteins, the starch digestibility of cooked sorghum flours increased 7–14%, while there was only 2% increase in normal maize; however, there was no effect of pepsin treatment on starch digestibility if the flours were first cooked and then digested. After cooking with reducing agent, 100 mM sodium metabisulfite, starch digestibility of sorghum flours increased significantly while no significant effect was observed for maize. Also, starch solubility of sorghum flours at 85 and 100°C was lower than in maize, and sodium metabisulfite increased solubility much more in sorghum than in maize. Differential scanning calorimetry results of the flour residue after α-amylase digestion did not show any peaks over a temperature range of 20–120°C, indicating that sorghum starches had all undergone gelatinization. These findings indicate that the protein in cooked sorghum flour pastes plays an important role in making a slowly digesting starch.     

Fermented Sorghum as a Functional Ingredient in Composite Breads

Whole sorghum flour was fermented (a five‐day natural lactic acid fermentation) and dried under forced draught at 60°C, and evaluated for its effect on sorghum and wheat composite bread quality. In comparison with unfermented sorghum flour, fermentation decreased the flour pH from 6.2 to 3.4, decreased total starch and water‐soluble proteins, and increased enzyme‐susceptible starch, total protein, and the in vitro protein digestibility (IVPD). Fermentation and drying did not decrease the pasting temperature of sorghum flour, but slightly increased its peak and final viscosity. In comparison with composite bread dough containing unfermented sorghum flour, fermented and dried sorghum flour decreased the pH of the dough from 5.8 to 4.9, increased bread volume by ≈4%, improved crumb structure, and slightly decreased crumb firmness. IVPD of the composite bread was also improved. Mixing wet fermented sorghum flour directly with wheat flour (sourdough‐type process) further increased loaf volume and weight and reduced crumb firmness, and simplified the breadmaking process. It appears that the low pH of fermented sorghum flour inactivated amylases and increased the viscosity of sorghum flour, thus improving the gas‐holding capacity of sorghum and wheat composite dough. Fermentation of sorghum flour, particularly in a sourdough breadmaking process, appears to have considerable potential for increasing sorghum utilization in bread.     

Protein biofortified sorghum: effect of processing into traditional african foods on their protein quality

Protein biofortification into crops is a means to combat childhood protein-energy malnutrition (PEM) in developing countries, by increasing the bioavailability of protein in staple plant foods and ensuring sustainability of the crop. Protein biofortification of sorghum has been achieved by both chemically induced mutation and genetic engineering. For this biofortification to be effective, the improved protein quality in the grain must be retained when it is processed into staple African foods. Suppression of kafirin synthesis by genetic engineering appeared to be superior to improved protein digestibility by chemical mutagenesis, because both the lysine content and protein digestibility were substantially improved and maintained in a range of African foods. For the genetically engineered sorghums, the protein digestibility corrected amino acid score was almost twice that of their null controls and considerably higher than the high protein digestibility sorghum type. Such protein biofortified sorghum has considerable potential to alleviate PEM.     

REVIEW: Developments in Our Understanding of Sorghum Polysaccharides and Their Health Benefits

The composition and structure of sorghum polysaccharides are remarkably similar to those in maize. Sorghum grain is rich in starch, cellulosic and noncellulosic polysaccharides (mainly glucuronoarabinoxylans [GAX]). Sorghum starch is similar to maize starch in terms of amylopectin, but the amylose may be more branched. This may account for sorghum starch having a generally slightly higher gelatinization temperature. The GAX in sorghum are highly substituted with glucuronic acid and arabinose, but the degree of these substitutions is lower when compared with maize GAX. Sorghum polysaccharides themselves are not sufficiently functional to allow the production of high‐quality baked goods. Sorghum has generally lower starch digestibility than maize. This is primarily due to the endosperm protein matrix, cell wall material, and tannins (if present) inhibiting enzymatic hydrolysis of the starch. Protein disulfide bond cross‐linking involving the kafirin prolamins in the protein matrix around the starch granules seems to be of major importance in reducing starch digestibility. It does not seem that sorghum polysaccharides, per se, have any unique health‐promoting effects. Any health‐promoting effects related to sorghum polysaccharides seem to be due to interactions between the polysaccharides and the endosperm matrix protein and phenolics.     

Qualitative Zymographic Studies of Major Proteinases from Malted Sorghum (Sorghum bicolor L.) and Effects of Cultivar

Proteolysis during cereal germination is vital both to seedling growth and the success of commercial malting and brewing. In this study, proteinases in proteolytic extracts from seeds and germinated grains of 11 Botswana sorghum cultivars were analyzed and partially characterized by one‐dimensional electrophoresis on SDS‐PAGE gels containing incorporated gelatin. Proteinase polymorphism was detected in both germinated and ungerminated sorghum grains. Fifteen distinct proteinase bands, with M_r values of 27,000–100,000 were detected in sorghum malt extract, while ungerminated sorghum displayed a maximum of four bands (M_r ≈ 78,000–100,000). Band numbers and identity varied markedly according to cultivar. More proteinase bands were detected at pH 4.6, than at pH 6.2 and 7.0, suggesting pH optima considerably below neutrality. Cysteine‐proteinases constituted a higher proportion (9 of 15) of the detected sorghum malt proteinases and were most detectable at pH 4.6. Multiple representatives were also detected for both serine‐ and metallo‐proteinases, although these were more active at pH 6.2 and 7.0. 1‐10 Phenanthroline inhibited malt metallo‐proteinase more strongly than EDTA, suggesting that these enzymes were most probably zinc‐dependent. Aspartyl‐proteinases were not detected, probably because of the substrate employed. Results indicate that the sorghum proteinase system is complex.     

Sequential in vitro pepsin digestion of uncooked and cooked sorghum and maize samples

An in vitro protein digestion study, using pepsin, was carried out in uncooked and cooked sorghum and maize flour samples. The digestibility values from the uncooked samples showed that sorghum presents digestibility values similar to those of maize. In the case of the cooked samples, it was found that a wet cooking procedure promotes a decrease in sorghum protein digestibility when compared to maize. Electrophoresis was used to follow the in vitro pepsin sequential digestion procedure, and infrared spectroscopy was applied to establish its efficiency. SDS-PAGE results showed that both uncooked samples (sorghum and maize) behave in a similar way. The wet cooking procedure increases the amount of high molecular weight aggregates and promotes the appearance of two nonreducible and nondigestible 45 and 47 kDa proteins. These two protein fractions are directly related to the loss of digestibility. It was also shown that in cooked sorghum the monomers (gamma-, alpha-, and beta-) are more resistant to digestion than the corresponding uncooked samples.     

Protein Quality and Physical Characteristics of Kisra (Fermented Sorghum Pancake‐like Flatbread) Made from Tannin and Non‐Tannin Sorghum Cultivars

Kisra is a naturally lactic acid bacteria‐ and yeast‐fermented sorghum thin pancake‐like flatbread produced in Sudan. Kisra has considerable potential as the basis for development of a gluten‐free sandwich wrap. To help direct cultivar selection for commercial production of these products, two white, tan plant non‐tannin Type I, one white Type II tannin, and one red Type III tannin sorghum cultivars were evaluated with respect to kisra protein quality and physical characteristics. Kisra from the non‐tannin sorghums were flexible and had an open‐textured structure with many regular gas cells, whereas those from the tannin sorghums were more brittle, denser in structure, and contained far fewer and smaller gas cells. Kisra from the tannin sorghums had the lowest reactive lysine content, in vitro protein digestibility, and Protein Digestibility Corrected Amino Score (PDCAAS), with values being lowest for the Type III sorghum. PDCAAS of kisra from the Type III sorghum was only 0.12, less than half of that from the Type I sorghums. As the tannins in tannin sorghums adversely affect kisra protein quality and physical characteristics, white tan plant, non‐tannin sorghum cultivars are most suitable for kisra production and for development of wrap‐type sorghum‐based baked goods.     

Characterization of Kafirins in Algerian Sorghum Cultivars

The prolamins in seven Algerian Sahara sorghum cultivars of varying seed shape and color were investigated. Protein contents ranged from 12 to 16%. Prolamins were the major protein fraction. They could be separated according to degree of disulfide cross‐linking. Kafirin monomers and low molecular weight polymers could be extracted with 70% ethanol, whereas highly cross‐linked kafirins additionally needed a reducing agent to become extractable. Kafirin monomers of α‐, β‐ and γ‐type were purified and N‐terminally sequenced. For the first time, δ‐kafirin was identified at the protein level. The study clearly revealed intercultivar differences between protein levels. The joint use of SDS‐PAGE, SE‐HPLC, and RP‐HPLC allowed discriminating among cultivars based on the differences in prolamin levels and composition.     

Formulation and Characterization of Drug‐Loaded Microparticles Using Distillers Dried Grain Kafirin

Kafirin, a protein extracted from sorghum grain, has been formulated into microparticles and proposed for use as a delivery system owing to the resistance of kafirin to upper gastrointestinal digestion. However, extracting kafirin from sorghum distillers dried grains with solubles (DDGS) may be more efficient, because the carbohydrate component has been removed by fermentation. This study investigated the properties and use of kafirin extracted from DDGS to formulate microparticles. Prednisolone, an anti‐inflammatory drug that could benefit from a delayed and targeted delivery system to the colon, was loaded into DDGS kafirin microparticles by phase separation with sodium chloride. Scanning electron micrographs revealed that the empty and prednisolone‐loaded microparticles were round in shape and varied in size. Surface binding studies indicated prednisolone was loaded within the microparticles rather than being solely bound on the surface. These findings demonstrate DDGS kafirin can be formulated into microparticles and loaded with medication. Future studies could investigate the potential applications of DDGS kafirin microparticles as an orally administered targeted drug‐delivery system.     

Effects of Brans from Specialty Sorghum Varieties on In Vitro Starch Digestibility of Soft and Hard Sorghum Endosperm Porridges

Brans of specialty sorghum varieties (high tannin, black, and black with tannin) were used to investigate the effects of sorghum phenolic compounds on starch digestibility of soft and hard sorghum endosperm porridges. Endosperms of varieties with the highest and lowest grain hardness index were mixed with brans of specialty sorghum varieties in the ratio of 85:15 and cooked into porridges with distilled water using a Rapid Visco Analyzer. Brans of condensed tannin containing sorghum varieties (high‐tannin and black with tannin sorghums) significantly (P < 0.05) decreased starch digestibility and estimated glycemic index (EGI) and increased resistant starch (RS) content of endosperm porridges. However, the addition of phenolic‐rich tannin‐free (mostly anthocyanins) black sorghum bran significantly (P < 0.05) increased starch digestibility and EGI but did not affect RS content of endosperm porridges. The disparate effects with black bran may, in part, result from its larger particle size and different bran structure compared with other sorghum varieties evaluated. Thus, our study showed that not only presence of phenolic compounds in the brans but also structural differences of specialty sorghum brans can have significant effects on starch digestibility.