Impacts of Fungal Stalk Rot Pathogens on Physicochemical Properties of Sorghum Grain.

Stalk rot diseases are among the most ubiquitous and damaging fungal diseases of sorghum (Sorghum bicolor (L.) Moench) worldwide. Although reports of quantitative yield losses to stalk rots are available, the impact of stalk rot on grain quality attributes is unknown. This study was conducted to test whether stalk rot diseases could affect grain mineral (N, P, K; Ca, Mg, Cu, Fe, Mn, and Zn) and macronutrient (protein, fat, and starch) content, ash content, and physical traits (unit grain weight, hardness, and diameter). A field experiment was conducted in 2013 and 2014 with four sorghum genotypes (two hybrids and two lines). Plants from each genotype were inoculated with four stalk rot pathogens (Fusarium andiyazi, F. proliferatum, F. thapsinum, and Macrophomina phaseolina) and mock-inoculated with phosphate-buffered saline (control). Grains collected from infected and control plants were analyzed for macronutrient and ash content using near-infrared reflectance spectroscopy, grain hardness and diameter using the single-kernel characterization system, and mineral content using the Rapid Flow Analyzer (Model RFA-300 for N) and inductively coupled plasma spectrometer (for P, K, Ca, Mg, Cu, Fe, Mn, and Zn). Although stalk rot pathogens significantly reduced unit grain weight, they did not significantly affect grain hardness and diameter and, therefore, may not affect milling quality. Pathogens significantly reduced all macronutrient and most mineral contents across genotypes and environments on a per-unit-grain basis, except N and Mg, which were affected in a genotype- and environment-specific manner, and Fe, which was not significantly affected. Most minerals tested were significantly and negatively correlated with disease severity (lesion length) and total grain weight per panicle. The hybrid tested (Pioneer 84G62) exhibited reduced mineral and macronutritional changes after stalk rot infection, providing insights into the possibility of producing high-yielding, nutritionally stable hybrids under stalk rot disease pressure through dedicated breeding efforts.

Development of a 96-well plate iodine binding assay for amylose content determination.

Cereal starch amylose/amylopectin (AM/AP) is critical in functional properties for food and industrial applications. Conventional methods of AM/AP are time consuming and labor intensive making it difficult to screen the large sample sets necessary for evaluating breeding samples and investigating environmental impact on starch development. The objective was to adapt and optimize the iodine binding assay in a 96-well plate format for measurement at both λ 620 nm and λ 510 nm. The standard curve for amylose content was scaled to a 96-well plate format and demonstrated R(2) values of 0.999 and 0.993 for single and dual wavelengths, respectively. The plate methods were applicable over large ranges of amylose contents: high amylose maize starch at 61.7±2.3%, normal wheat starch at 29.0±0.74%, and a waxy maize starch at 1.2±0.9%. The method exhibited slightly greater amylose content values than the Concanavalin A method for normal type starches; but is consistent with cuvette scale iodine binding assays.

Effect of nitrogen fertilization and cover cropping systems on sorghum grain characteristics.

Cover crop treatments and nitrogen (N) fertilization rates were investigated for their impact on sorghum grain quality attributes. Sorghum was planted in field plots treated with differing cover cropping systems and fertilization rates. The size (weight and diameter) and hardness of the kernels were influenced by both the cover crop and N rates. The protein content increased as the N rate increased and also with the addition of cover crops to the system. The protein digestibility values and starch granule size distributions were not affected by N rate or the cover cropping treatments. Soil properties were tested to determine relationships with grain quality attributes. The utilization of cover crops appears to increase the protein content without causing a deleterious effect on protein digestibility. The end-product quality is not hampered by the use of beneficial cropping systems necessary for sustainable agriculture.

Effect of HPMC on the quality of wheat-free bread made from carob germ flour-starch mixtures.

UNLABELLED: Carob germ proteins have been shown to have functional properties similar to wheat gluten enabling formulation and production of yeast leavened gluten-free baked goods from a true dough rather than a stiff batter. The purpose of this research was to optimize the production of wheat-free bread containing carob germ flour, corn starch, NaCl, sucrose, hydroxypropyl methylcellulose (HPMC), and H2O. A key criterion was to formulate viscoelastic dough similar to wheat dough. To that end, response surface methodology (RSM) was used to determine optimal levels of carob germ flour, H2O, and HPMC. Components varied as follows: 4.94%-15.05% for carob germ flour, 0.05%-3.75% HPMC, and 65.25%-83.75% H2O (percents are on a flour basis, where carob germ flour in combination with maize starch equals 100%). Sucrose, NaCl, and yeast were held constant at 2%. Bread parameters evaluated were specific volume and crumb hardness, where the largest specific volume and the lowest value for crumb hardness were considered most desirable. The optimum formula as determined by RSM consisted of 7% carob germ flour, 93% maize starch, 2% HPMC, and 80% H2O with predicted crumb hardness of ~200 g of force and a specific volume of ~3.5 cm³/g. When proof time was optimized, a specific volume of ~5.6 ml/g and crumb hardness value of ~156 g of force was observed. Carob germ flour may be used as an alternative to wheat flour in formulating viscoelastic dough and high quality gluten-free bread. PRACTICAL APPLICATION: Celiac disease affects approximately 1% of the world's population. Sufferers of the disease must consume a gluten-free diet. Currently, gluten-free baked products are made from batters and lack the ability to be made from dough based systems which limits the overall processability and product variety. This research is aimed at the utilization of carob germ protein and its ability to form dough to produce an optimal gluten-free bread formulation. This will help to alleviate problems in processability and product variety associated with gluten-free baked goods.

Separation of kafirins on surface porous reversed-phase high-performance liquid chromatography columns.

Surface porous high-performance liquid chromatography (HPLC) columns were investigated for the separation of kafirins, storage proteins of grain sorghum. Kafirins were successfully separated using C3, C8, and C18 surface porous stationary phases in less than 17 min. Separations using a monolithic C18 stationary phase were also developed and were slightly faster than those achieved on the surface porous C18 stationary phase. However, the resolution was higher on the latter column. Using an ammonium hydroxide/acetonitrile mobile phase, separations were performed on a novel, alkaline stable surface porous C18 stationary phase. The resolution at alkaline pH was not as high, however, as with the traditional acidic acetonitrile mobile phases. In comparison to fully porous stationary phases, the surface porous phases provided higher resolution with much lower separation times (17 versus 40 min). Total peak areas were correlated to total protein content of sorghum (r(2) = 0.96; n = 10), and a method to measure in vitro pepsin digestibility using reversed-phase (RP)-HPLC peak areas showed good correlation to the traditional nitrogen combustion method (r(2) = 0.82; n = 20). Thus, the surface porous stationary phases could be used not only for more rapid separations but also to provide simultaneous information on total protein content and digestibility.

Characterization of polymeric proteins from vitreous and floury sorghum endosperm.

Differences in protein content and composition between vitreous and floury endosperm were investigated using a number of different techniques. Differences in protein cross-linking between vitreous and floury endosperm were investigated using differential solubility, size exclusion chromatography (SEC), and analysis of sulfhydryl content and composition. Vitreous endosperm was found to have higher levels of total protein and kafirins, but floury endosperm had a higher proportion of gamma-kafirins than the vitreous. Floury endosperm was found to have higher levels of SDS-soluble proteins than SDS-insoluble proteins extracted using sonication than vitreous endosperm. Conversely, vitreous endosperm had a greater proportion of the insoluble proteins. SEC analysis of the polymeric proteins revealed that the insoluble proteins had more polymeric proteins than did the soluble proteins, indicating greater cross-linking and a larger Mw distribution. Vitreous endosperm was also found to have a greater percentage (i.e., a higher ratio of disulfide to total sulfhydryls) of disulfide bonds than floury endosperm. These results show that the proteins in vitreous endosperm have a higher degree of cross-linking and a greater Mw distribution than those found in floury endosperm.

Recent developments in high-performance capillary electrophoresis of cereal proteins.

Cereal proteins play important nutritional and functional roles in human foods and are also important components of animal feeds. As such, cereals are a major economic factor around the world. Because of their importance, cereal proteins have been widely studied. A new emerging technique for studying cereal proteins is high-performance capillary electrophoresis (HPCE). This review focuses mainly on new methods and applications of HPCE to cereal proteins that have been reported in the last three years.

High-performance capillary electrophoresis of meat, dairy, and cereal proteins.

Food proteins play important roles in food functionality, nutrition, and human health. For these reasons, new analytical methods are continually being developed to separate and characterize these important proteins. High-performance capillary electrophoresis (HPCE) is one of the latest analytical methods to be applied to the separation of food proteins. This review covers methods and applications for the separation of three major groups of food proteins, meat, dairy, and cereal proteins.

Electrophoresis of cereal storage proteins.

Cereal proteins have been studied by a number of analytical techniques over the years. One of the major methodologies utilized by cereal chemists has been electrophoresis. Starting with moving boundary electrophoresis and progressing to slab gels and high-performance capillary electrophoresis, innovative methods have been developed to provide high resolution separations of difficult to separate proteins. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), acid-PAGE, isoelectric focusing, free zone CE, and even high-resolution two-dimensional HPLC-HPCE methods have been developed to separate cereal proteins. This review focuses on electrophoretic methods for separating and characterizing cereal storage proteins.

Acetonitrile as a buffer additive for free zone capillary electrophoresis separation and characterization of maize (Zeamays L. ) and sorghum (Sorghum bicolor L. Moench) storage proteins.

An improved method for separating and characterizing maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) storage proteins by free zone capillary electrophoresis (FZCE) was developed. Previous electrophoretic methods for analyzing these proteins required high concentrations of urea to maintain protein solubility during separation. To overcome disadvantages of urea, we developed a FZCE method that mimicked reversed-phase high-performance liquid chromatography (RP-HPLC) in that it used high levels of acetonitrile (ACN) at low pH. The optimized FZCE buffer system consisted of 80 mM phosphate-glycine buffer, nominal pH 2.5, containing 60% ACN and a cellulose derivative to dynamically coat capillary walls. Resolution was similar to or higher than that previously achieved by FZCE buffers utilizing 8 M urea as a buffer additive. ACN concentrations of at least 50% were necessary to achieve acceptable separations; this ACN concentration is approximately that necessary to extract these storage proteins. ACN was equally effective as traditional ethanol solvents and 8 M urea for solubilizing maize and sorghum proteins. The ACN-based FZCE buffer system gave high repeatability (<0.3% relative standard deviation, measured over 15 consecutive injections) for migration time. Subclasses of maize and sorghum storage proteins were identified, and genotypes of each cereal were successfully differentiated using ACN-containing buffers. This FZCE method may be applicable for the analysis of other hydrophobic proteins without the use of urea.

Ultrafast capillary electrophoretic analysis of cereal storage proteins and its applications to protein characterization and cultivar differentiation.

Free zone capillary electrophoresis conditions have been improved to allow rapid (2-8 min) separations of grain proteins from several cereals (wheat, oats, rice, barley, and rye) with high resolution and reproducibility. This new method utilized the isoelectric compound iminodiacetic acid (IDA) in conjunction with 20% acetonitrile and 0.05% hydroxypropylmethylcellulose. Cultivars of all cereals tested could be differentiated in 3 min, including wheat, using either prolamin or glutelin protein patterns. Resolution was similar to or higher than that of separations in other acidic buffers. Migration time repeatability was excellent with run-to-run variability <1% RSD, day-to-day <1.4% RSD, and capillary-to-capillary <3.3% RSD. Because larger inner diameter capillaries (50 microm) could be used with this buffer, sensitivity was improved and capillary rinse times could be reduced when compared to smaller capillaries (25 microm i.d.). This also served to reduce total separation time so that the majority of cereal storage protein from several types of cereals could be analyzed with total analysis times of 2-8 min with extremely high resolution and repeatability. This method would allow unattended, high-throughput ( approximately 180-400 samples/24 h) analysis of cereal proteins without the generation of much organic solvent waste as well as automated data analysis and storage.

Sodium dodecyl sulfate capillary electrophoresis of wheat proteins. 1. Uncoated capillaries.

Four different polymer/buffer systems (a commercial polymer from Bio-Rad, dextran, poly(ethylene oxide) (PEO), and non-crosslinked poly(acrylamide)) were evaluated for use in sodium dodecyl sulfate capillary electrophoresis (SDS-CE) separations of wheat proteins. These polymers were chosen on the basis of published reports of their use in uncoated or dynamically coated capillaries. Each polymer was optimized (where possible) by manipulating the polymer concentration and buffer concentration, and through the use of organic modifiers such as methanol and ethylene glycol. The addition of ethylene glycol to the separation buffer was found to improve the resolution of the separations, despite dilution of the sieving polymers. When PEO was used as the sieving polymer, however, no improvement was seen when ethylene glycol was added. Despite producing similar separations of molecular mass markers, the polymers did not all produce similar wheat protein separations. The commercial reagent and dextran produced similar separations, while the poly(acrylamide) produced faster separations than either. The poly(acrylamide) displayed much lower resolution in the 40-60 kDa range than the other polymers, though this polymer was able to separate the high molecular mass glutenin subunits (HMM-GS) without the use of added organic solvent. PEO produced much different wheat protein separations than the other polymers, despite similar separations of the molecular weight markers. This may have been due to interaction between the wheat proteins and PEO. Each polymer system also predicted different molecular masses of the various wheat protein fractions separated, with the PEO and poly(acrylamide) grossly overestimating the masses for all protein classes. This could have been due to protein-polymer interactions. Further work was done with the Bio-Rad buffer modified by the addition of ethylene glycol. Several different wheat protein fractions as well as proteins extracted from several different cultivars were separated with this buffer and compared. SDS-CE separations were also compared to SDS-poly(acrylamide) gel electrophoresis (PAGE) and several differences in the migration pattern of HMM-GS were noted.

Separation and characterization of barley (Hordeum vulgare L.) hordeins by free zone capillary electrophoresis.

Extraction conditions, separation conditions, and capillary rinsing protocols were optimized for the separation of barley hordeins by free zone capillary electrophoresis. Stable hordein extracts were obtained with a single 5 min extraction after the albumins and globulins were removed. Hordeins had to be reduced for optimal resolution. Optimum separation conditions for hordein separations were 100 mM phosphate-glycine buffer containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose. The addition of zwitterionic sulfobetaine detergents containing hydrocarbon tails of eight and ten carbons slightly improved the resolution of the separations, but not enough to warrant their use on a routine basis. The migration positions of the hordein subclasses were determined by two- dimensional reversed-phase high-performance liquid chromatography x free zone capillary electrophoresis mapping. The hordein subclasses formed clusters similar to those of wheat gliadins. Separation-to-separation repeatability was good, with migration time relative standard deviations < 1% for a 15-run period. For routine discrimination of cultivars, a 2 min post-separation rinse with 500 mM acetic acid was necessary to prevent protein build-up on the capillary walls. An example of successfully differentiating barley cultivars using this technique is shown.

High-performance capillary electrophoresis of cereal proteins.

Cereal grains are widely used of human foods and animal feed throughout the world. Cereals provide dietary protein, which also often has a functional role, as wheat gluten does in bread. Cereal proteins are unique in many ways: they are highly complex and heterogeneous, are often difficult to extract, and aggregate readily, making them difficult to characterize. Because of the economic importance and widespread use of cereal proteins, however, many techniques have been used for their analysis. High-performance capillary electrophoresis (HPCE) is one of the newest techniques to be so used. This review describes the development of charge- and size-based HPCE methods for analysis of cereal grain proteins, and the use of these methods for cultivar identification, classification, and prediction of quality. HPCE is versatile, rapid, easily automated, readily quantified, and provides high-resolution separations. Clearly, HPCE is a valuable addition to other methods of cereal protein analysis and should, in time, be applicable to all protein classes from all cereals.

Faster capillary electrophoresis separation of wheat proteins through modifications to buffer composition and sample handling.

Studies were conducted to produce faster, simpler, more rugged protocols for separating wheat proteins by high performance capillary electrophoresis (HPCE). Three areas were targeted for improvement: initial capillary equilibration procedures, buffer composition, and post-separation rinsing procedures. For the initial equilibration of capillaries, a brief rinse with a hydroxypropylmethylcellulose (HPMC) solution was the most critical factor for successful separation of wheat proteins. To reduce separation time and maintain resolution, beta-alanine and glycine were each used in place of sodium phosphate as buffer ions. Two isoelectric buffers, aspartic acid and iminodiacetic acid (IDA) were also tested. Each of these four buffer systems generated substantially lower currents, and provided faster separations, than sodium phosphate-based buffers. Finally, post-separation rinsing procedures were re-examined with the goal of reducing the time necessary to rinse the capillary after each separation. A critical factor in achieving this goal was removal of albumins and globulins prior to separation. These proteins bind to the capillary wall and cause rising baselines and excessive peak tailing. Once these proteins were removed, capillaries could be rinsed with buffer for only 2 min between separations. Capillary equilibration procedures were shortened from 90 min to 30 min. Likewise, separation times were reduced by approximately 40% (25 min to 15 min) by using glycine in place of sodium phosphate in the separation buffer. Finally, post-separation times were reduced by 80% (10 min to 2 min). Overall, these factors resulted in a reduction in total separation time of 50% (35 to 17 min) and maintained high resolution separations and good run-to-run repeatability.