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.     

Sorghum Protein Extraction by Sonication and Its Relationship to Ethanol Fermentation

The objectives of this research were to develop a rapid method for extracting proteins from mashed and nonmashed sorghum meal using sonication (ultrasound), and to determine the relationships between the levels of extractable proteins and ethanol fermentation properties. Nine grain sorghum hybrids with a broad range of ethanol fermentation efficiencies were used. Proteins were extracted in an alkaline borate buffer using sonication and characterized and quantified by size‐exclusion HPLC. A 30‐sec sonication treatment extracted a lower level of proteins from nonmashed sorghum meal than extracting the proteins for 24 hr with buffer only (no sonication). However, more protein was extracted by sonication from the mashed samples than from the buffer‐only 24‐hr extraction. In addition, sonication extracted more polymeric proteins from both the mashed and nonmashed samples compared with the buffer‐only extraction method. Confocal laser‐scanning microscopy images showed that the web‐like protein microstructures were disrupted during sonication. The results showed that there were strong relationships between extractable proteins and fermentation parameters. Ethanol yield increased and conversion efficiency improved significantly as the amount of extractable proteins from sonication of mashed samples increased. The absolute amount of polymeric proteins extracted through sonication were also highly related to ethanol fermentation. Thus, the SE‐HPLC area of proteins extracted from mashed sorghum using sonication could be used as an indicator for predicting fermentation quality of sorghum.     

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.     

Fish species identification by thin layer isoelectric focusing.

Conventional electrophoretic techniques generally lack the resolution and reproducibility needed for the reliable identification of fish species. Variations in stabilizing media composition, sample application technique, separation time, applied voltage or current, and the analyst's skill all affect the protein pattern. Thin layer polyacrylamide gel isoelectric focusing (TLIEF), a high resolution protein separation technique, has been applied to the identification of fish species. Sarcoplasmic proteins are separated according to their isoelectric points in a stable, reproducible pH gradient. Protein patterns for 12 species of fish are compared in 4.0% polyacrylamide gels with pH 4.0--6.0 and pH 3.5--10 gradients. Similar patterns are shown in commercially prepared 5.0% polyacrylamide gels with pH 4.0--6.5 and pH 3.5--9.5 gradients (LKB PAG plates). The protein patterns are reproducible in each pH gradient and also correlate well between user-prepared and commercially prepared gels. The inherent high resolution and excellent reproducibility of TLIEF should allow the positive identification of fish species without the costly procedure of using known species as standards.     

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.     

小麦花药蛋白质组双向电泳技术体系的优化

以小麦单核期花药为材料,比较了两种不同蛋白质提取方法TCA/丙酮法和酚提取法及不同的蛋白质裂解液组成对双向电泳的影响,并在蛋白质上样量和SDS-PAGE胶浓度等方面也进行了探索与优化。结果表明,采用TCA/丙酮提取法比用酚提取法提取的蛋白质所得的2-DE胶图谱上蛋白质点数增加,图谱背景也比较清晰;样品溶解于含有硫脲和TBP的蛋白质裂解液Ⅱ中,可显著提高蛋白质的溶解性,在2-DE图谱上可分辨出554个蛋白质点,比用蛋白质裂解液Ⅰ提取的蛋白多39个点。以TCA-丙酮法提取小麦花药组织中的蛋白,用蛋白质裂解液Ⅱ[7mol/L尿素、2mol/L硫脲、4%CHAPS、2%TBP、65mmol/L DTT、0.2%载体两性电解质(其中0.1%pH 3~10,0.1%pH4~6)]溶解蛋白,以pH4~7线性17cm的IPG胶条进行双向凝胶电泳,在上样量为800μg,13%SDS-PAGE胶浓度下,蛋白质得到了更好的分离,2-DE图谱上可分辨出631个蛋白点,图谱质量最佳。优化后的双向电泳技术体系,适合于小麦花药全蛋白质的双向电泳分析。     

Rapid Isolation of Sorghum and Other Cereal Starches Using Sonication

High‐intensity ultrasound (sonication) was investigated as a method to rapidly purify starch from sorghum and other cereal grains. To improve the process, buffers were optimized to solubilize sorghum proteins in combination with the sonication. Protein content and starch color were determined to evaluate the efficiency of the extraction process. Sonication times, SDS concentration, different types and concentrations of reducing agents (sodium metabisulfite, dithiothreitol, and β‐mercaptoethanol), and centrifugation speeds of the starch washing procedure were tested. Protein content of isolated sorghum starch was reduced to 0–0.14% (db) after 2 min of sonication (using any of the reducing agents tested). Sodium metabisulfite was chosen as the preferred reducing agent because of its lower toxicity and odor compared with other reducing agents tested. The optimum conditions for producing high‐purity sorghum starches (0.06% protein) were obtained using the following conditions: 2 min of sonication time with 12.5 mM sodium borate buffer, pH 10, containing 0.5% SDS (w/v) and 0.5% sodium metabisulfite (w/v) using 1,500 rpm centrifugation speed during starch washing. Starches separated by this method showed significantly less protein content and b values (yellowness) compared with starches separated by enzymatic methods or methods using NaCl solutions and protein extraction buffers with multiple washing steps, both of which take several hours to complete. Differential scanning calorimetry thermogram values for starches isolated by three different methods showed similar patterns, except that starches obtained with the enzymatic method had slightly higher values of T_o, T_p, and ΔH. Other cereal starches from whole wheat meal, wheat flour, corn, rice, and barley were also obtained rapidly using sonication.     

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.     

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.     

Assessment of the effect of molecular size on the electrophoretic mobility of humic substances

The basic principles of the electrophoretic behaviour of humic substances (HS) still need to be systematically investigated. HS extracted from a Cryorthod (HS–1) and from an Haplumbrept (HS–2) were first fractionated by ultrafiltration to obtain two series of fractions of reduced polydispersity with a nominal mean relative molecular mass (M) of 7500, 20000, 40000, 75000 and 200 000 Da. Fractions extracted from the same soil behaved like an homologous series when tested by size exclusion chromatography, showing a linear relation of M with molecular size. When plotted against their mean M, the elution volumes of HS fractions extracted from the two different soils originated lines of different slope. Absolute electrophoretic mobilities of the fractions were determined in polyacrylamide gel slabs of increasing polyacrylamide concentration using a single buffer system; in all gels there was a close linear relation between the electrophoretic mobility and the logarithm of the mean M of HS fractions. Extrapolation of mobilities of HS–1 at zero gel concentration gave intercepts that did not differ significantly, showing that there was a constant mobility for all fractions in free solution. These results mean that charge differences have little effect on the electrophoretic mobilities of HS extracted from the same soil and imply the theoretical possibility of determining M distributions of HS by polyacrylamide gel electrophoresis in a single buffer system. Mobilities of HS–2 fractions were compared with those of HS–1: the latter fitted the regression equations of mobility against logarithm of the molecular weight obtained from HS–1 humic substances only in gels of small acrylamide concentration. Deviations were larger at small M, probably because of the increasing fulvic character of the fractions, and increased in gels of greater acrylamide concentration, indicating that charge differences may not be negligible when comparing humic substances extracted from different soils.     

Effects of Amylose,Corn Protein,and Corn Fiber Contents on Production of Ethanol from Starch‐Rich Media

The effects of amylose, protein, and fiber contents on ethanol yields were evaluated using artificially formulated media made from commercial corn starches with different contents of amylose, corn protein, and corn fiber, as well as media made from different cereal sources including corn, sorghum, and wheat with different amylose contents. Second‐order response‐surface regression models were used to study the effects and interactions of amylose, protein, and fiber contents on ethanol yield and conversion efficiency. The results showed that the amylose content of starches had a significant (P < 0.001) effect on ethanol conversion efficiency. No significant effect of protein content on ethanol production was observed. Fiber did not show a significant effect on ethanol fermentation either. Conversion efficiencies increased as the amylose content decreased, especially when the amylose content was >35%. The reduced quadratic model fits the conversion efficiency data better than the full quadratic model does. Fermentation tests on mashes made from corn, sorghum, and wheat samples with different amylose contents confirmed the adverse effect of amylose content on fermentation efficiency. High‐temperature cooking with agitation significantly increased the conversion efficiencies on mashes made from high‐amylose (35–70%) ground corn and starches. A cooking temperature of ≥160°C was needed on high‐amylose corn and starches to obtain a conversion efficiency equal to that of normal corn and starch.     

Phenolic Content,Antioxidant Activity,and Consumer Acceptability of Sorghum Cookies

Cookies were produced from different sorghum flours to determine their potential as vectors of antioxidants. Different sorghum cultivars and their flour extraction rates were evaluated for their effects on phenolic content and antioxidant activity of the cookies. Consumer acceptance of the sorghum cookies was compared with that of wheat flour cookies. For each sorghum cultivar, cookies of 100% extraction rate flours had two to three times more total phenolics compared with those of 70% extraction rate flours, while antioxidant activity was 22–90% higher. Cookies of the condensed tannin sorghum had two to five times more phenolics compared with those of condensed tannin‐free sorghum. Antioxidant activity was 145–227 μMol Trolox equivalents (TE)/g in cookies of condensed tannin sorghum compared with 10–102 μMol TE/g in those of condensed tannin‐free sorghum. The sorghum flours had slightly higher phenolic content and antioxidant activity values than their corresponding cookies. Cookies of the red tannin‐free sorghum flours (PAN 8564/8446) were equally liked as wheat flour cookies, except for texture. However, cookies of condensed tannin sorghum were least accepted compared with wheat flour cookies despite their high antioxidant activity.     

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.     

A Rapid Protein Digestibility Assay for Identifying Highly Digestible Sorghum Lines

Protein digestibility in sorghum (Sorghum bicolor (L.) Moench) lines was determined using two standard procedures (pepsin digestibility and pH‐stat) and compared with a newly developed, rapid electrophoresis‐based screening assay. The new assay was based on the rate of α‐kafirin disappearance after pepsin digestion. α‐Kafirin, the major sorghum storage protein, makes up ≈60–70% of the total protein in the grain. In the new assay, samples were first digested with pepsin for 1 hr, and undigested proteins were then analyzed by SDS‐PAGE. The intensitizes of the undigested α‐kafirin bands were measured. Higher band intensity indicated lower protein digestibility. The new assay was significantly correlated with the standard pepsin digestibility assay (r = −0.96, n = 16) after which it was patterned. The same was true of the pH‐stat procedure (r = −0.85, n = 16). This implies that the new assay is comparable to existing procedures and can be used for screening sorghum lines for protein digestibility. Two groups consisting of high‐protein digestibility and wild‐type sorghum lines were identified when the new assay was tested on 48 sorghum lines derived from crosses of wild‐type and mutant high protein digestibility lines, indicating that the new assay was efficient in differentiating between the two groups. Advantages of the new assay over the standard procedures include considerable reduction in analysis time and sample size required for the analysis. For example, analysis time was reduced by 20% and sample size by 10% when the new assay was used as compared with the pH‐stat procedure. We estimate that ≈60 sorghum lines can be screened in a day by a single operator using the new assay.     

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.     

Quantitative Glutenin Composition from Gel Electrophoresis of Flour Mill Streams and Relationship to Breadmaking Quality

Three samples of Nekota (hard red winter wheat) were milled, and six mill streams were collected from each sample. The 18 mill streams were analyzed separately as well as recombined to form three patent flours. The methods of multistacking (MS)‐SDS‐PAGE and SDS‐PAGE were used to separate the unreduced SDS‐soluble glutenins and the total reduced proteins, respectively. The separated proteins were quantified by densitometry. The quantity of unreduced SDS‐soluble proteins was significantly different among the mill streams at the 4% (largest molecular weight polymeric glutenins) and at the 10 and 12% (smaller molecular weight polymeric glutenins) origins of the MS‐SDS‐PAGE gels. The quantities of total HMW‐GS, LMW‐GS, 2*, 7+9, and 5+10 subunits and the ratio of HMW‐GS to LMW‐GS in polymeric protein samples isolated using preparative MS‐SDS‐PAGE and in total reduced protein extracts were significantly different among mill streams. The quantities of HMW‐GS, LMW‐GS, 2*, 7+9, and 5+10 subunits from total reduced proteins were positively and significantly correlated with loaf volume. The quantities of glutenin subunits (both HMW‐GS and LMW‐GS) from unreduced SDS‐soluble proteins were positively or negatively correlated with loaf volume at the various MS‐SDS‐PAGE gel origins but the levels of correlation were not significant. These results showed that the glutenin protein composition was different among the various mill streams and demonstrated that electrophoretic analysis of the proteins in these fractions is a useful tool for studying the variation in functional properties of flour mill streams.     

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.     

Detection of Proteins in Starch Granule Channels

Proteins were detected in channels of commercial starches of normal maize, waxy maize, sorghum, and wheat through labeling with a protein‐specific dye and examination using confocal laser scanning microscopy (CLSM). The dye, specifically 3‐(4‐carboxybenzoyl)quinoline‐2‐carboxaldehyde (CBQCA), fluoresces only after it reacts with primary amines in proteins, and CLSM detects fluorescence‐labeled protein distribution in an optical section of a starch granule while it is still in an intact state. Starch granules in thin sections of maize kernels also had channel proteins, indicating that proteins are native to the channels and not artifacts of isolation. Incubation of maize starch with protease (thermolysin) removed channel proteins, showing that channels are open to the external environment. SDS‐PAGE analysis of total protein from gelatinized commercial waxy maize starch revealed two major proteins of about M_r 38,000 and 40,000, both of which disappeared after thermolysin digestion of raw starch. Commercial waxy maize starch granule surface and channel proteins were extracted by SDS‐PAGE sample buffer without gelatinization of the granules. The major M_r 40,000 band was identified by MALDI‐TOF‐MS and N‐terminal sequence analysis as brittle‐1 (bt1) protein.     

Optimizing Quantitative Reproducibility in High‐Performance Capillary Electrophoresis (HPCE) Separations of Cereal Proteins

High‐performance capillary electrophoresis (HPCE) is capable of producing high‐resolution, rapid separations of cereal proteins. Furthermore, HPCE is highly reproducible in terms of migration time. However, little work has focused on the quantitative reproducibility of cereal protein separations. Several factors such as sample matrix, sample evaporation, voltage ramp‐up time, sample injection time, and capillary end‐cut were evaluated for involvement in quantitative reproducibility. These experiments showed that preventing sample evaporation, using optimum injection times, and ensuring a clean, square cut on the capillary all improved the reproducibility of peak areas. Combining these factors into an optimized procedure produced reproducibility with peak areas varying by 1.76% relative standard deviation (RSD). Migration time was also excellent under these conditions, varying by only 0.45% RSD. Other variables such as peak area percent, peak height, and peak height percent also showed good reproducibility with RSD < 4%. Increasing the voltage ramp‐up time from 0.17 to 0.68 increased peak efficiency by ≈150%. This factor had no effect on quantitative reproducibility, however. The gradual buildup of contaminants on the capillary walls occurred over time and decreased both separation efficiency and reproducibility. Rinsing capillaries periodically with appropriate solvents delayed this effect. Peak efficiency was a good marker for capillary performance and lifetime.     

Effect of Moisture Content and Tempering Method on the Functional and Sensory Properties of Popped Sorghum

Red and white sorghum were tempered using two methods (direct and indirect) to adjust moisture content (MC) to 12, 14, and 17% and popped using a hot‐air popper. Kernel size, pericarp thickness, bulk density, popped kernel expansion volume and ratio, popped volume, and surface color were recorded. Forty‐six untrained panelists evaluated popped sorghum using a 9‐point hedonic scale and expressed preference using a paired comparison test. Sorghum with thicker pericarp (red) had higher percent popped, expansion ratio, and popped volume. In white sorghum, indirect tempering increased percent popped and tempering to 17% MC increased popped volume. Panelists significantly preferred popcorn in appearance (8.0 ± 1.0) and size (7.9 ± 1.0) to sorghum (6.6 ± 1.7 appearance and 5.6 ± 2.2 size for 12 and 17% MC tempering). Panelists equally preferred the taste of popcorn (7.1 ± 1.5) and popped sorghum tempered to 12% MC (6.4 ± 1.8) and 17% MC (6.9 ± 1.4). Sorghum pops were brighter, vividly whiter, and 5× smaller than popcorn.