Rhett C. Kaufman

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.

Position of modifying groups on starch chains of octenylsuccinic anhydride-modified waxy maize starch

Octenylsuccinic anhydride (OSA)-modified starches with a low (0.018) and high (0.092) degree of substitution (DS) were prepared from granular native waxy maize starch in aqueous slurry. The position of OS substituents along the starch chains was investigated by enzyme hydrolysis followed by chromatographic analysis. Native starch and two OS starches with a low and high DS had β-limit values of 55.9%, 52.8%, and 34.4%, respectively. The weight-average molecular weight of the β-limit dextrin from the OS starch with a low DS was close to that of the β-limit dextrin from native starch but lower than that of the β-limit dextrin from the OS starch with a high DS. Debranching of OS starches was incomplete compared with native starch. OS groups in the OS starch with a low DS were located on the repeat units near the branching points, whereas the OS substituents in the OS starch with a high DS occurred both near the branching points and the non-reducing ends.

Variability in tannin content, chemistry and activity in a diverse group of tannin containing sorghum cultivars.

BACKGROUND Tannins are large polyphenolic polymers and are known to bind proteins, limiting their digestibility, but are also excellent antioxidants. Numerous studies investigating the functional properties of sorghum tannin have been conducted by comparing grain samples from different sorghum lines without considering the other intrinsic characteristics of the grain. The purpose of this study was to remove the confounding intrinsic factors present in the endosperm so the effect of the tannins could be evaluated utilizing a unique decortication/reconstitution procedure. RESULTS The tannin content of the 14 cultivars tested ranged from 2.3 to 67.2 catechin equivalents. The bran fractions were studied for their impact on protein binding and antioxidant capacity. Protein digestibility by pepsin ranged from 8% to 58% at the highest tannin level addition. Protein binding ranged from 3.11 to 16.33 g blue bovine serum albumin kg⁻¹ bran. Antioxidant capacity ranged from 81.33 to 1122.54 µmol Trolox equivalents g⁻¹ bran. High-performance size-exclusion chromatography detailed molecular size distributions of the tannin polymers and relationship to tannin functionality. CONCLUSION The tannin content and composition play a significant role in determining tannin functionality. These differences will allow for selections of high-tannin sorghums with consideration of the biological activities of the tannins.

Structure of Waxy Maize Starch Hydrolyzed by Maltogenic α-Amylase in Relation to Its Retrogradation.

Maltogenic α-amylase is widely used as an antistaling agent in bakery foods. The objective of this study was to determine the degree of hydrolysis (DH) and starch structure after maltogenic amylase treatments in relation to its retrogradation. Waxy maize starch was cooked and hydrolyzed to different degrees by a maltogenic amylase. High-performance anion-exchange chromatography and size exclusion chromatography were used to determine saccharides formed and the molecular weight (Mw) distributions of the residual starch structure, respectively. Chain length (CL) distributions of debranched starch samples were further related to amylopectin (AP) retrogradation. Differential scanning calorimetry (DSC) results showed the complete inhibition of retrogradation when starches were hydrolyzed to >20% DH. Mw and CL distributions of residual AP structure indicated that with an increase in %DH, a higher proportion of unit chains with degree of polymerization (DP) ≤9 and a lower proportion of unit chains with DP ≥17 were formed. A higher proportion of short outer AP chains that cannot participate in the formation of double helices supports the decrease in and eventual inhibition of retrogradation observed with the increase in %DH. These results suggest that the maltogenic amylase could play a powerful role in inhibiting the staling of baked products even at limited starch hydrolysis.

Grain sorghum proteomics: integrated approach toward characterization of endosperm storage proteins in kafirin allelic variants

Grain protein composition determines quality traits, such as value for food, feedstock, and biomaterials uses. The major storage proteins in sorghum are the prolamins, known as kafirins. Located primarily on the periphery of the protein bodies surrounding starch, cysteine-rich β- and γ-kafirins may limit enzymatic access to internally positioned α-kafirins and starch. An integrated approach was used to characterize sorghum with allelic variation at the kafirin loci to determine the effects of this genetic diversity on protein expression. Reversed-phase high performance liquid chromatography and lab-on-a-chip analysis showed reductions in alcohol-soluble protein in β-kafirin null lines. Gel-based separation and liquid chromatography-tandem mass spectrometry identified a range of redox active proteins affecting storage protein biochemistry. Thioredoxin, involved in the processing of proteins at germination, has reported impacts on grain digestibility and was differentially expressed across genotypes. Thus, redox states of endosperm proteins, of which kafirins are a subset, could affect quality traits in addition to the expression of proteins.

Impacts of kafirin allelic diversity, starch content, and protein digestibility on ethanol conversion efficiency in grain sorghum.

ABSTRACT 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...

Improved characterization of sorghum tannins using size-exclusion chromatography.

L. Moench) is a native grass spe-cies of the arid and semi-arid regions of Africa (Kimber 2000). Sorghum is a genetically diverse crop and contains some geno-types that have a pigmented testa and therefore contain tannins (Rooney et al 1980; Rooney and Miller 1982). Such lines have the dominant B1_B2_ genes and are the only sorghum types with tannins (Blakeley et al 1979), despite common misperceptions that all sorghums have tannins or that the presence of tannins is linked to seed color (Boren and Waniska 1992). Due to the high antioxidant activity, sorghum tannins have recently been exam-ined for potential as a nutraceutical, especially cancer prevention (Awika and Rooney 2004). Generally, tannins which are polymers, are characterized on the basis of their monomeric composition, and their degree of polym-erization, both of which can impact their functionality (Asquith et al 1983). Many analytical methods have been used to study their composition such as reversed phase (RP) HPLC (Putman and Butler 1989; Prior and Gu 2005), normal-phase (NP) HPLC (Gu et al 2002; Awika et al 2003), size-exclusion chromatography (SEC) (Williams et al 1983; Karchesy et al 1988; Kennedy and Taylor 2003), capillary electrophoresis (CE) (Bae et al 1994; Ci-fuentes et al 2001; Bonoli et al 2004), and mass spectrometry (Cheynier and Fulcrand 2003; Kruger et al 2003). It is generally agreed that sorghum tannins are composed of monomeric flavan-3-ols. Thus, recent research has focused on the degree of polymerization of these subunits, with one limitation being how well the largest polymers (i.e., a high degree of polym-erization [DP]) can be resolved. Recently, NP-HPLC has resolved tannins to a DP of >10 and has significantly improved the separa-tion and characterization of tannins from a number of sources, including sorghum (Hammerstone et al 1999; Gu et al 2002). While NP-HPLC was capable of excellent resolution, run times were lengthy (40 min) and NP-HPLC must be conducted in the total absence of water, which complicates switching instruments between types of HPLC such as RP-HPLC. Thus, the objectives of this study were to investigate the use of improved SEC procedures for analyzing sorghum tannins that required no special sample preparation (such as derivatization, tannin purification, etc.) and that would provide information on the M

A Rapid Method to Determine Starch Damage in Sorghum

As a major component of cereal grains, including sorghum, starch plays an important role not only in grain development but also in post-harvest processing and end-product quality. Since milling can lead to the inadvertent disruption of starch granules, negatively affecting dough rheology, monitoring of starch damage is an important part of flour formulation. As the existing methods for quantifying starch damage are time-consuming, an alternative non-enzymatic methodology was sought. This study described the use of SDmatic—an instrument developed for determining starch damage in wheat flour—for assessment of sorghum flour via generation of a standard calibration. The known starch damage values were integrated to the SDmatic Ai%, and the starch damage was calculated using the following equation: Starch Damage = aAi%2 + bAi% + c. The following variables were developed for calibrating sorghum starch damage: a = 0.168, b = −30.123, and c = 1349.648. Using this calibration developed specifically for sorghum flour increased our linear regression from initial R2 = 0.38 to R2 = 0.95.