Helen L. Beasley

Synergistic and Additive Effects of Three High Molecular Weight Glutenin Subunit Loci. II. Effects on Wheat Dough Functionality and End-Use Quality

ABSTRACT Understanding the relationship between basic and applied rheological parameters and the contribution of wheat flour protein content and composition in defining these parameters requires information on the roles of individual flour protein components. The high molecular weight glutenin subunit (HMW-GS) proteins are major contributors to dough strength and stability. This study focused on eight homozygous wheat lines derived from the bread wheat cvs. Olympic and Gabo with systematic deletions at each of three HMW-GS encoding gene loci, Glu-A1, Glu-B1, and Glu-D1. Flour protein levels were adjusted to a constant 9% by adding starch. Functionality of the flours was characterized by small-scale methods (2-g mixograph, microextension tester). End-use quality was evaluated by 2-g microbaking and 10-g noodle-making procedures. In this sample set, the Glu-D1 HMW-GS (5+10) made a significantly larger contribution to dough properties than HMW-GS coded by Glu-B1 (17+18), while subunit 1 coded by Glu-A1 made ...

Synergistic and Additive Effects of Three High Molecular Weight Glutenin Subunit Loci. I. Effects on Wheat Dough Rheology

ABSTRACT The high molecular weight glutenin subunits (HMW-GS) play an important role in governing the functional properties of wheat dough. To understand the role of HMW-GS in defining the basic and applied rheological parameters and end-use quality of wheat dough, it is essential to conduct a systematic study where the effect of different HMW-GS are determined. This study focuses on the effect of HMW-GS on basic rheological properties. Eight wheat lines derived from cvs. Olympic and Gabo were used in this study. One line contained HMW-GS coded by all three loci, three lines were each null at one of the loci, three lines were null at two of the loci and one line null at all three loci. The flour protein level of all samples was adjusted to a constant 9% by adding starch. In another set of experiments, in addition to the flour protein content being held at 9%, the glutenin-to-gliadin ratio was maintained at 0.62 by adding gliadin. Rheological properties such as elongational, dynamic, and shear viscometric ...

The characterisation and mapping of a family of LMW-gliadin genes: effects on dough properties and bread volume

Abstract.Analysis of a cDNA library from wheat cv Wyuna endosperm, indicated a significant size and sequence variation among seed-endosperm protein genes. In this study, a family of low-molecular-weight seed protein genes are analysed that are related to the gliadins and the low-molecular-weight glutenin subunits. Sequence analysis and comparison of these proteins showed that they are closely related to a 17-kDa protein from barley, ε hordein, which plays a role in beer foam stability in the brewing industry. Mapping of these genes in wheat shows that they are located on group 7 and 4 chromosomes, as opposed to a group 1 and 6 location for the glutenins and gliadins. It is possible that this family of proteins forms a new class of seed-endosperm proteins important in defining the quality characteristics of wheat flour. Therefore, a representative gene from this family was expressed in Escherichia coli and the purified protein was supplemented into a base wheat flour. Rheological analysis showed that the protein effected dough strength and resistance break down during mixing of the dough, and provided a 20% increase in loaf height after baking.

A new technique to measure bird’s dietary exposure to pesticides

Abstract Current methods to analyse pesticide residues in wild animals are essentially forensic examination procedures. This paper describes a new approach to obtain samples from live birds for pesticide residue analysis without harm to the animal involved. The technique measures the dietary exposure and has been tested successfully in birds exposed to farm chemicals in Australia. Stomach flushing with warm water was used to obtain ‘flushing’ samples (stomach and droppings combined) from 11 species of birds at four locations near or away from cotton farms. Water samples from all sites were also taken. Immunoassays were used to analyse these samples for residues of DDE, DDT, diuron, endosulfan and parathion-methyl. ELISA is suited to environmental residue analysis because of its high sensitivity, small sample volume requirements, low cost and speed. Prior to the residue analysis, optimization of the assays and elimination of matrix effects are essential. Positive residues were found in 90% of the birds, their amounts varying up to four-orders of magnitude amongst individuals and species, with predators and insectivorous having higher levels than granivorous and nectivores. The technique can be applied to the same animal over a period of time, thus providing a useful tool for monitoring programs in environmental studies. Its application to ecological risk assessment and exposure are discussed.

Preparative Method for In Vitro Production of Functional Polymers from Glutenin Subunits of Wheat

ABSTRACT An in vitro method for preparative-scale production of artificial glutenin polymers utilizes a controlled environment for the oxidation of glutenin subunits (GS) isolated from wheat flour to achieve high polymerization efficiency. The functionality of in vitro polymers was tested in a 2-g model dough system and was related to the treatment of the proteins before, during, and after in vitro polymerization. When added as the only polymeric component in a reconstituted model dough (built up from gliadin, water solubles, and starch fractions), in vitro polymers could mimic the behavior of native glutenin, demonstrating properties of dough development and breakdown. Manipulating the high molecular weight (HMW)-GS to a low molecular weight (LMW)-GS ratio altered the molecular weight distribution of in vitro polymers. In functional studies using the 2-g mixograph, simple doughs built up from homopolymers of HMW-GS were stronger than those using homopolymers of LMW-GS. These differences may be accounted ...

Applications and limitations of immunochemical analysis of biopolymer quality in cereals

The mixing and baking properties of wheaten doughs are determined largely by the content, composition and interactions of the major groups of flour proteins, the disulphide‐bonded glutenin subunits and monomeric gliadins. Prediction of dough and bread quality is currently based on medium‐scale rheological and baking tests, but the slow throughput of such tests limits their use both by millers and baking companies and in early‐generation screening by plant breeders. Thus identification and quantification of specific flour proteins by immunoassay has the potential advantages of speed, simplicity and applicability to small samples in breeding. Technical problems can arise from the low solubilities of these proteins and their high degrees of sequence homology (which often give rise to extensive antibody cross‐reaction). These problems can be minimized by modifications to methods and combining monoclonal antibodies with selected extraction conditions to enhance the functional specificity of the assay. Limitati...

Sample Matrix Interference in Immunoassays for Organochlorine Residues in Plant-derived Foods and Some Strategies for Their Removal

Immunoassays for two groups of organochlorine insecticides, cyclodienes (endosulfan and heptachlor) and DDT were applied to the analysis of a diverse range of plant-derived foods. Water-miscible solvent extracts of high-moisture, low-fat foods such as cauliflower, cabbage, green and red blue grapes and tomato caused little or no interference with the assays, enabling methanol or acetonitrile extracts of the foods to be analysed directly by immunoassay, after dilution in assay buffer. Reasonable recoveries of spikes of these pesticides were obtained by direct analysis of extracts of spiked commodities, with reliable detection down to 0.025 mg kg−1 heptachlor or endosulfan and 0.1 mg kg−1 DDT in the commodities. Acetonitrile extracts of milk could also be analysed directly for DDT. In contrast, extracts of low moisture, non-fatty (rice) and fatty (cottonseed) food commodities interfered appreciably with the assays, reducing assay colour and detection sensitivity. Some simple cleanup methods were developed to remove interference and enable detection of spiked organochlorines in these foods. Extracts of coloured foods, such as tea, coffee and spinach caused similarly major interference in the assays, and a number of simple clean-up methods were ineffective in removing interference. However, use of an immunoaffinity chromatography method for cyclodienes enabled quantitative recoveries to be obtained in extracts of several of these foods when analysed by either ELISA or gas chromatography. Direct analysis was suited for screening purposes but immunoaffinity chromatography results were more quantitative. These results indicate that ELISAs can be applied under developing country conditions to a range of diverse foods, but that cleanup strategies need to be tailored to different types of foods.

An Enzyme Immunoassay for the Organochlorine Insecticide Hexachlorocyclohexane (HCH), Through Conversion to Trichlorophenols

A method for immunoassay analysis of the organochlorine insecticide, hexachlorocyclohexane (HCH) has been developed, based upon alkaline conversion in standards and samples to trichlorobenzenes. The trichlorobenzenes were detected through antisera developed to haptens containing either a trichlorobenzene or trichlorpyridine moiety, developed using the herbicides, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and triclopyr, respectively. An enzyme conjugate based on 2,4,5-trichlorophenol provided most sensitive and specific detection. Although the assays cross-reacted with the herbicides, they did not suffer from the major disadvantage of extremely strong cross-reaction with cyclodiene organochlorines reported for a commercial HCH assay. The most sensitive assay had a lower detection limit of 20 w g l−1 in drinking water and was applied to water and soil matrices.

Rapid field tests for the organophosphorus pesticides, fenitrothion and pirimiphos-methyl—Reliable estimates of residues in stored grain

Abstract In order for grain handlers and traders to reliably estimate residues of grain protectants in the field, antibody-based tests were developed for the organophosphorus pesticides, fenitrothion and pirimiphos-methyl. To complement the rapid analysis, rapid but efficient extraction techniques were developed. In these tests, a pesticide-containing methanol extract of the grain sample and an enzyme-labelled component are added dropwise to precoated tubes containing buffer. After a brief incubation, the tubes are rinsed out in tap water and a substrate/chromogen for the enzyme is added. The colour developed is stabilized by acidification and the test result read either by eye or in a portable field photometer. Significant levels of the particular pesticide result in a pale colour compared to a dark green pesticide-free control. No calculations were required to provide a quantitative estimate of residue as this could be read directly from a graph of colour yield (absorbance) vs logarithm of pesticide concentration, using standard solutions of pesticide. For fenitrothion, the test had a limit of detection of 4 ng/ml (0.1 ppm in grain) and gave quantitative estimates in the range 0.5–15 ppm (in the grain), while the pirimiphos-methyl test had a limit of detection of 1 ng/ml (0.03 ppm in grain) and gave quantitative estimates over the range 0.1–15 ppm. Thus both tests can be used to segregate “pesticide-free” grain, with residues below 0.1 ppm. Data obtained using the field test correlated well with those obtained using laboratory methods, including both gas-liquid chromatography and immunoassay using microwell plates. The field immunoassay reagents were formatted into a small prototype test kit, and the components stabilised for field use.

Validation of Pyrithiobac Sodium (Staple® Herbicide) ELISA for Australian Cotton Soils

An enzyme‐linked immunosorbent assay (ELISA) for pyrithiobac‐sodium (Staple®) produced by DuPont was validated in Australian soils. This pyrithiobac‐sodium ELISA was shown to be highly sensitive with the limit of detection of 4–5 ppt. Soil samples were extracted either in PBS buffer by shaking or by accelerated solvent extraction (ASE). While pyrithiobac sodium can be analyzed directly by ELISA after ASE extraction with 1/10 or more dilutions, the analysis of PBS extract required filtration and dilution 1/20 or more depending on the concentration. Immunoassay results compared favorably with GC‐MS results for both ASE and PBS extract of incurred residue of pyrithiobac sodium in soil samples, indicating that this ELISA can be an inexpensive and reliable alternative to conventional residue analysis methods for quantification of pyrithiobac‐sodium. This validation provided the basis for applying the ELISA to a field study of pyrithiobac‐sodium.