Differential effects of a post-anthesis fertilizer regimen on the wheat flour proteome determined by quantitative 2-DE.

BACKGROUND: Mineral nutrition during wheat grain development has large effects on wheat flour protein content and composition, which in turn affect flour quality and immunogenic potential for a commodity of great economic value. However, it has been difficult to define the precise effects of mineral nutrition on protein composition because of the complexity of the wheat flour proteome. Recent improvements in the identification of flour proteins by tandem mass spectrometry (MS/MS) and the availability of a comprehensive proteome map of flour from the US wheat Butte 86 now make it possible to document changes in the proportions of individual flour proteins that result from the application of mineral nutrition. RESULTS: Plants of Triticum aestivum 'Butte 86' were grown with or without post-anthesis fertilization (PAF) and quantitative 2-dimensional gel electrophoresis (2-DE) was used to analyze protein composition of the resulting flour. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, high molecular weight glutenin subunits (HMW-GS) and serpins as well as some alpha-gliadins increased in proportion with PAF. In contrast, alpha-amylase/protease inhibitors, farinins, purinins and puroindolines decreased in proportion. Decreases were also observed in several low molecular weight glutenin subunits (LMW-GS), globulins, defense proteins and enzymes. The ratio of HMW-GS to LMW-GS in the flour increased from 0.61 to 0.95 and the ratio of gliadins to glutenins increased from 1.02 to 1.30 with PAF. Because flour protein content doubled with PAF from 7 to 14%, most protein types actually increased in absolute amount (µg/mg flour protein). Data further suggest that flour proteins change with PAF according to their content of sulfur-containing amino acids Cys + Met. CONCLUSIONS: A 2-DE approach revealed changes in the wheat flour proteome due to PAF that are important for flour quality and immunogenic potential. The work forms a baseline for further studies of the effects of environmental variables on flour protein composition and provides clues about the regulation of specific flour protein genes. The study also is important for identifying targets for breeding programs and biotechnology efforts aimed at improving flour quality.

Effects of environmental stress on stability of tandem repeats in Escherichia coli O157:H7.

Multilocus variable-number tandem-repeat analysis (MLVA) is used for source tracking Escherichia coli O157:H7 in agricultural environments. Tandem repeats were stable after limited replication but changed after exposure to irradiation, elevated temperatures, and starvation conditions. The pO157 plasmid was frequently lost under these stress conditions. Environmental stresses may increase phylogenetic diversity as measured by MLVA.

Studies on the aroma of five fresh tomato cultivars and the precursors of cis- and trans-4,5-epoxy-(E)-2-decenals and methional.

Three tasty (BR-139, FA-624, and FA-612) and two less tasty (R-144 and R-175) fresh greenhouse tomato cultivars, which significantly differ in their flavor profiles, were screened for potent odorants using aroma extract dilution analysis (AEDA). On the basis of AEDA results, 19 volatiles were selected for quantification in those 5 cultivars using gas chromatography-mass spectrometry (GC-MS). Compounds such as 1-penten-3-one, ( E, E)- and ( E, Z)-2,4-decadienal, and 4-hydroxy-2,5-dimethyl-3(2 H)-furanone (Furaneol) had higher odor units in the more preferred cultivars, whereas methional, phenylacetaldehyde, 2-phenylethanol, or 2-isobutylthiazole had higher odor units in the less preferred cultivars. Simulation of the odor of the selected tomato cultivars by preparation of aroma models and comparison with the corresponding real samples confirmed that all important fresh tomato odorants were identified, that their concentrations were determined correctly in all five cultivars, and that differences in concentration, especially of the compounds mentioned above, make it possible to distinguish between them and are responsible for the differential preference. To help elucidate formation pathways of key odorants, labeled precursors were added to tomatoes. Biogenesis of cis- and trans-4,5-epoxy-( E)-2-decenals from linoleic acid and methional from methionine was confirmed.

Effects of methyl bromide concentration, fumigation time, and fumigation temperature on Mediterranean and oriental fruit fly (Diptera: Tephritidae) egg and larval survival.

The effects of methyl bromide (MB) concentration (16, 32,48, or 64 g/m3), fumigation temperature (15, 20, 25, or 30 degrees C), and fumigation time interactions on the survival of Mediterranean fruit fly, Ceratitis capitata (Wiedemann), and oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), eggs and first and third instars were recorded. Increasing the fumigation temperature from 15 to 20 degrees C or from 20 to 25 degrees C resulted in a significant reduction in fumigation time required for equivalent egg and larval mortalities at all studied MB concentrations; no further reductions in fumigation time resulted from increasing the temperature from 25 to 30 degrees C. Conversely, increasing temperature and time allowed for a reduction in MB concentration to obtain equivalent mortality. Thus, the optimum fumigation temperature for Mediterranean and oriental fruit fly eggs and larvae was 25 degrees C. Reducing MB concentrations required for phytosanitary fumigations would save time and expense, and reduce the amount of MB released into the atmosphere during aeration. Mediterranean fruit fly was as or more tolerant to MB than oriental fruit fly in MB tolerance for eggs and first instars. The egg stage was generally more tolerant to MB regardless of concentration. However, Mediterranean fruit fly eggs showed similar tolerance to first instars at 25 degrees C for the three highest concentrations and to third instars at 25 and 30 degrees C for the highest concentration, with no significant difference between them. Therefore, eggs alone can be used to obtain MB fumigation efficacy and quarantine security data at fumigation temperatures between 15 and 30 degrees C for Mediterranean and oriental fruit fly.

Optimization of a supercritical fluid extraction/reaction methodology for the analysis of castor oil using experimental design.

The aim of this work was to optimize a supercritical fluid extraction (SFE)/enzymatic reaction process for the determination of the fatty acid composition of castor seeds. A lipase from Candida antarctica (Novozyme 435) was used to catalyze the methanolysis reaction in supercritical carbon dioxide (SC-CO(2)). A Box-Behnken statistical design was used to evaluate effects of various values of pressure (200-400 bar), temperature (40-80 degrees C), methanol concentration (1-5 vol %), and water concentration (0.02-0.18 vol %) on the yield of methylated castor oil. Response surfaces were plotted, and these together with results from some additional experiments produced optimal extraction/reaction conditions for SC-CO(2) at 300 bar and 80 degrees C, with 7 vol % methanol and 0.02 vol % water. These conditions were used for the determination of the castor oil content expressed as fatty acid methyl esters (FAMEs) in castor seeds. The results obtained were similar to those obtained using conventional methodology based on solvent extraction followed by chemical transmethylation. It was concluded that the methodology developed could be used for the determination of castor oil content as well as composition of individual FAMEs in castor seeds.

Analysis of fenbendazole residues in bovine milk by ELISA.

Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.

Prevalence of Escherichia coli associated with a cabbage crop inadvertently irrigated with partially treated sewage wastewater.

Preharvest contamination of field crops may have many sources, including feces, soil, and irrigation water. In March 2000, a sewage spill released unchlorinated tertiary-treated effluent into a creek used to irrigate commercial produce. A field of young cabbage transplants was irrigated with creek water as the contaminated water flowed past this land. Cabbage samples were taken from plots within this field, and Escherichia coli was isolated from the roots of these plants but not from the edible portion of the cabbage. No E. coli was isolated from water samples or from control samples taken from a nearby cabbage field watered with chlorinated municipal water. The cabbage field under study had not been fertilized with manure for at least 2 years prior to the contamination incident. Six different E. coli serotypes were identified, although none of them proved to be pathogenic. These serotypes were separated into five groups by a RiboPrinter; the resulting groups correlated well with the serotypes and the locations in the field from which these strains were isolated. We previously found that certain nonpathogenic E. coli strains displayed lower levels of adherence to lettuce seedling roots in a hydroponic adherence assay. The E. coli field strains displayed variable patterns of adherence to lettuce seedlings: strain MW421 showed significantly lower root and shoot adherence levels than did the other field strains, while strains MW423 and MW425 showed significantly higher root and shoot adherence levels. These data suggest that water quality is of paramount importance for the food safety of growing crops.

Association of Escherichia coli O157:H7 with preharvest leaf lettuce upon exposure to contaminated irrigation water.

Recent foodborne outbreaks have linked infection by enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 to the consumption of contaminated lettuce. Contamination via food handler error and on-the-farm contamination are thought to be responsible for several outbreaks. Though recent studies have examined the application of EHEC to store-bought lettuce, little is known about the attachment of EHEC to growing plants. We investigated the association of lettuce seedlings with EHEC O157:H7 strains implicated in lettuce or fruit outbreaks using hydroponic and soil model systems. EHEC strains that express the green fluorescent protein were observed by stereomicroscopy and confocal laser scanning microscopy to determine adherence patterns on growing lettuce seedlings. Bacteria adhered preferentially to plant roots in both model systems and to seed coats in the hydroponic system. Two of five nonpathogenic E. coli strains showed decreased adherence to seedling roots in the hydroponic system. EHEC was associated with plants in as few as 3 days in soil, and contamination levels were dose-dependent. EHEC levels associated with young plants inoculated with a low dose suggested that the bacteria had multiplied. These data suggest that preharvest crop contamination via contaminated irrigation water can occur through plant roots.