1a.Objectives (from AD-416)
1. Survey samples of tree nuts, dried fruits, and spices from retail and other markets for ochratoxin contamination.
2. Isolate and identify ochratoxin-producing fungi from contaminated samples.
1b.Approach (from AD-416)
Samples will be purchased from sources across the U.S. and transported to WRRC. Tree nuts will be ground using a food processor; dried fruits will be ground using a meat grinder (or similar machine). Ground nuts, ground fruits, and dried spices will be extracted with methanol and subjected to immunoaffinity chromatography to concentrate any ochratoxin from the samples. Ochratoxin in purified extracts will be quantified using high-performance liquid chromatography (HPLC). Samples with high levels of ochratoxin will be used to isolate fungi, using standard microbiological methods. Production of ochratoxin by individual fungi will be assessed using HPLC of methanol extracts of fungal material. Genus and species identification of ochratoxin-producing fungi will be determined by DNA sequence analyses of calmodulin and beta-tubulin gene sequences, as well as large-subunit ribosomal RNA gene sequence analysis.
This project is just underway, as the extramural funding was just received with only one month to go in the reporting period. Nevertheless, the development of protocols for assessing ochratoxin content and identification of microbes that could serve as biological control agents is underway.