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.
We have developed protocols for recovery of ochratoxin from dried fruits and nuts based on previously published methods for raisins and currants. To determine percent ochratoxin recovery from each commodity to be surveyed, we spiked food samples with known amounts of authentic ochratoxin. Following extraction and immunoaffinity chromatography, levels of recovered ochratoxin from each food and at each spiked concentration were measured. Recovery rates from food were between 70 and 80%, and were consistent for all spiking levels (2 parts per billion to 10 parts per billion) for each food. These recovery rates are similar to those published in other studies. Percent recovery from each commodity will be used to calculate ochratoxin levels in survey samples. Supermarket samples of raisins, figs, dates, prunes, almonds, pistachios and walnuts have been received from collaborating scientists at University of Nebraska, North Dakota State University, and Texas Woman’s University. Samples from collaborators at Illinois Institute of Technology and University of Pittsburgh will be received shortly. This research is in direct support of objective 4 of the parent project, "Develop biological control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments."