The proposed research has several goals. The long term objective is to provide a tool for the protection of United States food and feed grains from contamination by aflatoxins, potent hepatocarcinogens produced by Aspergillus flavus. Though this fungus is generally an ubiquitous soil saprophyte, it can infect globally important grains, particularly oilseeds such as corn under certain conditions. These toxins are frequently carcinogenic and represent a direct threat to human health. The work will broaden the toolset for study of these important materials and provide a basis for a "real-time" sensor detecting volatile organic compounds generated by the aflatoxin producing microorganism Aspergillus flavus. <P>
The specific aims of this work are: <OL> <LI> Identify unique volatile compound(s) associated with aflatoxin production by Aspergillus flavus growing on a defined liquid growth medium such as agar and then on corn. <LI> Establish and optimize norms for stability, reproducibility, and ruggedness of the sensor. <LI> Improve sensor response and device reliability, and develop device software. <LI> Operate the sensor device to obtain selective analysis of volatiles over toxigenic-inoculated corn. </OL> A number of A. flavus strains (toxigenic and atoxigenic) will be employed in this study. Microbiological growth assays on defined medium will determine the volatiles produced by each of the test fungi on corn meal and agar. During a three-week incubation period seeds will be removed to determine weight and aflatoxin levels. A series of seed controls will be used (no fungal inoculum) to determine the volatiles present, if any. Aflatoxin-Volatile profile determinations will be made using separate experiments utilizing nonsterile and sterilized corn and cottonseed as growth medium for the test fungi. Assays will be performed separately on non-sterile and sterile seeds with inoculations of an equal mixture of atoxigenic and toxigenic strains of A. flavus. This will determine the effect of the growth of the atoxigenic strain, which is competing for the same nutrients as the toxigenic strain, on aflatoxin production and volatile profiles of the toxin producing strain. <P>
The most prevalent volatiles with the highest concentrations produced will be used as test markers in the Sensor Development Corporation (SDC) test system. Using these gases the SDC sensor device will be designed to analyze selected test gases. Significant upgrades will be added to the sensor device used in the Phase I project. These will include state-of-the-art application of the tin oxide coating on the wafer and screen-printing the circuitry on each side of the sensor wafer. In addition the sensor device will have enhanced software management of the conductance data from the individual wafers. <P>
At the completion of this project the specific marker volatiles from A. flavus will be known, as well as the common interferant gases that exist in a corn bin. The project will be a success with the demonstrated repeated performance of a robust sensor device which can selectively detect with high sensitivity the identified marker gases for toxigenic A.flavus.
NON-TECHNICAL SUMMARY: Aflatoxin was discovered in groundnut meal which killed over 10,000 turkeys in England. Poultry are extremely sensitive to aflatoxin B1 with turkeys being more sensitive than chickens. Later research discovered that poultry are the most susceptible food animal species to the toxic effects of aflatoxin. Based on epidemiological data and knowledge of liver biochemistry it is suggested that humans fall somewhere in the middle of this lethality range. Lethality in humans has also been documented. In 1974, nearly 10 per cent of 1,000 patients died from suspected acute aflatoxin poisoning. Aflatoxicosis outbreaks (2004) in eastern Kenya resulted in 317 cases and 125 deaths. Specific medical implications of aflatoxin are related to their carcinogenic properties. Aflatoxin is listed as a Group I carcinogen by the International Agency for Research on Cancer. It is has been demonstrated in animal species to be the most potent liver carcinogen known, and is implicated as a cause of human primary heptatocellular carcinoma. This form of cancer is one of the most common forms of cancer in China, Saharan Africa, and Southeast Asia and causes at least 250,000 deaths annually worldwide, especially in developing countries. The human body also metabolizes aflatoxin to several compounds including aflatoxin M1 which is secreted in both mother?s milk and urine. Studies in Africa confirm infant exposure to aflatoxin via mother?s milk as well as the ability of aflatoxin in blood to cross the human placenta. Approximately 4.5 billion people around the world are exposed to virtually unregulated amounts of the toxin on a daily basis. The substance is so toxic that it is one of 19 contaminants along with mercury and DDT for which the US Food and Drug Administration imposes strict tolerance levels with only trace amounts allowed. In addition to the health impact of detecting the mycotoxins early, there is a significant economic impact. The Midwest drought in 2005 triggered an outbreak of poisonous aflatoxin at thousands of corn farms, spurring regulators and food companies to greatly expand testing of grain and milk. During the last US drought, many dairies were forced to dump aflatoxin-contaminated milk from cows fed corn contaminated with this toxin. The concern for aflatoxin-contaminated corn led company officials at the Quaker Oats breakfast cereal plant in Cedar Rapids, Iowa, to assay every truckload of corn entering the plant for aflatoxin. The value of this solution is evident by the fact that two major grain storage companies are committed to serve as beta test sites for the product.
APPROACH: Several techniques have been used to identify volatile compounds in different samples including tenax cartridge trapping methods and Solid \Phase Micro-Extraction (SPME). Two commercially available SPME fibers suitable for volatile analysis available from Supelco ( Bellefonte, PA) will be examined for this study. These are poly(dimethylsiloxane) (PDMS; 100 ƒÝm), and divinylbenzene/carboxen/poly(dimethylsiloxane) (DCP, 50/30 ƒÝm). The SPME fiber is inserted into the headspace above the fungal sample manually. Adsorption is timed for 1 h. SPME fibers are desorbed at 230C for 2 min in the injection port of an HP5890/5989A GC-MS (Hewlett-Packard, Palo Alto, CA) with a HP-5 (cross-linked 5% phenyl methyl silicone, Hewlett Packard, Palo Alto, CA) column (50 m, 0.2 mm i.d., 0.5 ƒÝm film thickness). Positive identification of a component will be performed in two corn varieties inoculated with two molds by comparison of its retention time or Retention Index (RI) and mass spectrum with that of an authentic compound (when available). The RI for each identified compound is calculated using a series of straight-chain alkanes (C5-C20). Tentatively identified compounds will be uniquely identified on the basis of the mass spectra from the Wiley (v.7 NIST98) library of mass spectral database (Palisade Corp., Newfield, NY). A. flavus, whose toxigenic strains produce aflatoxin, exhibits a marker target gas associated with those toxigenic strains, the sesquiterpene MVOC, C15H24, Humulene. To confirm the conclusion regarding selectivity, will use sensor response data to conduct a simulated 2-gas demonstration by sequentially testing two different sensor wafers in the same chamber using the same pair of gases at identical concentrations in each instance. Various combinations of four gases and two sensor wafers (one un-catalyzed and the other catalyzed), at three operating temperatures. Though these sensors will be tested simultaneously in one test chamber, the sequential procedure essentially represented that condition. These data will be used to calculate the "measured" concentration of each individual gas. A mixture of inteferant gases and susquiterpenes will also be tested using two catalyzed sensors at different temperatures. Combinatorial Chemistry techniques may be used to select 2-catalyst combinations.