Douglas L. Park

Feasibility of Immunodiagnostic Devices for the Detection of Ricin, Amanitin, and T-2 Toxin in Food

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values > or = 0.1 mg/kg of body weight. Ricin (oral LD50 > 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 > 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically < or = 0.020 microg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

Isolation and identification of dihydrocitrinone, a urinary metabolite of citrinin in rats

Dihydrocitrinone, 3,4-dihydro-6,8-dihydroxy-3,4,5-trimethylisocoumarin-7-carboxylic acid, was isolated and identified as a urinary metabolite after oral administration of citrinin to rats. Male and female Osborne-Mendel rats received 30 mg citrinin/kg body weight by oral intubation. The metabolite dihydrocitrinone was present in urine collected at 0-2, 2-4, 4-6, 6-8, and 8-24 h after treatment. Only unchanged citrinin was found in blood collected 24 h after administration of the compound. The metabolite had a blue fluorescence and the same Rf on thin-layer chromatography, the same retention time on reverse-phase high-pressure liquid chromatography, and the same mass spectrum as an authentic sample of dihydrocitrinone.

Mutagenic potential of ammonia-related aflatoxin reaction products in a model system

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the U.S. Department of Agriculture determined the mutagenic potential of aflatoxin reaction products following ammoniation of aflatoxin B 1 in a pressure chamber used to decontaminate aflatoxin-contaminated cottonseed meal. Uniformly ring-labeled (14C)-aflatoxin B1 was added to nonlabeled B1, distributed on an inert carrier and treated with 4% ammonia at 40 psi, 100 C, for 30 min. Aflatoxin-derived decontamination reaction products were separated, and fractions having a high specific activity were tested for mutagenic activity using the Salmonella/mammalian-microsome mutagenicity assay (Ames test). When concentrations ranging from 3.3 to 100 μg per plate were tested, all fractions exhibited a similar mutagenic response. The observed mutagenic activity was 2,000-20,000 times less than that observed with nonammoniated aflatoxin B1.

Comparison of the Microbial Quality of Raw Shrimp from China, Ecuador, or Mexico at Both Wholesale and Retail Levels

Aerobic plate counts (APC), Listeria spp., and Vibrio spp. and antibiotic resistance patterns of Vibrio spp. were determined for imported shrimp from China, Ecuador, and Mexico obtained from wholesale/frozen and retail/previously frozen markets. Statistically significant differences in APC were observed among source countries and wholesale/frozen versus retail/previously frozen samples. Wholesale/frozen shrimp products were consistently excellent quality with respect to APC; higher contamination levels were observed in retail/previously frozen samples. Listeria spp. and L. monocytogenes were isolated from 16.7 and 6.7% of shrimp samples, respectively. Vibrio spp. were present in 63.3% of the samples, more often isolated from shrimp from Mexico or China than Ecuador. The majority of isolates were identified as V. parahaemolyticus (36.7%), V. alginolyticus (26.7%), or V. vulniftcus (16.7%), and 53.7% were resistant to at least one antibiotic. These data reveal that important differences in microbial quality occur in raw shrimp products as a function of source and between retail and Wholesale products.

Direct application of a new hypochlorite sanitizer for reducing bacterial contamination on foods

The efficacy of the buffered sodium hypochlorite solution, Bionox, in controlling bacterial contamination was evaluated on fresh-cut poultry (chicken, light and dark meat), fish fillets, fruit, and vegetables. Food products were immersed in a nutrient broth suspension of Salmonella enteritidis (ATCC #13067) for 30 s and allowed to drip/air dry for 10 s prior to exposure to the sanitizing agent. Food products were then each vigorously washed with 100 ml of buffered peptone which was plated in serial dilution on XLD-N agar and incubated at 37°C for 24 h. Typical Salmonella colonies as well as non- Salmonella colonies growing on the XLD-N plates were counted and identified. Results showed the sanitizing solution to be effective in reducing S. enteritidis on all test foodstuffs. Count reductions of 4, 3, and 2 logs per gram on chicken, vegetables, and fruit, respectively, were achieved. Salmonella reductions of two logs were also achieved on fish fillets, but the sanitizer performance depended to some extent on the background bacterial flora present prior to the addition of the Salmonella test organism. The effect of the sanitizing solution on protein functionality, lipid oxidation, and starch degradation was determined using protein dispersibility and solubility assays, peroxide and iodine values, and changes in reducing sugars levels, respectively. Results showed no adverse effects on these parameters after exposure of the food products to the sanitizing solution.

Distribution of ammonia-related aflatoxin reaction products in cottonseed meal

The fate of aflatoxin during ammoniation of contaminated cottonseed meal was studied under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled14C-aflatoxin B1 was added to 27.7 kg meal (14% moisture) that contained ca. 4000 µg naturally incurred aflatoxin B1/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B1 after treatment with 4% ammonia at 40 psi, 100 C for 30 min. This treatment reduced the chemically detected aflatoxin B1 to less than 4 µg/kg. In control nonammoniated meals, 90% of the radiolabeled material was accounted for in the methylene chloride extract. Duplicate 2-kg samples of the ammoniated meal were fractionated and the distribution of radioactivity was determined. Ca. 86% of the radioactivity was detected in the meal after initial air-drying. Ca. 25% of the added radioactivity was extracted from the air-dried meal with methylene chloride and another ca. 5% was extracted from this residue with methanol. Weak acid released 3% of the added radioactivity from the residue after methanol extraction, bicarbonate released 1% and Pronase digestion, including methylene chloride extraction of the residue, accounted for nearly 19% of the total added radioactivity. Only 37% of the added radioactivity remained in the meal matrix following solvent extractions and chemical and enzymic treatments.

Mutagenic potential of ammonia-related aflatoxin reaction products in cottonseed meal

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the US Department of Agriculture studied the mutagenic potential of aflatoxin reaction products following ammoniation of contaminated cottonseed meal under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled14C-aflatoxin B1 was added to cottonseed meal that contained ca. 4000 µg naturally incurred aflatoxin B1/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B1 after treatment with ammonia. The radioactivity-to-weight ratio of the fractions isolated by solvent extractions and chemical and enzymic treatments was used to measure the relative concentration of an aflatoxin decontamination product. All extract fractions having a radioactivity-to-weight ratio ≥1 were tested for mutagenic activity using theSalmonella/microsome mutagenicity test (Ames test). Purified aflatoxin B1 was mutagenic at a concentration of ca. 0.005 µg/plate. The methylene chloride extract of the ammoniated meal after Pronase digestion exhibited a similar response when 180 µg of this fraction was applied to each plate. This fraction represented 0.16% of the original added radioactivity. The other fractions produced no detectable mutagenic response at the concentrations tested (10–1000 µg/plate) onSalmonella tester strain TA100. Ammonia treatment of aflatoxin-contaminated cottonseed meal significantly decreased aflatoxin levels, and the aflatoxin decontamination products formed by the treatment had little or no mutagenic potential.

DECONTAMINATION OF AFLATOXIN-CONTAMINATED PEANUT MEAL USING MONOMETHYLAMINE: CA (0H)2

Radiolabeled aflatoxin B1 was added to naturally contaminated peanut meal, and the fate of aflatoxin-related by-products after decontamination by monomethylamine:Ca(OH)2 was monitored. The decontamination process resulted in a 94-100% reduction in aflatoxin levels, depending on the level of contamination and the chemical structure of the aflatoxin. Following air drying and extractions with chloroform/water, methanol, acetic acid and water, the majority (69-86%) of the,decontamination by-products remained with the residue. Significant amounts of aflatoxin B1, (12%) and aflatoxin-related compounds (27%) were liberated from the residue after enzymic digestion. Isolation and identification studies revealed a wide variety of aflatoxin-related decontamination by-products. High performance liquid and thin layer Chromatographie separation of chloroform-soluble compounds revealed compounds that exhibited some degree of toxicity. Comparative toxicity tests showed that, although some decontamination by-products exhibited elevated responses to specific toxicity tests, the relative toxicity was inferior to aflatoxin B1. The moisture, protein and nitrogen levels did not change significantly. There was a significant change in the mold flora following the decontamination process, and the product was susceptible to recontamination.

Reduction of Mutagenic Potentials in Milk: Effects of Ammonia Treatment on Aflatoxin-Contaminated Cottonseed

Milks obtained from cows fed rations containing aflatoxin-contaminated cottonseed, ammonia-treated aflatoxin-contaminated cottonseed, and uncontaminated cottonseed were tested for mutagenic potential using the Salmonella /mammalian microsome mutagenicity assay using Salmonella typhimurium strains TA98 and TA100. Standard assay protocol was used with S-9 liver homogenate added. Samples including whole milk, nonfat dry milk powder, cream, and reconstituted whole milk were applied directly to the plates in triplicate. As a control, samples of whole milk, reconstituted whole milk, and nonfat dry milk powder from cows fed uncontaminated feed were spiked with aflatoxin B1 and tested for mutagenic activity. High levels of mutagenic activity were observed in all samples from cows exposed to aflatoxin-contaminated cottonseed and the aflatoxin-spiked milks. This high activity was not evident in whole milk and whole milk component samples from cows fed the ammonia-treated aflatoxin-contaminated cottonseed or nonaflatoxin containing cottonseed. A low level of mutagenic potential was evident in whole milk from the ammonia treated group using TA100 tester strain.

Anti-mutagenic compounds from corn.

In previous studies with aflatoxin-contaminated corn an uncharacteristic response for AFB1 in the Salmonella/microsomal mutagenicity assay (Ames test) was observed and the presence of anti-aflatoxin factors in the corn was suggested. In the current study, corn was extracted and fractionated using thin layer chromatography (TLC) using different developing solvent systems and the Ames test was used to monitor for anti-mutagenic activity in the corn fractions. Both Salmonella tester strains TA98 and TA100 with metabolic activation (S9) were used. Several corn fractions, at different stages in the isolation and purification process, showed anti-mutagenic dose-responses when exposed to pure AFB1. Corn extracts were non-toxic to the tester strains and TLC fractions that showed the best anti-mutagenic dose-responses were selected for further partial characterization analyses.