Evaluation and Characterization of Reaction Products from Ozonated Aflatoxin Contaminated Corn

Evaluation and Characterization of Reaction Products from Ozonated Aflatoxin Contaminated Corn

Summary

Two batches of corn samples from previous studies were used in the experiments. Treatment protocol included untreated clean corn, ozone-treated clean corn, contaminated corn and ozone-treated contaminated corn. Analysis showed that untreated corn in the first batch contained 644 ppb AFB1 and 38 ppb AFB2 while the second batch contained 143 ppb AFB1 and 25 ppb AFB2. TLC analysis showed three major zones where the bands are present. Blue and yellow bands were observed close to the solvent front while AFB1 and AFB2 bands were at the middle. Faint yellow bands were observed between the aflatoxins and the origin.

Objectives/Performance Targets

General Objective
To determine the safety of the ozonation procedure to reduce aflatoxin hazards in corn.

Specific Objectives
1. To evaluate the formation and distribution of ozone-aflatoxin and ozone-corn reaction products.
2. To characterize and identify ozone-aflatoxin and ozone-corn reaction products.
3. To determine the mutagenic potential of reaction products using the Salmonella / microsomal mutagenicity assay.

Accomplishments/Milestones

Sample Preparation

Corn sample from each treatment was ground using a Romer Hammer Mill. It was ground further using a Brinkmann mill to pass a No. 20 mesh sieve. Samples were transferred to clean plastic bags, labeled and were stored at 4°C until further analysis.

Aflatoxin Determination

Aflatoxin determination in samples was carried out using the AOAC approved Multifunctional Column (Mycosep) method (AOAC Official Method 994.08, 1995). Aflatoxin levels were determined using a Waters 510 High Performance Liquid Chromatograph (HPLC) equipped with Waters 470 fluorescence detector, and a Microsorb-mv C-18 reverse phase column using water-acetonitrile (8:2 v/v) as a mobile phase at a flow rate of 2 ml/min. Initial results of the aflatoxin analysis showed that untreated corn in the first batch contained 644 parts per billion (ppb) AFB1 and 38 ppb AFB2 while in the second batch, 143 ppb AFB1 and 25 ppb AFB2 were detected.
Sequential Fractionation of Corn
Sequential fractionation of corn samples was done through a series of extraction, partition and digestion procedures.

a. Methylene chloride extraction
Extraction was carried out on first batch of samples. Four hundred grams of corn sample were extracted with dichloromethane (CH2Cl2) using a 1:5 (w/v) ratio. The mixture was shaken for 30 min and filtered under vacuum. The extract was concentrated to about 300 ml (volume recorded), sealed and kept in the freezer until further analysis. The residue were air-dried for 30 minutes in a chemical hood and then dried overnight in an oven at 45°C to remove residual solvent.

b. Thin layer chromatographic analysis
The volume of dichloromethane extracts were adjusted to 400 ml. Ten ml of the extract were transferred to a pre-weighed vial, evaporated to dryness under stream of nitrogen and weighed. The dry materials were re-suspended with 1 ml CH2Cl2 and were spotted on the TLC plate. Initial results of the experiment showed that 10 µl of extract is the ideal volume for spotting and anhydrous ether-methanol-water (96+3+1) is the ideal developing solvent instead of chloroform-acetone (9+1). Using this solvent resulted in good resolution and separation of individual components (Rf=4-7). Results also showed that there are three major zones where bands are present. Blue and yellow bands were observed close to the solvent front while aflatoxin B1 and B2 bands were in the middle. Faint yellow bands were observed between the aflatoxin bands and the origin. Further evaluation of these bands will be conducted.

c. Methanol extraction
The corn meal remaining after the CH2Cl2 was extracted with methanol (1:5 w/v). The methanol extract was concentrated and adjusted to 500 ml and a 50 ml aliquot was transferred to a separatory funnel. Fifty ml acetone-water (3:7), 100 ml of CH2Cl2 and 40 ml MeOH were added, shaken and allowed to equilibrate. These solvents were used instead of those stated in the original proposal because clear separations of aqueous and organic layers were obtained. The aqueous phase (upper layer) was transferred to another separatory funnel and fifty ml of acetone was added, shaken and filtered under gravity. The filtrate was evaporated to dryness under a stream of nitrogen. The organic phase (lower layer) was concentrated to ca. 20 ml and 100 ml of hexane was added. The solution was mixed and filtered. The filtrate was evaporated and transferred with hexane to a pre-weighed vial and dried under a stream of nitrogen. The precipitate (if present) was air-dried in a chemical hood and then oven-dried overnight at 70°C. Weights of precipitates and corresponding soluble fractions were recorded and kept at 4°C until further analysis.

Future Activities

Continuation of other extraction, partition and digestion procedures will be carried out on both batches of corn samples. Mutagenicity testing using the Ames assay will be carried out after all the extractions are completed. Determination of distribution of ozone-aflatoxin-corn reaction products will also be carried out. There is a delay in this portion of the project since aflatoxin-contaminated corn is not available. In addition, we had difficulty in requesting for radiolabelled aflatoxin to be used in this study. There is a regulation prior to February 7th, 2003, that restricts the use of these materials.

Impacts and Contributions/Outcomes

Aflatoxin contamination of corn is a perennial problem in corn producing areas in the United States, especially in the Southern Region. It is an important problem and other methods of managing this would be very beneficial to the whole corn industry. Ozonation, a physical/chemical method of decontamination, has been found to be effective in reducing aflatoxin levels in contaminated corn. It is seen as a potential alternative method of addressing the aflatoxin problem instead of using chemical methods. However, it is necessary to study and determine the potential toxicity and carcinogenicity of ozone-aflatoxin-corn reaction products. The evaluation and characterization of these reaction products will be very important in assessing the suitability and acceptability of the ozonation process.

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