Evaluation and Characterization of Reaction Products from Ozonated Aflatoxin Contaminated Corn

Evaluation and Characterization of Reaction Products from Ozonated Aflatoxin Contaminated Corn

Summary

Fractionation of corn samples was done following a series of extraction, partition and digestion procedure. Each extract collected was subjected to preliminary thin layer chromatographic analysis to determine the distribution of aflatoxin(s). Result of the analysis showed that aflatoxin B1 is present in methylene chloride, methanol, acetone, pronase soluble solid and pronase organic fractions of untreated contaminated corn. Aflatoxin was also observed on extracts from treated contaminated corn but to a lesser extent. No aflatoxin was observed on extracts from untreated and treated clean corns. No aflatoxin was also observed in hexane extracts from all samples.

Objectives/Performance Targets

To determine the distribution of aflatoxin in corn using a series of extraction, partition, and digestion procedure.

Accomplishments/Milestones

Methodology
Extract was dissolved with an appropriate solvent and spotted on a 10 x 20 cm silica gel plate (Sigma-Aldrich, Z18, 531-01). Ten µL of dissolved extract was spotted on an imaginary line 1.5 cm from the bottom edge of the plate. Ten µL of mixed aflatoxin reference standard (B1, B2, G1 and G2) was spotted as an external standard. The plate was first developed with petroleum ether and then with ether-methanol-water (96+3+1). The plate was developed for 45-60 min or until aflatoxins reach Rf of > 0.4. The plate was air-dried and was viewed under a UV cabinet. The plate was examined for fluorescent spots having Rf’s close to those of the reference standards. The extracts were either re-diluted or the volume of spot was increased to adjust the intensity of the fluorescent spots.

Results

Methylene chloride Extract
Extracts were diluted with 5 ml of CH2Cl2. Ten and 20 µL of each extract were spotted on the TLC plate. Ten, 20 and 30 µL of mixed standard were also spotted as reference. After development, the presence of very intense blue fluorescent spot/band was observed in untreated contaminated samples. These spots have Rf’s close to that of the reference standard. A faint blue fluorescent band was also observed in treated contaminated corn. The intensity of the spots was less than those of the standard. No blue fluorescent spots/bands were observed in untreated clean and treated clean corns. The presence of the blue fluorescent spots/bands indicated the presence of aflatoxin in the sample.

Methanol Extract
Twenty mL of methanol extract from each treatment was transferred into a scintillation vial and evaporated to dryness under a stream of nitrogen. The dried extract was re-dissolved with 2 mL of methanol. Ten µL of each extract was spotted on the TLC plate and developed first with petroleum ether and then with ether-methanol-water. Result showed the presence of numerous fluorescent bands in all of the samples. Bands were observed between the origin and AFG2, between AFB1 and AFG2, and between AFB1 and solvent front. A very intense blue fluorescent spot with an Rf close to that of AFB1 was observed in untreated contaminated corn extract. A less intense blue spot with Rf close to that of AFB1 was also observed in treated contaminated corn extract.

Acetone Extract
Acetone extracts were diluted with 5 ml acetone. Twenty mL of the extract and 10 mL of the standard were spotted on the plate. The plate was developed first with petroleum ether and then with ether-methanol-water. Result showed the presence of faint blue fluorescent band in untreated contaminated corn and treated contaminated corn extracts. No fluorescence was observed in both the treated and untreated clean corn. Fifty µL of extracts from untreated and treated contaminated corn were re-spotted to confirm the presence of aflatoxin B1. Result show a very intense blue fluorescent spots with Rf close to that of standard AFB1 in untreated contaminated corn extracts. For the treated contaminated corn, the intensity of the blue fluorescence did not change compared to the previous.

Hexane Extract.
Extracts were diluted with 1 mL hexane. Twenty µL of the extract and 10 µL of the standard were spotted on the plate. After development with petroleum ether and ether-methanol water, no fluorescent spots/bands were observed in all sample extracts.

Pronase Soluble Solid Fraction
Sample extracts were diluted with CH2Cl2 to give a final concentration of 10,000 µg/ml. Ten µL each of the extracts was spotted on two separate TLC plates. Ten µL of mixed standard was spotted as an external standard. Plates were first developed with petroleum ether until it reached the top edge of the plate. One plate was developed with ether-methanol-water (96+3+1) and the other plate with chloroform-acetone-water (88+12+1.5). Result of the first plate showed that the Rf’s for B1, B2, G1 and G2 were 0.88, 0.77, 0.68, and 0.55. Presence of blue fluorescent spot with an Rf of 0.88 was observed in D1 indicating the presence of aflatoxin B1. No fluorescent spots were observed on other samples. Yellowish streaks were observed on the path of all samples. For the second plate, the Rf’s were 0.81, 0.76, 0.71 and 0.67 for AF- B1, B2, G1, and G2, respectively. Presence of blue fluorescent spot with an Rf of 0.86 was observed in untreated contaminated corn extract. Obviously, this is similar to AFB1. The Rf was greater than the standard due to uneven solvent migration.

Pronase Soluble – Organic Fraction
1st trial -The same procedure as above was done except that only ether-methanol-water was used as developing solvent. Ten µL each of the extracts and standard were spotted on the TLC plate. The plate was developed with petroleum ether and ether-methanol-water. Result showed that Rf’s for B1, B2, G1, and G2 were 0.92, 0.82, 0.74 and 0.6. A faint blue fluorescent spot with an Rf of 0.92 was observed in untreated contaminated corn but none in other samples.
2nd trial – The same procedure as above was followed but the amount of sample spotted was increased to 20 µL. Result showed that Rf’s were 0.82, 0.72, 0.64 and 0.51 for AFB1, B2, G1 and G2. Blue fluorescent spots with Rf’s of 0.85 and 0.75 were observed in D1. The intensities of the spots were similar with that of the std. AFB1 and B2. No fluorescent spots were observed in other samples.
3rd trial. The same procedure was followed. Twenty µL of samples and 10 µL of standard were spotted on the plates. The plate was first developed with petroleum ether then with ether-methanol-water. Result showed that Rf’s for the standard were 0.75, 0.66, 0.58, and 0.49 for AF-B1, B2, G1 and G2, respectively. Yellow streaks were observed in all of the samples. Dark, yellowish spots were also observed between Rf 0.23 and 0.34 in all of the samples. Blue fluorescent spots with Rf of 0.75 and 0.66 were observed in untreated contaminated corn extract indicating the presence of aflatoxin B1 and B2. The aflatoxin B1 in sample was more intense than the standard while the B2 was less intense compared to the standard.

Future Activities
Continuation of TLC analysis for acid and bases extracts. Determination of distribution of ozone-aflatoxin-corn reaction products will be carried out by using radiolabelled aflatoxin. Extraction and identification of the reaction products will be carried out using GC/MS. Mutagenicity testing using the Ames assay will be carried out after all the extractions are completed.

Impacts and Contributions/Outcomes

The results of the recent experiment supported the findings of the previous work done by the participants. The presence and absence of aflatoxin(s) in the extracts supported the result of the Ames mutagenicity assay from previous work. Noteworthy is the result for the pronase soluble and organic fractions. Result of the Ames assay showed a positive correlation between the presence of aflatoxin in the extract and the mutagenic response of tester strains in the Ames assay.

Collaborators: