Evaluation and Characterization of Reaction Products from Ozonated Aflatoxin Contaminated Corn

2004 Annual Report for GS02-015

Project Type: Graduate Student
Funds awarded in 2002: $10,000.00
Projected End Date: 12/31/2006
Region: Southern
State: Louisiana
Graduate Student:
Major Professor:
Joan King
LSU Agricultural Center

Evaluation and Characterization of Reaction Products from Ozonated Aflatoxin Contaminated Corn

Summary

Ozonation of aqueous solution of aflatoxin B1 and mixed aflatoxins were carried out. Aflatoxin B1 was totally degraded after ozonation for 30 seconds or longer. Aflatoxin B2 and G2 were not affected by ozonation. Aflatoxin analysis was conducted for a 2nd batch of corn samples and results showed that aflatoxins were not present in treated and untreated clean corn. Aflatoxins B1 and B2 were present in untreated contaminated corn at levels of ~140 ppb and ~23 ppb, respectively. Aflatoxins were not present in treated contaminated corn indicating 100% degradation of aflatoxin by ozonation. Ozonation of a 3rd batch of corn samples was also conducted. Preliminary TLC analysis showed that aflatoxin was present, but subsequent HPLC analysis did not detect aflatoxin in both ozonated and untreated contaminated corn samples of batch 3. It will be necessary to inoculate the corn to attain aflatoxin levels that are detectable. Two unidentified compounds were observed in all corn samples that have retention times between AFG2 and AFB2.

Objectives/Performance Targets

1.Fractionation of corn samples from the 2nd batch.
2.Inoculation of batch 3 corn with Aspergillus flavus followed by ozonation.
3.Evaluation of extracts by TLC and HPLC.

Accomplishments/Milestones

Ozonation of Aqueous Solution of Aflatoxin/s (A Model System)

A standard solution of AFB1 was prepared by diluting 1 mg of AFB1 (Sigma, A6636) with 1 ml acetonitrile to give a concentration of 1mg/ml. One-hundred µl of the standard solution containing 0.1 mg AFB1 was added into 9.9 ml HPLC grade water in a vial and was sealed with septa. Seven solutions were prepared and were kept at 4 C. The treatment protocol included ozonation for 0, 10, 20, 30, 40, 50, and 60 seconds. The same procedure was done using a standard mixture of AFB1, AFB2, AFG1, and AFG2. The solution was transferred into a separatory funnel and aflatoxins were extracted with 10 ml methylene chloride. The methylene chloride layer was collected and transferred into a scintillation vial and evaporated to dryness under nitrogen gas. The extracts were re-diluted with 1 ml methylene chloride and 20 µl of each was spotted into a TLC plate. The plate was developed with ether-methanol-water (96+3+1) and viewed in a UV cabinet. Result showed that AFB1 was not present in extracts ozonated for 30 seconds or longer. No visible blue fluorescent spots close to the Rf of AFB1 were observed. Similar result was obtained for the mixed aflatoxins. However, aflatoxins B2 and G2 were not affected by ozonation since visible bluish and greenish spots close to the Rf of B2 and G2 were observed in all extracts.
HPLC analysis using a photodiode array detector (210 ~ 500 nm) was done for all the extracts. The same extracts were dried and re-diluted with 2 ml acetonitrile. Ten-µl each of the extracts was injected and passed through a reverse phase column (Microsorb-MV, C18, 4.6 x 150 mm). The extracts were eluted with acetonitrile-methanol-water (1+1+4) at a flow rate of 1 ml/min. Results showed that no peaks were present in all extracts. This must be due to the small amount of aflatoxins present in the extracts or the small amount of sample injected resulting in concentrations below the detection limit.
HPLC Analysis of 2nd Batch of Corn Samples
HPLC of corn samples from previous study was conducted. Aflatoxin determination in samples was carried out using the AOAC approved Multifunctional Column (Mycosep) method (AOAC Official Method 994.08, 1995). Treatment protocols include untreated clean corn, treated clean corn, untreated contaminated corn, and treated contaminated corn. Result of the analysis showed that no aflatoxin was present in clean corn both for ozone-treated and untreated corn. Untreated contaminated corn contained ~140 ppb AFB1 and ~23 ppb AFB2 whereas treated contaminated corn contained 0 ppb AFB1 and ~4 ppb AFB2. This result indicated that the ozonation process totally degraded AFB1 in corn. Two unidentified compounds that had retention times between AFG2 and AFB2 were observed. These compounds were degraded after ozonation.
Ozonation of Corn Samples (3rd Batch)
Corn in cobs was kindly provided by Dr. Manjit Kang of the Agronomy Department. Corns were manually threshed and sound grains were separated from damaged or visibly contaminated grains. Two 5-kilogram damaged/contaminated grains were prepared. One part was kept for ozonation and the other part was kept as untreated sample. The moisture content of corn was adjusted from 11.65% to ~13% by adding the required amount of water and tumbled overnight. Five hundred grams was randomly drawn from the sample to determine the presence of aflatoxins. TLC analysis showed that aflatoxins were present in corn. The remaining corn samples were treated with 17.17 wt % ozone gas for 96 hrs at a flow rate of 175 ml/min and mixed every 12 hrs. The moisture content after ozonation increased to about 14% due to the moisture coming out from the ozonator. The corn was air-dried overnight inside the fumehood. Color analysis using a photospectrometer (Minolta CM-500D Series) showed that ozonated corn was lighter and had less redness and yellowness compared to non-ozonated corn. Treated and untreated corns were ground using a Brinkman mill and kept at 4°C. Aflatoxin analysis of samples using HPLC was done by Dr. Janet Simonson of the Agricultural Chemistry Dept. Results showed that no aflatoxins are present in both treated and untreated corn. However, there was a compound present in both corn samples that has a retention time between AFG2 and AFB2. The area of the compound was lower in treated corn than in untreated corn. It is hypothesized that this compound is another mycotoxin or a compound that is intrinsic in corn.
Future Activities
Continuation of TLC and HPLC analyses of corn extracts using different solvent systems. Carry out extraction, purification, and identification of isolates from corn extracts. Mutagenicity testing using the Ames assay will be carried out after all the extractions are completed. Carry out inoculation of corn with Aspergillus flavus to obtain contaminated corn samples.

Impacts and Contributions/Outcomes

Ozonation was further confirmed to destroy AFB1 in corn, but there was some slight resistance of AFB2. There were some unidentified compounds in the corn that were also suceptible to ozone treatment. The levels of these compunds were lower in untreated contaminated corn than untreated clean corn. They may be desirable corn constituents that are affected by ozonation.

Collaborators:

Alfredo Prudente

aprude1@lsu.edu
student
LSU Agricultural Center
Department of Food Science
111 Food Science Bldg., Louisiana State University
Baton Rouge, LA 70803
Office Phone: 2255785186