Evaluation and Characterization of Reaction Products from Ozonated Aflatoxin Contaminated Corn

Final 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
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Project Information

Summary:

Ozonation of clean corn, AFB1-contaminated corn, and pure AFB1 was carried out to evaluate the formation of reaction products. Results of TLC and HPLC analyses of aflatoxin in the model system showed seven possible reactions products. Results also indicated that the products formed are water-soluble. Further study to isolate and identify these compounds was conducted but no data were generated. Although these compounds are present in the model system, attempts to determine their presence in ozonated corn did not get any positive result. The presence of other materials in the extracts interferes with the analysis.

Introduction

Aflatoxins are a group of closely related bis-dihydrofurano secondary metabolites that have been epidemiologically implicated as environmental carcinogens in humans. They are considered one of the most toxic compounds that have ubiquitously contaminated cereal grains, oilseed crops and milk worldwide (CAST, 1989). Aflatoxins are produced primarily by Aspergillus flavus Link: Fr. and A. parasiticus Speare. These fungi have the capability of invading various agricultural commodities such as corn, peanuts, cottonseed, and various tree nuts, during maturation in the field and/or after harvest while the products are stored.
The aflatoxin that has caused the most concern is AFB1. It has been a focus of considerable research since its discovery because of its widespread occurrence, its prevalence among the four naturally occurring aflatoxins, and its acute toxicity and carcinogenicity (McKenzie et al., 1997). Human exposure to aflatoxins can be from direct consumption of contaminated commodities, or consumption of foods from animals previously exposed to aflatoxins through feeds. Exposure to aflatoxin B1 is generally considered a major factor in the high incidence of hepatocellular carcinoma, a malignant neoplasm of hepatic cells, commonly referred to as primary liver cancer. Apart from its effect on health, aflatoxin contamination also impacts the agricultural economy through the loss of produce and the time and cost involved in monitoring and decontamination efforts. It is estimated that as much as one-quarter of the world’s yearly food and feed crops are contaminated with mycotoxins (FAO, 1996). The Council for Agricultural Sciences and Technology (1989) estimated that in the United States alone, twenty million dollars is lost annually on peanuts contaminated with aflatoxins.
In an effort to limit human exposure to these toxins, prevention and control programs have been continuously being studied and established. These include monitoring of commodities susceptible to aflatoxin contamination, the establishment of limits and regulations that are legally enforced, and decontamination procedures designed to remove or inactivate the toxicant in food or feed (Park, 1993). Methods to decontaminate aflatoxin-affected foods and feed are constantly being studied and evaluated in order to optimize those that already exist, or to obtain more efficient and safer methods.
The most rational and practical approach to control human and animal exposure to aflatoxin contamination is by prevention (Park, 1993). However, prevention is not always possible under certain agronomic and storage practices (Samarajeewa, et al., 1990). Once the contamination has occurred, other control measures must be established and applied to reduce the risk of exposure to these toxins. Some of the necessary approaches include physical, chemical or biological removal, or use of chemical or physical inactivation. The use of chemical treatments to decontaminate aflatoxin-containing commodities is currently the most practical approach. Although these chemical treatments are effective, through their direct and indirect interaction with either mold or aflatoxins, however, decontamination products are still the points of contention and extensive investigations. The numbers of chemicals that are effective without leaving deleterious residues or without excessive damage to nutrients appear to be small. For foods, in addition to nutrients retention, it is essential that odor and flavor, color, texture, and functional properties be acceptable to consumers (Goldblatt and Dollear, 1977).
One method of decontamination for aflatoxin-affected commodities that has been a focus of attention is ozonation, a physical/chemical oxidation method. Several studies undertaken previously have established the effectiveness of ozonation as a decontamination process. It has been found to be effective in reducing aflatoxin levels by as much as 95%. However, few or limited studies have been done on the potential toxicity and possible carcinogenicity of ozone-aflatoxin reaction products. These aspects are very important in assessing the suitability and acceptability of the ozonation process.

Project Objectives:

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.

Cooperators

Click linked name(s) to expand
  • Alfredo Prudente

Research

Materials and methods:

A. Chemicals
Standard aflatoxins (B1, B2, G1, and G2) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Chloroform, dichloromethane, acetonitrile, petroleum ether, diethyl ether, benzene, methanol, dimethyl sulfoxide, hexane, trifluoroacetic acid, and water were HPLC-grade and were purchased from Fisher Scientific, Raleigh, NC.

B. Corn Samples
Two batches (Batch 1 and Batch 2) of corn samples provided by Dr. Kenneth S. McKenzie of Lynntech, Inc., College Station, Texas were used in the preliminary studies. Ten kilograms each of corn samples with and without aflatoxin contamination were treated with ozone. Corn sample was placed into a 30-gallon polyethylene reactor with false bottom. A 10-15” headspace was allowed to achieve even ozone dispersion though the corn. The reactor lid was fitted with ¼ “ Teflon bulkheads. Ozone gas, 10-12 wt%, was flowed in through the top at approximately 2 L/min. A 2.5 L/min vacuum was placed at the bottom. All corn samples were treated for 96 hours at 12-15 hour intervals with mixing occurring every 30 hours. The treatment protocol included untreated clean corn (control), ozone-treated clean corn, naturally contaminated corn and ozone-treated naturally contaminated corn. This allowed the determination of the efficacy of the ozonation process to degrade aflatoxin and to determine the effect of ozone on the quality of the corn from a safety perspective. Ten (10) kilograms of corn sample from each treatment was ground using a Romer Hammer Mill and was ground further using a Brinkmann mill to pass a 1.0 mm sieve. Samples were transferred to clean plastic bags, labeled and were stored at 4°C until further analysis.
A third batch (Batch 3) of corn was kindly provided by Dr. Manjit Kang of the LSU-Agronomy Department. Freshly harvested 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 of corn was randomly drawn from the sample to determine the presence of aflatoxins. The remaining corn 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 corn was air-dried overnight inside the fumehood. Treated and untreated corns were ground using a Brinkman mill and kept at 4°C. Additional corn (Batch 4 and Batch 5) were kindly provided by Dr. Kenneth Damann and they were used to produce artificially contaminated corn.

C. Inoculation of corn with A. flavus.
Conidial suspensions of A. flavus were prepared by following the method used by Tubajika and Damann (2001). Briefly, conidia of A. flavus (A53) suspended in 0.01% Triton X-100 was streaked/plated on a V8 juice agar (5% V-8 juice and 2% agar) and incubated for 10 days at 38°C. After incubation, the colonies were scraped and washed several times with 0.01% Triton X and transferred into scintillation vials. The concentrations were determined using a counter chamber (2/10 mm depth, 1/16 sq. mm). The concentrations were calculated to be 9.65E7 and 9.45E7 cells/ml.
Batch 3 – Five kg of corn with an initial MC of ca. 13% was transferred into a 5-gal capacity Nalgene container. The moisture content of the corn was adjusted to ca. 28% by adding an appropriate amount of sterile distilled water. Six ml of conidial suspension (9.65E7) was added and the corn was tumble mixed overnight to ensure an even distribution of conidial cells and even rehydration of corn. Corn was transferred into an autoclavable biohazard bag and incubated at 30°C for 10 days. A pan with distilled water was put inside the incubator to maintain a 100% RH. The corn was mixed everyday to avoid too much increase in grain temperature and to make sure that A. flavus cells are distributed. The corn was removed from the incubator after 10 days and put into a 60°C oven overnight to kill the fungi.
Batch 4 – the same procedure was followed as mentioned previously. Six ml of conidial suspension (9.45E7 cells/ml) was added. After incubation, the presence of non-A. flavus colonies, especially black colonies, was observed. This can be due to non-sterilized corn samples and/or too much water added. This was corrected by treating a 3rd batch of corn
Batch 5 – the same procedure was followed except that the corn was first sterilized for 15 min at 121°C and the moisture content was adjusted to ca. 20%. The same amount of conidial suspension (9.45E7 cells/ml) was added. Growth was observed after 3 days and there were no other colonies present. After 10 days, the whole lot was heavily contaminated with greenish colonies (A. flavus). The corn was put inside a 60°C oven overnight to kill the fungi.

C. Ozonation of Artificially-contaminated Corn

Batch 3 – Prior to ozonation, the presence of aflatoxin was determined by TLC. Extraction was done following the Mycosep column method. Dried extracts were re-diluted with 500 µl dichloromethane and 20µl was spotted on a 10 x 20 silica gel plate. Ten-µl of standard AFB1 was spotted to serve as external standard. The plate was developed first with 50 ml petroleum ether for 45 min. Plate was removed from the developing chamber and air-dried for 2-3 min. Plate was re-developed with 50 ml ether-methanol-water (96:3:1) for 45 min. Plate was air-dried inside the fume hood and viewed in a UV cabinet (365 nm). Blue fluorescent spots were observed that have similar Rf with that of the external standard. It was assumed that aflatoxin is present in the sample. Two and a half kg of “contaminated corn” was used in the experiment. Corn was treated with 17.17 wt% ozone at a flow rate of ca. 175 cc/min for 96 hrs. Corn had a moisture content of 11.65 and 14.63 % before and after ozonation, respectively.

Batch 4 – Five kg corn each of clean and inoculated corn was used in the experiment. Corn samples were treated with 12-13 wt% ozone at a flow rate of ca. 125 cc/min for 96 hrs. After ozonation, the samples were kept at 4°C for further analysis. Visual observation showed that ozonated corn were lighter (discolored) compared to untreated corns.

D. Aflatoxin Analyses
Aflatoxin determination in samples was carried out using the AOAC approved Multifunctional Column (Mycosep) method (AOAC Official Method 994.08, 1995). Fifty grams of ground sample were combined with 100 ml acetonitrile-water (9:1) solution and blended for 2 minutes at high speed. After blending, the extract was filtered through Whatman No.1 filter paper under vacuum. Fifty ml of the filtrate was collected into a 50-ml disposable centrifuge tube. A 3 ml aliquot of the filtrate was collected and applied into the Mycosep multifunctional cleanup (MFC) column and was collected into a 20-ml scintillation vial. Two hundred µL of the purified extract was transferred into a derivatization vial and 700 µl of derivatization solution (trifluoroacetic acid + glacial acetic acid + water, 20:10:70) were added. The vial was heated in a 65°C water bath for 8.5 minutes to complete derivatization of aflatoxin B1 and/or G1. The vial was then transferred to an auto-sampler (Waters 717). Aflatoxin levels were determined using a Waters 510 High Performance Liquid Chromatograph 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.
Thin layer chromatographic analysis of samples was accomplished using normal phase TLC on 20 x 20 cm or 10 X 20 cm general purpose silica gel plates (Sigma). Mobile phase used were ether-methanol-water (96:3:1) and/or chloroform-acetone (9:1). Plates were examined under long wave (365 nm) UV light.

E. 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.

F. Ozonation of Aqueous Solution of Aflatoxin B1 (A Model System)

Trial 1. 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. Treatment protocol includes ozonation for 0, 10, 20, 30, 40, 50, and 60 seconds. The same procedure was done using a standard mixture of AF-B1, -B2, G1, and G2. 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. 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.

Trial 2. Five-hundred µL of a standard solution containing 100µg of AFB1 were transferred into scintillation vials and evaporated to dryness. Dry materials were suspended in 10 ml distilled water and treated with 12-13 wt.% ozone from 0 to 60 sec at 10 minute intervals. Dichloromethane extracts of the solutions were dried under nitrogen. Water portions were freeze dried, re-dissolved in methanol, transferred into vials, and dried. Extracts were evaluated with single and 2-dimentional TLC using ether:methanol:water (96:3:1) and chloroform:acetone (9:1) as developing solvents. MALDI-MS analysis was conducted also to partially identify reaction products using 2,5-Dihydroxybenzoic acid (DHB) as matrix.

Trial 3. The same amount of AFB1 was used as mentioned in the previous trials except that the dried materials were not suspended in distilled water. After ozonation, extracts were reconstituted with 500 µl acetonitrile and evaluated by HPLC equipped with a tunable absorbance detector (365 nm). Ten to 20 µl of extracts was passed through a reverse phase column and eluted with acetonitrile-methanol-water (8:1:1 or 7:1.5:1.5).

G. Method development.
Methods were developed by following procedures from published papers. HPLC was accomplished using Waters Associates systems including: Waters 2690 separation module equipped with Waters 996 Photodiode Array Detector,
Numerous runs were made using different chromatographic systems (RP and NP), solvent systems, columns (Waters/NovaPak C18, Rainin/Microsorb C18 and Phenomenex/Sphereclone Silica), amount of samples injected, run times, flow rates, wavelengths. Isocratic and gradient elution were also performed in some samples.

Research results and discussion:

A. Aflatoxin Content in Corn Samples
Result of the HPLC analysis showed that aflatoxin B1 and B2 are present in all contaminated samples except for Batch 3. Table 1 summarizes the amount of aflatoxins in each batch. TLC analysis of samples from Batch 3 showed the presence of aflatoxin, however, further analysis using HPLC did not show the presence of aflatoxins.

Table 1. Aflatoxin content in corn samples.
Corn AFB1 (ppb) AFB2 (ppb)
Batch 1 644 38
Batch 2 140-143 23-25
Batch 3 ND ND
Batch 4 572 58
Batch 5 8151 871

B. Sequential Fractionation of Corn

a. 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.

b. 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.

c. 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.

d. 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.
e. 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 is greater than the standard due to uneven solvent migration.

f. Pronase Soluble – Organic Fraction

Trial 1 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.

Trial 2 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.

Trial 3 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 is more intense than the standard while the B2 was less intense compared to the standard.

The results of these experiments 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.

C. Ozonation of Aqueous Solution of Aflatoxin B1 (A Model System)

Trial 1. 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 Rf ‘s 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.

Trial 2. Analysis of dichloromethane extracts showed the presence of AFB1 after ozonation for 50 sec and it was totally degraded after 60 sec. Conversely, analysis of the water portion extracts showed the presence of seven compounds having Rf values of 0, 0.07, 0.14, 0.07, 0.25, 0.5, and 0.39, respectively, after ozonation for 60 sec. In comparison, Rf values for AFB1, B2, G1, and G2 were 0.71, 0.62, 0.56, and 0.46, respectively. Results of the study suggested the formation of polar compounds. Result of MALDI-MS showed presence of compounds that have higher molecular weights than AFB1. Mass spectra of dichloromethane extracts from samples ozonated for 50 and 60 sec showed base peaks with molecular weights of ca 459 and 439, respectively. Extracts from water portions showed based peaks with molecular weights of ca 475 and 494. The discovery of these compounds produced from aflatoxin is important because they may be formed in ozonated corn and they may have toxic properties.

Trial 3. Result of the HPLC analysis showed the presence of six peaks with retention times of 1.26, 3.42, 4.19, 6.15, 8.18, and 11.85 min. Isolation of individual peaks was attempted using a fraction collection. However, subsequent TLC and HPLC analysis of collected fractions did not show any positive result. This maybe due to the small amount of materials collected.

In addition, numerous TLC and HPLC analyses were conducted to determine if these compounds are present in contaminated treated samples. Materials collected from the sequential fractionation were examined but no positive result was obtained. This is due to the presence of other materials extracted from corn that interfere with the analysis. It is suggested that further clean-up be conducted on the extracts.

D. Method Development
One particular method that was followed in most of the analyses was a RP system using Microsorb C18 column (5µm, 4.6 x 150 mm) and water-methanol-acetonitrile (4:1:1) as mobile solvent. Other solvent systems that were used include water-methanol-acetonitrile (5:1:1), hexanes-isopropanol (2:1), methanol-acetone (9:1, 98:2 and 95:5), and water-0.05% TFA in acetonitrile (gradient, 90:10 – 10:90).
1. Initial runs were made on mixed standards of aflatoxin B1, G1, B2 and G2. Runs were also made using standard AFB1 only.
System: Reverse Phase
Detector: PDA, 210-500 nm
Mobile Phase: Water-methanol-acetonitrile (4:1:1), isocratic
Column: Microsorb C18
Injection Volume: 10 µl
Flow rate: 1.5 ml/min
Run time: 30 min
Standard conc: Mixed: B1 and G1 = 45 ppb, B2 and G2 = 13.5 ppb AFB1: 12 ppm and 100 ppm
Six major peaks were eluted and their retention times and areas were as follows:
Peak RT Area ID
1 2.588 66241 ?
2 3.419 44506 ?
3 4.913 96412 G2
4 6.263 179743 B2
5 7.088 75418 G1
6 9.152 148714 B1

The presence of two unidentified peaks was unexpected since the standard is supposed to have the 4 major aflatoxins only. This result was reported to Sigma-Aldrich and they said that they had the same complaint from other laboratories. The standard was kept for future reference.
For 12 ppm standard AFB1, the retention times and area were 9.252 min and 355561. For the 100 ppm standard, RT and area were 9.338 and 3418795.

2. Aflatoxin analysis for 2nd Batch of Samples from Dr. McKenzie. Trial 1.
System: Reverse phase
Detector: FLD, ex: 360 nm, em: 440 nm
Mobile Phase: Water-methanol-acetonitrile (4:1:1)
Column: Microsorb C18, 5 µm, 4.6 x 150 mm
Injection Volume: 50 µl
Flow rate: 1.0 ml/min
Run time: 20 min
Standard Conc: Mixed: B1 and G1 = 37 ppm, B2 and G2 = 11 ppm
Standards and samples were derivatized first with TFA following the Mycosep column method prior to injection.

Sample ID MajorPeakNo. RT Area ID

Mixed Std. 1 3.54 9620106 G1
2 4.652 25948278 B1
3 6.793 4687615 G2
4 9.787 10706258 B2
2A1 1 1.633 968347 solvent
2 7.414 27410 ?
3 8.125 84171 ?
2B1 1 1.595 454259 solvent
2 6.338 17844 ?
3 7.089 163874 ?
4 7.750 849572 ?
2C1 1 1.595 440888 solvent
2 4.125 12082 B1
3 8.616 97707 B2 ?
2D1 1 1.578 501389 solvent
2 3.095 29691 G1 ?
3 4.014 2309714 B1
4 6.59 37205 G2 ?
5 7.178 199065 ?
6 8.282 478805 B2 ?

Result of the analysis showed that aflatoxin B1 is present in untreated contaminated corn (2D1) and was reduced after treatment with ozone (2C1). No other compounds were observed. This maybe due to the small amount of samples injected.

3. Aflatoxin analysis for 2nd Batch of Samples from Dr. McKenzie. Trial 2
System: Reverse phase
Detector: FLD, ex: 360 nm, em: 440 nm
Mobile Phase: Water-methanol-acetonitrile (5:1:1), isocratic
Column: Microsorb C18, 5 µm, 4.6 x 150 mm
Injection Volume: 50 µl
Flow rate: 1.0 ml/min
Run time: 20 min
Standard conc: Mixed: S0 = 37 ppm B1 and G1, 11 ppm B2 and G2, S1 = 45 ppb and 13.5 ppb, S2 = 29.7 ppb and 9.0 ppb, S3 = 15.3 ppb and 4.5 ppb
The same result was observed as in Trial 1 except that the compounds were eluted faster.

4. Pronase Soluble Solid
System: Reverse phase
Detector: PDA, 210-500 nm
Mobile Phase: Water-methanol-acetonitrile (4:1:1), isocratic
Column: Microsorb C18
Injection Volume: 10 µl
Flow rate: 1.5 ml/min
Run time: 30 min
Standard conc: Mixed: B1 and G1 = 45 ppb, B2 and G2 = 13.5 ppb, AFB1: 12 ppm and 100 ppm

Two sets of samples were run. The first set of sample was dissolved in 1 ml acetonitrile while the second set was dissolved in 1 ml methanol. Standard aflatoxin G2, B2, G1 and B1 were eluted after 3.062, 3.805, 4.262, and 5.4 min, respectively. For samples dissolved in acetonitrile, no major peak was observed in 1A1 but for the rest of the samples, there was a peak that was eluted after ~7 min that is not aflatoxins. For samples dissolved in methanol, this peak is present only in 1A1 but not in the other samples. In addition, there were peaks observed that eluted between 0 and 6 min. Noteworthy was the two peaks that eluted after ~3.9 and ~5.5 min. They have close retention times with those AFB2 and AFB1, respectively. The other peaks cannot be compared to the standards.

5. Hexane extracts
a.System: Normal phase
Detector: UV-Vis, 254 nm
Mobile Phase: Hexane-isopropanol, gradient, 90:10 to 10:90, Hexane-isopropanol (2:1), isocratic
Column: Sphereclone Si, 5 µm, 4.6 x 150 mm
Injection Volume: 10 µl
Flow rate: 1.5 ml/min
Run time: 50 min
No standard was run in this experiment. Extracts were diluted with 1 ml hexane prior to injection. Gradient use was 90:10 that was linearly changed to 50:50 within 30 min, changed to 10:90 within 10 min and went back to 90:10 within 10 min. Results show that there is a peak that eluted after 17.847 min present in 1A1 that is noteworthy. This peak is not present in 1B1. Similar result was observed in 1C1 and 1D1 except that the peak has larger area compared to that with 1A1. This should be investigated further for it might be a reaction product formed between aflatoxin and ozone or corn and ozone.
Isocratic elution using the same sample was also done but there was no significant result that was observed.

b. Test for presence of lutein in hexane extracts.
System: Non-aqueous reverse phase
Detector: UV-Vis, 470 nm
Mobile Phase: Methanol-acetone (9:1)
Column: Nova Pak C18, 5 µm, 4.6 x 150 mm
Injection Volume: 10 µl
Flow rate: 1 ml/min
Run time: 15 min
Five-hundred ppb of standard lutein was used in the experiment. Lutein was eluted after 2.9 min. Lutein was also found present in all the samples injected. There are other compounds present in the samples but no comparison can be made since standard aflatoxins were not used in the experiment.

6. Other test runs were conducted using different solvent systems such as, water + 0.05% TFA in acetonitrile and methanol + acetone (92:8 and 95:5), but no significant results were generated. Test runs were also conducted to establish the appropriate conditions for the experiments conducted.
As a general comment, the amounts of samples used in these experiments are too small to discern their differences. It is suggested that similar experiments be conducted using higher amount of samples.

Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:

King, J.M. and A.D. Prudente. 2005. Chemical Detoxification of Aflatoxin in Foods and Feeds in Aflatoxins and Food Safety, ed. Hamed Abbas. Chp. 26. pp. 543-553.

Prudente, A. and King, J.M. Effects of Ozone on the Degradation of Aflatoxin B1 in a Model System. Presented at the IFT annual meeting, Orlando, FL, June, 2006. Abstract #003F-07.

Prudente, A. and King, J. Distribution of aflatoxin in corn after ozonation. Presented at the IFT annual meeting, New Orleans, July, 2005. Abstract #54I-18.

Prudente, A. and King, J. Mutagenic Potential of Ozone-Related Aflatoxin Reaction Products in Corn. Presented at the National American Oil Chemists’ Society (AOCS) annual meeting, Cincinnati, OH, May, 2004. Abstract in PCP 5: General Proteins and Co-Products.

Project Outcomes

Project 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.

The preliminary results generated from this study are a breakthrough in this area of aflatoxin research since no previous work has been done on the determination of reaction products between aflatoxin and ozone. The studies conducted demonstrate that there are products being formed in the reaction of ozone with aflatoxin B1. These reaction products are very stable that it is possible for these compounds to be isolated, identified and further investigated. Noteworthy about these studies is the conversion of AFB1 to polar compounds thereby reducing its toxicity. These preliminary results further support the potential of ozonation process as an alternative method to destroy and/or degrade aflatoxins.

Recommendations:

Areas needing additional study

The main objectives of this study were not fulfilled. It is suggested that further investigation be conducted to isolate, purify, and identify the reaction products from ozonated AFB1 standard and from AFB1-contaminated corn. It is suggested that an efficient extraction method be develop that will produce clean extracts that are free of interferences. In addition, reaction products are to be tested for mutagenicity or toxicity potentials.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.