Use of Ozonation to Degrade Certain Chemicals in Foods and Animal Feeds

NON-TECHNICAL SUMMARY: Undesirable chemicals, whether natural or food-borne, require some cost effective method of decontamination before food or feed products can be sold. This project examines the effectiveness of ozonation in destroying these chemicals without producing toxic products or destroying benefical nutrients.

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APPROACH: The goal of this research will be to investigate the use of ozonation to destroy unwanted chemicals in foods and animal feeds. New gaseous and/or liquid based ozonation processes, that maximize ozone penetration into the food or feed, will be developed and will be tested to determine their effectiveness in destroying target chemical compounds. Existing and newly developed ozonation processes that are effective will be tested to determine if any new mutagenic toxins are being formed, by safety evaluation through the use of the Ames test. The effects of the ozonation process on nutrients, specifically lipids and proteins, in the food or feed products will also be studied through analytical analyses. The main parameters of importance are the time of ozonation and ozone concentration, which are correlated to the initial level of contamination. These parameters will be adjusted to minimize nutrient loss while maintaining effectiveness of the ozonation procedure for destroying target chemical compounds.

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PROGRESS: 2001/05 TO 2007/04 <BR>
Research was done on the effectiveness of ozone to destroy aflatoxin in corn. Ozonation released lutein from clean corn, while in the contaminated corn, ozonation destroyed some of lutein in the corn. However, ozonation did not change the antimutagenic potential of lutein extracts against AFB1. Protein can be destroyed by ozone, which influences the nutritious value of corn. 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 reaction 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 result in any positive result. The presence of other materials in the extracts possibly interferes with the analysis or the compounds are not concentrated enough. Further work will be done beyond the scope of this project to determine if aflatoxin is affected by ozonation directly to produce potentially new toxic compounds by following radiolabled aflatoxin through the process. The effects of ozone treatment on depolymerization and decolorization of chitosan were investigated. Molecular weight of ozone-treated chitosan in acetic acid solution decreased appreciably as the ozone treatment time increased. Ozonation for 20 minutes reduced the molecular weight of the chitosan by 92% (104 KDa) compared to the untreated chitosan (1333 KDa) with a decrease in viscosity of the chitosan solution. Ozonation for 5 min markedly increased the whiteness of chitosan; however, further ozonation resulted in development of yellowness. In case of the ozonation in water, there were no significant differences of the molecular weight and color between ozone-treated chitosans. However, results showed that ozone treatment of chitosan in both water and acetic acid solution was not effective in removing acetyl groups (deacetylation) in chitosan molecules. This study showed that ozone can be used to modify molecular weight and remove pigments of chitosan without chemical use in a shorter time with less cost. The effects of ozone on off-flavors in catfish was studied. The study showed that oxygen and ozone treatments did differ, indicating that ozone treatment was successful in reducing catfish off-flavor. Moisture was unaffected, however, color was significantly changed, and it was unclear if ozone was the cause of changes in fat.
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IMPACT: 2001/05 TO 2007/04<BR>
Current processes for removing chemicals from food or feeds require large quantities of costly, toxic solvents, create a solvent waste disposal problem and are also time consuming.Ozonation is an inexpensive, rapid procedure that creates no waste disposal problem. Development of methods utilizing ozone to replace the solvents and to destroy undesirable toxins, off-flavors or colors and to degrade compounds will be of value.Aflatoxin contamination in crops that include corn, peanuts and cottonseed continues to plague the agricultural and food industries particularly in warm and humid regions around the world. Research emphasis has focused on both pre-harvest prevention and post-harvest removal of aflatoxin from grains. Ozonation may be the best new choice because of potential economic benefits and effectiveness of treatment compared to other detoxification techniques. Off-flavor of catfish is a problem for the aquaculture industry in the southern United States.Development of methods to reduce off-flavor compounds to a level below the threshold of 0.7 ppb will be of great importance.Chitosan has antibacterial properties that depend on molecular weight.Currently, depolymerization and decolorization of chitosan are done by chemical or enzymatic methods which are time consuming and expensive.Ozone has been shown to be able to degrade macromolecules and remove pigments due to its high oxidation potential and is a new alternative to chemical methods for decreasing chitosan molecular weight.Ozonation methods must be effective without forming new toxins or destroying nutrients.
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PROGRESS: 2006/01/01 TO 2006/12/31<BR>
The purpose of this research was to determine the effects of ozone on off-flavors in catfish. The study indicated that oxygen and ozone treatments did differ, indicating that ozone treatment was successful in reducing catfish off-flavor. Moisture was unaffected, however, color was significantly changed although it was unclear if ozone was the cause of changes in fat. The effects of ozone treatment on depolymerization and decolorization of chitosan were also investigated. Molecular weight of ozone-treated chitosan in acetic acid solution decreased appreciably as the ozone treatment time increased. Ozonation for 20 minutes reduced the molecular weight of the chitosan by 92% (104 KDa) compared to the untreated chitosan (1333 KDa) with a decrease in viscosity of the chitosan solution. Ozonation for 5 min markedly increased the whiteness of chitosan; however, further ozonation resulted in development of yellowness. In the case of ozonation in water, there were no significant differences in molecular weight and color between ozone-treated chitosans. However, results showed that ozone treatment of chitosan in both water and acetic acid solution was not effective in removing acetyl groups (deacetylation) in chitosan molecules. This study showed that ozone can be used to modify molecular weight and remove pigments of chitosan without chemical use in a shorter time with less cost.
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IMPACT: 2006/01/01 TO 2006/12/31<BR>
Off-flavor of catfish has posed a problem for the aquaculture industry in the southern part of the United States. Every year, many catfish are not marketable because of their off-flavor. If we can develop a method to reduce 2-MIB and GSM to a level below the threshold of 0.7 ppb or completely destroy them, the method will be of great importance. Chitosan has been shown to have antibacterial properties that depend on molecular weight. Currently, depolymerization and decolorization of chitosan are achieved by chemical or enzymatic methods which are time consuming and expensive. Ozone has been shown to be able to degrade macromolecules and remove pigments due to its high oxidation potential and is a new alternative to chemical methods for decreasing chitosan molecular weight. It is important that these new ozonation methods be effective without resulting in new toxins being formed or destroying nutrients.

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PROGRESS: 2005/01/01 TO 2005/12/31<BR>
The overall goal of this research is to determine the safety and efficacy of ozonation as a procedure to reduce aflatoxin in corn. Ozonation reduced aflatoxin levels by 92% and no reversion to the parent compound was observed. Identification of reaction products between ozone and aflatoxin B1 (AFB1) is very important in assessing the suitability, and acceptability of the ozonation process to degrade aflatoxin. The effect of ozone on the degradation of AFB1 in an aqueous model system was determined. Standard solutions containing AFB1 were dried and suspended in water and treated with ozone for up to 60 secs. Dichloromethane extracts and the remaining water solution of the samples were evaluated with 2-D thin layer chromatography (TLC). MALDI-MS analysis was conducted to partially identify reaction products. Dichloromethane extracts analysis showed that AFB1 was totally degraded after 60 sec. Water portion extracts showed the presence of seven compounds having Rf values unrelated to aflatoxin standards. Results of the study suggested the formation of polar compounds. MALDI-MS showed that these compounds have higher molecular weights than AFB1. Mass spectra of dichloromethane extracts from samples ozonated 60 sec showed a base peak of 439. Extracts from water portions showed a based peak of 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.
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IMPACT: 2005/01/01 TO 2005/12/31<BR>
The economic losses suffered by producers of aflatoxin-susceptible crops could be reduced and the availability of foods and feeds increased. Ozonation is a less expensive alternative to ammoniation without the solvent waste problem. Off-flavor of catfish has posed a problem for the aquaculture industry in the southern part of the United States. Every year, many catfish are not marketable because of their off-flavor. If we can develop a method to reduce the level of 2-MIB and GSM to a level below the threshold of 0.7 ppb or completely destroy them, the method will be of great importance. It is important that new methods for decontamination of food and feeds be effective without resulting in new toxins being formed or destroying nutrients.
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PROGRESS: 2005/01/01 TO 2005/12/31<BR>
The overall goal of this research is to determine the safety and efficacy of ozonation as a procedure to reduce aflatoxin in corn. Ozonation reduced aflatoxin levels by 92% and no reversion to the parent compound was observed. Identification of reaction products between ozone and aflatoxin B1 (AFB1) is very important in assessing the suitability, and acceptability of the ozonation process to degrade aflatoxin. The effect of ozone on the degradation of AFB1 in an aqueous model system was determined. Standard solutions containing AFB1 were dried and suspended in water and treated with ozone for up to 60 secs. Dichloromethane extracts and the remaining water solution of the samples were evaluated with 2-D thin layer chromatography (TLC). MALDI-MS analysis was conducted to partially identify reaction products. Dichloromethane extracts analysis showed that AFB1 was totally degraded after 60 sec. Water portion extracts showed the presence of seven compounds having Rf values unrelated to aflatoxin standards. Results of the study suggested the formation of polar compounds. MALDI-MS showed that these compounds have higher molecular weights than AFB1. Mass spectra of dichloromethane extracts from samples ozonated 60 sec showed a base peak of 439. Extracts from water portions showed a based peak of 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.
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IMPACT: 2005/01/01 TO 2005/12/31<BR>
The economic losses suffered by producers of aflatoxin-susceptible crops could be reduced and the availability of foods and feeds increased. Ozonation is a less expensive alternative to ammoniation without the solvent waste problem. Off-flavor of catfish has posed a problem for the aquaculture industry in the southern part of the United States. Every year, many catfish are not marketable because of their off-flavor. If we can develop a method to reduce the level of 2-MIB and GSM to a level below the threshold of 0.7 ppb or completely destroy them, the method will be of great importance. It is important that new methods for decontamination of food and feeds be effective without resulting in new toxins being formed or destroying nutrients.

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PROGRESS: 2004/01/01 TO 2004/12/31<BR>
The overall goal of this research is to determine the safety and efficacy of ozonation as a procedure to reduce aflatoxin in corn. Ozonation reduced aflatoxin levels by 92% and no reversion to the parent compound was observed. 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. 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 in extracts from treated contaminated corn but to a lesser extent. No aflatoxin was observed in extracts from untreated and treated clean corns. No aflatoxin was observed in hexane extracts from all samples. Hexane extracts from ozone-treated contaminated corn had lower inhibitory effects. This suggested that a fat-soluble mutagen was being formed or natural inhibitors of mutagenicity were being destroyed. There were results indicating the possible formation of new mutagenic compounds and the presence of anti-mutagenic or inhibitory compounds depending on the solvent. Further investigation into the products being formed and the inhibitory compounds being destroyed is underway. Preliminary results indicated that lutein is not destroyed by ozonation and the molecular weight of the proteins is not altered. Further study is necessary to verify these results and to determine if the lutein extracted still shows antimutagenic capability against aflatoxin as reported in the literature.
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IMPACT: 2004/01/01 TO 2004/12/31<BR>
The economic losses suffered by producers of aflatoxin-susceptible crops could be reduced and the availability of foods and feeds increased. Ozonation is a less expensive alternative to ammoniation without the solvent waste problem. Off-flavor of catfish has posed a problem for the aquaculture industry in the southern part of the United States. Every year, many catfish are not marketable because of their off-flavor. If we can develop a method to reduce the level of 2-MIB and GSM to a level below the threshold of 0.7 ppb or completely destroy them, the method will be of great importance. It is important that new methods for decontamination of food and feeds be effect without resulting in new toxins being formed or destroying nutrients.
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PROGRESS: 2003/01/01 TO 2003/12/31<BR>
The overall goal of this research is to determine the safety and efficacy of ozonation as a procedure to reduce aflatoxin in corn. Results thus far have suggested either that a new-fat soluble mutagen is being formed or that the ozonation process is destroying natural inhibitors of mutagenicity in corn. We also found oxidation of some unsaturated lipids in the ozone treated contaminated corn. The current objectives are: 1) to determine if fat-soluble mutagens are being produced in non-aflatoxin contaminated and aflatoxin contaminated corn by ozonation; 2) to determine if anti-mutagens or inhibitors of mutagenicity are being destroyed in non-aflatoxin and aflatoxin contaminated corn by ozonation; and 3) to determine if nutrients in the corn are being destroyed by ozonation. Ozonation may be a valuable method for the decontamination of aflatoxin containing corn, but further analysis is required on the mutagenic and inhibitory effects of the reaction products formed. Planned work includes using radio labeled aflatoxin to follow its distribution more carefully to see if it is bound to the protein and to separate and identify the new mutagens that may be forming. Proteins and anti-mutagen components in the corn will also be tested to determine if ozone has any affect on their stability. Work is also continuing on developing an ozonation procedure for removing off-flavors from catfish fillets.
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IMPACT: 2003/01/01 TO 2003/12/31<BR>
The economic losses suffered by producers of aflatoxin-susceptible crops could be reduced and the availability of foods and feeds increased. Ozonation is a less expensive alternative to ammoniation without the solvent waste problem. Off-flavor of catfish has posed a problem for the aquaculture industry in the southern part of the United States. Every year, many catfish are not marketable because of their off-flavor. If we can develop a method to reduce the level of 2-MIB and GSM to a level below the threshold of 0.7 ppb or completely destroy them, the method will be of great importance. It is important that new methods for decontamination of food and feeds be effect without resulting in new toxins being formed or destroying nutrients.
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PROGRESS: 2002/01/01 TO 2002/12/31<BR>
The present study was undertaken to determine the distribution of ozone-aflatoxin reaction products in corn and to isolate these compounds using a sequential fractionation method. Additionally, the purpose was to determine the mutagenic potentials of the isolates in the Ames assay using tester strains TA 98 and TA 100. Clean and aflatoxin contaminated corn were treated with ozone gas (10-12 wt%). Ozonation reduced aflatoxin levels by 92 percent and no reversion to the parent compound was observed. Ground corn was solvent fractionated and mutagenicity of isolates was determined by the Ames assay. Dichloromethane, methanol and acetone fractions showed no dose-response or mutagenic potential. For hexane fraction isolates, the number of revertants decreased for both TA 98 and TA 100, with TA 100 showing a dose response. These findings provide evidence of antimutagenic compounds in corn that interfered with the assay. The organic fraction after digestion with Pronase E showed mutagenic potential for TA 100 for ozone treated contaminated sample, but inhibitory effects for TA 98. Dichloromethane extraction of the solids after enzymatic digestion showed increased mutagenic potential against TA 98. This might be an indication of the possible formation of reaction products that have relatively low mutagenic potential. These results suggest either a new-fat soluble mutagen is being formed or the ozonation process is destroying natural inhibitors of mutagenicity in corn. Ozonation may be a valuable method for the decontamination of aflatoxin containing corn, but further analysis is required on the mutagenic and inhibitory effects of the reaction products formed. Planned work includes using radio labeled aflatoxin to follow its distribution more carefully to see if it is bound to the protein and to separate and identify the new mutagens that may be forming. Work is also in progress on developing an ozonation procedure for removing off-flavors from catfish fillets.
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IMPACT: 2002/01/01 TO 2002/12/31<BR>
The economic losses suffered by producers of aflatoxin-susceptible crops could be reduced and the availability of foods and feeds increased. Ozonation is a less expensive alternative to ammoniation without the solvent waste problem. Off-flavor of catfish poses a problem for the aquaculture industry in the southern part of the United States. Every year, many catfish are not marketable because of their off-flavor. If we can develop a method to reduce the level of 2-MIB and GSM to a level below the threshold of 0.7 ppb or completely destroy them, the method will be of great importance. It is important that new methods for decontamination of food and feeds be effective without resulting in new toxins being formed or destroying nutrients.

<BR> <BR> PROGRESS: 2001/01/01 TO 2001/12/31<BR>
Our study aimed to determine the efficacy and safety of ozonation to reduce aflatoxin hazards in corn and peanuts. Other ozonation studies are in progress. Ozonation reduced aflatoxin levels by 92 percent and no reversion to the parent compound was observed. Ozonation had minimal effect on fatty acids of uncontaminated corn, but had significant effect on fatty acids of contaminated corn. Crude extracts showed no mutagenic potential in the Ames assay using TA98 and TA100. Clean-up using hexane increased their mutagenic potentials. Clean-up using Mycosep columns increased the mutagenic potentials 18 to 617 percent. Hexane extracts from ozone-treated contaminated corn had lower inhibitory effect. This suggested that a fat-soluble mutagen is being formed or natural inhibitors of mutagenicity are being destroyed. Ground corns were then solvent fractionated and mutagenicity of isolates was determined by the Ames assay. No isolates from the dichloromethane extraction had mutagenic potential against both tester strains up to a concentration of 10,000 ug/plate. Subsequent extraction using methanol and acetone showed a slight increase in mutagenic response for TA 98 but not for TA 100. Isolates from hexane fractions had mixed results. The number of revertants using TA 98 slightly increased but not for TA 100. The organic fraction after digestion with Pronase E did not show mutagenic potentials against TA 98 and TA 100. Conversely, extracts after enzymatic digestion followed by dichloromethane extraction showed strong mutagenic potential against TA 98. This might be an indication of the possible formation of reaction products that have relatively low mutagenic potential. After 9 hours of ozone treatment aflatoxin content in peanuts was reduced by 58.8 percent. After 72 hours of ozonation AFB1 level decreased 92.7 percent, therefore, greater than 72 hours of exposure time is required for complete degradation of AFB1. None of the solvent fractionation extracts showed mutagenic response for TA 100, but all extracts except methylene chloride (MC) and organic Pronase phase showed lower revertants than the natural numbers indicating that there were interfering compounds, especially for methanol extracts. MC extracts showed mutagenicity for TA 98, but lower than natural revertant numbers for all other extracts. These findings provide evidence of antimutagenic compounds in corn that interfered with the assay. These results suggested either that a new-fat soluble mutagen is being formed or that the ozonation process is destroying natural inhibitors of mutagenicity in corn. Ozonation may be a valuable method for the decontamination of aflatoxin containing peanuts, but further analysis is required on the mutagenic and inhibitory effects of the reaction products formed. Ozonation shows promise as a process for destroying 2-MIB and GSM in off-flavor catfish in preliminary experiments. Further study is needed to optimize the process for enhancing degradation of these compounds.
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IMPACT: 2001/01/01 TO 2001/12/31<BR>
The economic losses suffered by producers of aflatoxin-susceptible crops could be reduced and the availability of foods and feeds increased. Ozonation is a less expensive alternative to ammoniation without the solvent waste problem. Off-flavor of catfish has posed a problem for the aquaculture industry in the southern part of the United States. Every year, many catfish are not marketable because of their off-flavor. It is known that the off-flavor of catfish is mainly caused by two chemicals: 2-methylisoborneol (2-MIB) and geosmin (GSM). The human threshold for sensing the level of the off-flavor is 0.7 ppb. If we can develop a method to reduce the level of 2-MIB and GSM to a level below 0.7 ppb or completely destroy them, the method will be of great importance. It is important that new methods for decontamination of food and feeds be effect without resulting in new toxins being formed.