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Aflatoxin Contaminated Corn Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2010 Comparing detection methods of aflatoxin and exploring aflatoxin decontamination methods Rebecca Burgett Singleterry Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Singleterry, Rebecca Burgett, "Comparing detection methods of aflatoxin and exploring aflatoxin decontamination methods" (2010). Theses and Dissertations. 1103. https://scholarsjunction.msstate.edu/td/1103 This Graduate Thesis - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. COMPARING DETECTION METHODS OF AFLATOXIN AND EXPLORING AFLATOXIN DECONTAMINATION METHODS By Rebecca Burgett Singleterry A Thesis Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Agricultural Life Science (Biochemistry) in the Department of Biochemistry and Molecular Biology Mississippi State, Mississippi December 2010 COMPARING DETECTION METHODS OF AFLATOXIN AND EXPLORING AFLATOXIN DECONTAMINATION METHODS By Rebecca Burgett Singleterry Approved: ________________________________ ________________________________ Ashli Brown Ty Schmidt Assistant Professor of Biochemistry Assistant Professor of Animal and Molecular Biology Animal and Dairy Science (Major Professor) (Committee Member) ________________________________ ________________________________ Scott Willard Din-Pow Ma Professor and Head of Biochemistry Professor of Biochemistry and Molecular Biology and Molecular Biology (Committee Member) (Graduate Coordinator of the Department of Biochemistry and Molecular Biology) ________________________________ George Hopper Interim Dean of the College of Agriculture and Life Sciences Name: Rebecca Burgett Singleterry Date of Degree: December 10, 2010 Institution: Mississippi State University Major Field: Agriculture and Life Science (Biochemistry) Major Professor: Dr. Ashli Brown Title of Study: COMPARING DETECTION METHODS OF AFLATOXIN AND EXPLORING AFLATOXIN DECONTAMINATION METHODS Pages in Study: 81 Candidate for Degree of Master of Science Ethanol fermentation of highly concentrated aflatoxin-contaminated corn (Zea mays) was conducted on a lab scale to determine if aflatoxin concentrated in the distilled ethanol and/or dry distillers grain end-products. Alliquots of fermented mash, distilled ethanol, stillage, and dry distillers grain (DDG) were analyzed via LC-MS/MS and immunoassay detection methods for aflatoxin. Results indicate that aflatoxin does not greatly concentrate during fermentation in the DDGs and is undetectable in distilled ethanol. Addition of binders, MTB-100®, to aflatoxin-contaminated DDGs showed great reduction in aflatoxin concentrations when analyzed via LC-MS/MS. Also, an experiment investigating detoxification of aflatoxin using Clorox® was conducted. Results obtained from LC-MS/MS showed a positive correlation of decreased aflatoxin levels with increasing Clorox® levels following a logarithmic trend. Key words: aflatoxin, ethanol fermentation, decontamination methods ACKNOWLEDGMENTS I cannot thank enough the encouragement and prodding of my major professor Dr. Ashli Brown to whom I am forever humbly indebted for the completion of this degree in her willingness to see me through it. Thank you for your guidance and sense of humor through my years as an undergraduate and graduate student. Next, I would like to thank Dr. Scott Willard and Dr. Ty Schmidt for advising me through this process. I have so much enjoyed getting to know you. It is professors like you that make a world of difference in shaping the careers of students, and I am so blessed to have been under your guidance. Also, I would like to say a special thank you to Dr. Brian Baldwin for the use of his lab and sharing his experience in the field. Thank you for taking the time to help me with the set-up of my experiments and for answering all my questions. Your sense of humor made it all worth while. Next, I would like to thank all the staff from the mass spectrometry lab of Mississippi State Chemical Laboratory which became my family during my years at the lab. You are great co-workers from whom I learned much about the “art” of science. I now realize that the special atmosphere present at the lab will never be replicated elsewhere. I will miss you guys greatly. Finally, I would like to thank my family. I am not sure if I would have finished if it were not for your faithful encouragement. Most of all, I would like to ii thank my heavenly Father and Savior for His love towards me. Thank you for your grace and forgiveness, of which none I deserve. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................. ii LIST OF TABLES ........................................................................................................ vi LIST OF FIGURES ...................................................................................................... vii CHAPTER I. LITERATURE REVIEW ....................................................................... 1 Aflatoxin .................................................................................................. 1 Introduction into Aspergillus ................................................................... 4 Introduction into Mycotoxins .................................................................. 7 History of Aflatoxin’s Discovery ............................................................. 9 Biological Activity ................................................................................... 10 Factors Influencing Aflatoxin Formation ................................................ 11 Toxicity .................................................................................................... 13 International Legislation .......................................................................... 18 Sampling Method ..................................................................................... 18 Detection Methods ................................................................................... 20 Ethanol Fermentation ............................................................................... 25 Detoxification of Aflatoxin ...................................................................... 27 LITERATURE CITED ........................................................................... 30 II. INVESTIGATION OF AFLATOXIN INOCULATED CORN (Zea mays) DURING ETHANOL FERMENTATION USING LC-MS/MS AND IMMUNOASSAY DETECTION METHOD, A STRATEGY FOR SALVAGING AFLATOXIN- CONTAMINATED DDGS USING BINDERS .......................... 41 Abstract ................................................................................................... 41 Introduction ............................................................................................ 42 Materials and Methods ............................................................................ 46 Corn Sample Information ........................................................... 46 Preparation and Fermentation of Corn ........................................ 47 Distillation of Ethanol ................................................................. 49 iv Preparation, Extraction, and Analysis of Samples ...................... 50 Corn................................................................................. 50 DDGs .............................................................................. 51 Fermented Mash .............................................................. 51 Distilled Ethanol ............................................................. 51 Stillage ............................................................................ 52 Immunoassay Conditions ............................................................ 52 Immunoaffinity column assay .......................................... 52 Enzyme-link immunosorbent assay ................................. 53 LC-MS/MS Conditions ............................................................... 53 Standards used for LC-MS/MS Analysis ........................ 55 Preparation and Analysis of DDGs mixed with Binders ............ 55 Results ...................................................................................................... 56 Discussion ............................................................................................... 62 Conclusion .............................................................................................. 65 LITERATURE CITED ........................................................................... 67 III. DETOXIFICATION OF AFLATOXIN USING CLOROX® ANALYZED BY LC-MS/MS .................................................... 72 Abstract ................................................................................................... 72 Introduction ............................................................................................. 72 Materials and Methods ............................................................................ 74 Standards Information ................................................................. 74 Sample Preparation ..................................................................... 74 LC-MS/MS Conditions
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