Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2015 The Evaluation of Adsorbents for the Removal of Aflatoxin M1 from Contaminated Milk Erika D. Womack Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Womack, Erika D., "The Evaluation of Adsorbents for the Removal of Aflatoxin M1 from Contaminated Milk" (2015). Theses and Dissertations. 4456. https://scholarsjunction.msstate.edu/td/4456 This Dissertation - 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]. Automated Template B: Created by James Nail 2011V2.1 The evaluation of adsorbents for the removal of aflatoxin M1 from contaminated milk By Erika D. Womack A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Molecular Biology in the Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology Mississippi State, Mississippi December 2015 Copyright by Erika D. Womack 2015 The evaluation of adsorbents for the removal of aflatoxin M1 from contaminated milk By Erika D. Womack Approved: ____________________________________ Darrell L. Sparks, Jr. (Major Professor) ____________________________________ Ashli Brown-Johnson (Minor Professor) ____________________________________ Janice DuBien (Minor Professor) ____________________________________ Stephanie H. Ward (Committee Member) ____________________________________ Xueyan Shan (Committee Member) ____________________________________ Kenneth O. Willeford (Graduate Coordinator) ____________________________________ George Hopper Dean College of Agriculture and Life Sciences Name: Erika D. Womack Date of Degree: December 11, 2015 Institution: Mississippi State University Major Field: Molecular Biology Major Professor: Darrell L. Sparks, Jr. Title of Study: The evaluation of adsorbents for the removal of aflatoxin M1 from contaminated milk Pages in Study: 210 Candidate for Degree of Doctor of Philosophy Taking precautions to restrain aflatoxin M1 (AFM1) from milk is critical, particularly due to the health and economic impact AFM1 imposes. The predominant post-harvest means of reducing AFM1 in milk includes the addition of sequestering agents to feed to diminish the bioavailability of aflatoxin B1 (AFB1), the parent compound of AFM1 found in contaminated feed. Still, residual AFM1 has been found in the milk. Using sequestering agents added to raw milk, we found that activated carbon was the most effective binder to reduce AFM1 contamination. The combination of 0.75% granular activated carbon (GAC) and a flow rate of 0.4 mL/min to pump contaminated milk through a glass column were chosen as optimum conditions for the removal of AFM1. These conditions obtained a 98.4% reduction of 0.75 ng/mL AFM1 from raw milk. The treated milk was also analyzed to assess the effects of GAC on milk constituents. The results determined that GAC had no significant effect on major nutritive milk constituents: total protein, lactose, minerals, and fat. Additionally, we optimized an extraction method coupled to high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) that minimized matrix effects, lowered the levels of detection, and reduced analysis costs. The optimized extraction method was based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe). Results determined 5 mL milk (15°C) with 10 mL acetonitrile, 3200 g centrifugation, and 0.2 µm syringe filter were the optimum conditions for the extraction of 0.5 ng/mL AFM1 from raw milk. The method was validated according to AOAC guidelines. This study reports experimental results on AFM1 remediation from raw bovine milk. The use of GAC for the removal of AFM1 in raw milk has reduced the amount of AFM1 below the FDA action limit and European Union maximum regulatory level. This method could have a global health impact, particularly, for people in developing nations and for infants and children who are more susceptible to the adverse effects of AFM1. DEDICATION My graduate work is dedicated to my mom, Lenna B. Womack. Her strength and faith surpassed anything I could ever hope to accomplish. I really would not have made it this far without her encouragement and love. It has impacted who I am today and for that I am eternally grateful. Rest in Heaven. ii ACKNOWLEDGEMENTS I would first like to thank my major professor, Dr. Darrell L. Sparks. Without his guidance and knowledge throughout my entire doctoral career, this dissertation would not be possible. Even through my “…so Dr. Sparks…” questions, he’d effortlessly guide me through any problem. I also would like to thank Dr. Ashli Brown who has guided me as well through this degree process. She has offered tremendous support and introduced me to a number of opportunities I may have never experienced. I would like to thank the rest of my committee members, Dr. Janice DuBien, Dr. Stephanie H. Ward, and Dr. Xueyan Shan, for providing guidance and mentoring me through this process. I would especially like to thank Dr. DuBien for helping me through some difficult statistical analysis. I would like to thank Kenneth Graves for supplying me with limitless fresh raw milk from MSU’s dairy farm. I would like to thank the Mississippi State Chemical Laboratory for, not only analyzing samples for me but also teaching me to use very expensive laboratory equipment and instruments. Thank you for putting a little trust in me that I may not break your instruments. I would like to thank the Mississippi Agricultural and Forestry Experiment Station (MAFES) for supporting my graduate assistantship. Lastly, I would like to thank my family and friends for their love and support. iii TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES .............................................................................................................x CHAPTER I. GENERAL INTRODUCTION ..........................................................................1 Background of the Problem ...............................................................................1 Statement of the Problem and Study Justification .............................................4 Significance, Objectives, and Goals of the Project ............................................5 II. LITERATURE REVIEW ..................................................................................7 Mycotoxin-Producing Fungi ..............................................................................7 Aspergillus .......................................................................................................11 Aflatoxin ..........................................................................................................13 Background ................................................................................................13 Economic and health implications of aflatoxin .........................................15 Factors influencing aflatoxin contamination .............................................18 Aflatoxin biosynthesis ...............................................................................22 Pre-harvest Management of Aflatoxin .......................................................28 III. A RECENT REVIEW OF NON-BIOLOGICAL REMEDIATION OF AFLATOXIN-CONTAMINATED CROPS .......................................31 Abstract ............................................................................................................31 Introduction ......................................................................................................31 Sorting of aflatoxin-contaminated grains .........................................................34 Binding agents for the reduction of aflatoxins .................................................38 Irradiation for the reduction of aflatoxins ........................................................41 Chemical methods for the detoxification of aflatoxins ....................................46 Conclusion .......................................................................................................50 IV. AFLATOXIN M1 IN MILK AND MILK PRODUCTS..................................52 iv Abstract ............................................................................................................52 Introduction ......................................................................................................53 Toxicity ............................................................................................................55 Analytical methodology ...................................................................................57 Sample preparation and clean-up ...............................................................57 Detection and quantitative techniques .......................................................62 Occurrence .......................................................................................................66