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Mid-Infrared Spectral Characterization of Aflatoxin Contamination in Peanuts

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Mid-Infrared Spectral Characterization of Aflatoxin Contamination in Peanuts MID-INFRARED SPECTRAL CHARACTERIZATION OF AFLATOXIN CONTAMINATION IN PEANUTS Hande Kaya Celiker Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biological Systems Engineering Parameswarakumar Mallikarjunan, Chair Justin R. Barone Maria E. Christie Archileo N. Kaaya David G. B. Schmale September 11, 2012 Blacksburg, VA Keywords: Peanut; Aflatoxin; Aspergillus species; PLS regression; Discriminant Analysis; Fourier Transform Infrared Spectroscopy; Attenuated Total Reflectance; Photoacoustic Spectroscopy Copyright 2012, Hande Kaya Celiker MID-INFRARED SPECTRAL CHARACTERIZATION OF AFLATOXIN CONTAMINATION IN PEANUTS Hande Kaya Celiker ABSTRACT Contamination of peanuts by secondary metabolites of certain fungi, namely aflatoxins present a great health hazard when exposed either at low levels for prolonged times (carcinogenic) or at high levels at once (poisonous). It is important to develop an accurate and rapid measurement technique to trace the aflatoxin and/or source fungi presence in peanuts. Thus, current research focused on development of vibrational spectroscopy based methods for detection and separation of contaminated peanut samples. Aflatoxin incidence, as a chemical contaminant in peanut paste samples, was investigated, in terms of spectral characteristics using FTIR-ATR. The effects of spectral pre-processing steps such as mean-centering, smoothing the 1 st derivative and normalizing were studied. Logarithmic method was the best normalization technique describing the exponentially distributed spectral data. Spectral windows giving the best correlation with respect to increasing aflatoxin amount led to selection of fat associated spectral bands. Using the multivariate analysis tools, structural contributions of aflatoxins in peanut matrix were detected. The best region was decided as 3028-2752, 1800-1707, -1 2 1584-1424, and 1408-1127 cm giving correlation coefficient for calibration (R C), root mean square error for calibration (RMSEC) and root mean square error for prediction (RMSEP) of 98.6%, 7.66ppb and 19.5ppb, respectively. Applying the constructed partial least squares model, 95% of the samples were correctly classified while the percentage of false negative and false positive identifications were 16% and 0%, respectively. Aspergillus species of section Flavi and the black fungi, A. niger are the most common colonists of peanuts in nature and the majority of the aflatoxin producing strains are from section Flavi . Seed colonization by selected Aspergillus spp. was investigated by following the chemical alterations as a function of fungal growth by means of spectral readouts. FTIR-ATR was utilized to correlate spectral characteristics to mold density, and to separate Aspergillus at section, species and strain levels, threshold mold density values were established. Even far before the organoleptic quality changes became visually observable (~10,000 mold counts), FTIR distinguished the species of same section. Besides, the analogous secondary metabolites produced increased the similarity within the spectra even their spectral contributions were mostly masked by bulk peanut medium; and led to grouping of species producing the same mycotoxins together. Aflatoxigenic and non-aflatoxigenic strains of A. flavus and A. parasiticus were further studied for measurement capability of FTIR-ATR system in discriminating the toxic streams from just moldy and clean samples. Owing to increased similarity within the collected spectral data due to aflatoxin presence, clean samples (having aflatoxin level lower than 20 ppb, n=44), only moldy samples (having aflatoxin level lower than 300 ppb, n=28) and toxic samples (having aflatoxin level between 300-1200 ppb, n=23) were separated into appropriate classes (with a 100% classification accuracy). iii Photoacoustic spectroscopy (PAS) is a non-invasive technique and offers many advantages over more traditional ATR system, specifically, for in-field measurements. Even though the sample throughput time is longer compared to ATR measurements, intact seeds can be directly loaded into sample compartment for analysis. Compared to ATR, PAS is more sensitive to high moisture in samples, which in our case was not a problem since peanuts have water content less than 10%. The spectral ranges between: 3600-2750, 1800-1480, 1200-900 cm - 1 were assigned as the key bands and full separation between Aspergillus spp. infected and healthy peanuts was obtained. However, PAS was not sensitive as ATR either in species level classification of Aspergillus invasion or toxic-moldy level separation. When run for separation of aflatoxigenic versus non-aflatoxigenic batches of samples, 7 out of 54 contaminated samples were misclassified but all healthy peanuts were correctly identified (15 healthy/ 69 total peanut pods). This study explored the possibility of using vibrational spectroscopy as a tool to understand chemical changes in peanuts and peanut products to Aspergillus invasion or aflatoxin contamination. The overall results of current study proved the potential of FTIR, equipped with either ATR or PAS, in identification, quantification and classification at varying levels of mold density and aflatoxin concentration. These results can be used to develop quality control laboratory methods or in field sorting devices. iv ACKNOWLEDGEMENTS I am greatly indebted to the chair of my dissertation committee, Dr. Kumar Mallikarjunan, for his guidance and support during my doctoral studies. His experience and knowledge in the field of food science and emerging non-destructive technologies motivated me to go into the doctoral level research and also shaped my future research in the same field. I am also thankful to him for his sincere, thoughtful approach and his constructive suggestions throughout this process. I would also like to thank my other committee members for their invaluable assistance and support. I am appreciative of Dr. Justin Barone’s help for enhancing the research idea thorough his experience in the field. I would like to thank Dr. David Schmale III for providing me with his lab resources, also the time that he dedicated for me, whenever I needed it . I would also like to thank Dr. Maria Elisa Christie for her genuine and helpful approach throughout the research process. I am also thankful to Dr. Archileo Kaaya for his hospitality during my Uganda visit and helping me see other possible views on my research subject. I am thankful to the faculty and staff in the Department of Biological Systems Engineering. I extend my special thanks to Amy Egan for her help at different points in time during the last four years, including the initial set-up of the lab and FTIR at CRC. I am also appreciative of Govindraj Dev Kumar for coordinating with me during my work in the Food Microbiology Lab in the Department of Food Science and Technology. I also thank Harriet Williams for her help in using GC/MS. I am also grateful to Fatma Sahmurat who has been with me in several instances through her sincere friendship. I would also like to thank Chris Whysong for her continuous help and v answering my questions patiently. I also thank Kevin Scott Richter for working with me in harmony in the same lab. I am also thankful to Stephen Nimitz for his friendship. Finally, it would not have been possible without my family. I thank my mother and sister for their continuous support and encouragement. My deepest appreciation goes to my husband for his patience and making my job much easier in numerous occasions during this journey. vi TABLE OF CONTENTS TITLE PAGE ................................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................................ v TABLE OF CONTENTS .............................................................................................................. vii LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi CHAPTER 1 INTRODUCTION .......................................................................................................................... 1 CHAPTER 2 LITERATURE REVIEW .......................................................................................................... 8 INTRODUCTION ..................................................................................................................... 8 2.1 The genus Aspergillus .......................................................................................................... 9 2.2 Aflatoxins ........................................................................................................................... 13 2.2.1 Chemistry of Aflatoxin ...............................................................................................13 2.2.2 Some of the Outbreaks Associated with Aflatoxin .....................................................15 2.2.3 Aflatoxicosis ...............................................................................................................15
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