Citation
Inactivation of Aflatoxins B1, B2 in Peanuts by Pulsed Light (Pl)

Material Information

Title:
Inactivation of Aflatoxins B1, B2 in Peanuts by Pulsed Light (Pl)
Creator:
Abuagela, Manal Othman
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (138 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science
Food Science and Human Nutrition
Committee Chair:
SARNOSKI,PAUL J
Committee Co-Chair:
GU,LIWEI
Committee Members:
WANG,YU
SMITH,MATTHEW E

Subjects

Subjects / Keywords:
aflatoxins -- pulsedlight
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Food Science thesis, Ph.D.

Notes

Abstract:
Aflatoxins (AFTs) are secondary metabolites of Aspergillus flavus and Aspergillus parasiticus molds. They are potent toxigenic, carcinogenic, and immunosuppressive compounds commonly found in groundnuts and groundnut products. Many methods have been studied to provide food free of AFTs. Pulsed light (PL) treatment is one of the novel food preservation techniques, which showed promising results in the degradation of pure AFT in dilution (97% reduction). The first section in this study was conducted to determine the ability of PL treatment to reduce AFB1 and AFB2 in A. flavus inoculated peanuts (with skin and without skin). PL treatment was using three distances (5, 7, and 10 cm) from the PL strobe and different exposing times. The second section was conducted to optimize all parameters which were attributed to the efficiency of the treatment, and trying to establish a PL optimum treatment for peanuts' treatment. Five treatment methods were tested peanuts in an aluminum plate on a conventional conveyor; peanuts on a shaker for shaking treatment, peanuts in a glass tube for rotating treatment, sliced peanuts, and finally an ice tray treatment. In addition, a hurdle technique was conducted by soaking the peanuts in a citric acid previous to the PL treatment in the third section of this study. AFB1 and AFB2 contents were detected using an enzyme-linked immunosorbent assay (ELISA) and liquid Chromatography Mass Spectrometry (LC-MS/MS). The temperatures of the treated peanuts were monitored during the study using an infrared thermometer and thermo-sensors to determine the actual temperature inside the kernels during PL treatment. Furthermore, the last section was an investigation the effect of storage time on some of the PL treated peanuts' quality parameters such as peroxide value, free fatty acids, and acid value. Results showed that in-plate treatment of with-skin and without-skin peanuts for 240s at a 7 cm distance degraded AFTs by 62.4% and 86% respectively. PL treatment in-plate for 300s at 5 cm from the strop reduced AFT by 82% for with skin and 95.3% for without skin. However, the surfaces of the peanuts were burned. Surface burning was avoided by shaking or rotating the peanuts during the treatment. In addition to keeping the peanuts' quality, shaking treatment had the best reduction percentage compared with all other PL treating methods due to exposing all peanut sides to the light during the treatment. The hurdle techniques' results achieved 100% reduction in an approximately short time comparing with PL treatment solo. Peroxide value and free fatty acid were under the restricted levels after three months of storage. This study indicates that PL illumination could degrade the AFTs in the peanuts as a result of PL's photo-chemical and photo-thermal effects. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2017.
Local:
Adviser: SARNOSKI,PAUL J.
Local:
Co-adviser: GU,LIWEI.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2018-06-30
Statement of Responsibility:
by Manal Othman Abuagela.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
6/30/2018
Classification:
LD1780 2017 ( lcc )

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1 INACTIVATION OF AFLATOXINS B 1 B 2 IN PEANUTS BY PULSED LIGHT (PL) By MANAL OTHMAN ABUAGELA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D EGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017

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2 2017 Manal Othman Abuagela

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3 To God, who created me as a being filled with hope, faith, and persistence. To my Mother, whose spirit watches over me through the good and bad. To my kids, who bring happiness into my life. To my people, who have supported me over the course of this difficult, but worthy, journey of my career. Last of all, to my country, for believing in my abilities and giving me the opp ortunity to study abroad despite the competition.

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4 ACKNOWLEDGMENTS without the help of a few. I would like to show gratitude and appreciation to my chair Dr. Paul Sarnoski ; his feedback, advic e and support helped me through my times of need. I would also like to address and appreciate the help of my previous chair Dr. Wade Yang for providing me the opportunity to study in his lab ; he opened my eyes to so much and I still carry his knowledge with grace. Also, I would like to thank Dr. Liwei Gu for his patience, academic support, and uncountable advice ; and Dr. Matthew Smith for his support and understanding and the strong knowledge he provided me in a very short time as he provid e d me with a key of a huge sealed door. I would also like to thank Dr. Harry S i tr e n for his wisdom, as his way of seeing things made the most difficult of problems seem so easy to figure out. I am truly appreciative of Dr. Susan Percival for providing me t he funds to support my research for the last 2 years. I would like to thank Dr. Charles Sims for the amount of support he gave me is what made this tough journey a bit bearable. I would like to thank my friend Marianne Mangon e for the unlimited support a nd help. I would also like to thank Dr. Yavuz Yagis ; he helped me out with some experiments and without his help I would have been lost. I would also like to tha nk my lab mates: Abeer Al H endi and Hussein Mostafa Awad for the endless amount of support th ey gave me in my time of need. Also Basheer Iqdam and M ohammed Gomaa for their advice and appreciative of my other colleagues: Devin Lewis, Tara Faidhalla, Senem Guner, Rasha Al Mansori, Bhaskar A Janve, Xingyu Zhao, Sara M arshal ; and Dr. Asli Kirli,

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5 Samantha Amos, Bri ttany Xu, Mary Ann Spitzer and S h elia Parker fo r their help, love, and support ; and Chi Gao for helping with my LC/MS results. I would like to thank my Mother, Fatima Bezan ; she is the one who motiv ated me to aspire for greatness. S he raised me to be a fighter like she was and to never give up no matter how many obstacles that come in my way. My Mother may not be with me physically, but her spirit will always be by my side I did this for her and only her, and I know I made her proud. I would also like to thank my Father, Othman Abuagela, and my siblings: Hossam Abuagela, Essra Abuagela, Amani Abuagela, and Asma Abuagela, for their endless support. I would also like to thank my husband Fouad Abuhalala, and my supportive kids: Nazek Abuhalala, Nawres Abuhalala, Shames Abuhalala, Shahed Abuhalala, and Sulieman Abuhalala. I would like to thank them for their support and understanding of the situation and the pressure we went through as a family I would like to th ank them for their strength and wisdom as they endured too much throughout this journey.

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6 TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INT RODUCTION ................................ ................................ ................................ .... 17 2 LITERATURE REVIEW ................................ ................................ .......................... 21 Aflatoxins Regulation ................................ ................................ .............................. 23 AFT Detoxification Methods ................................ ................................ .................... 26 Chemical Approach ................................ ................................ .......................... 26 Using the ozone in AFT detoxification ................................ ........................ 27 Ammonia NH 3 treatment ................................ ................................ ............ 29 Addition of sorbents ................................ ................................ ................... 31 Citric acid treatment ................................ ................................ ................... 31 Biological Approach ................................ ................................ .......................... 32 Adsorption of mycotoxins by yeasts ................................ ........................... 33 Adsorption of mycot oxins by lactic acid bacteria (LAB) .............................. 34 Enzymes for detoxification ................................ ................................ ......... 35 Physical Approach ................................ ................................ ............................ 35 Extraction ................................ ................................ ................................ ... 35 Heat ................................ ................................ ................................ ........... 36 Irradiation ................................ ................................ ................................ ... 37 Microwave ................................ ................................ ................................ .. 37 Ultraviolet (UV) light ................................ ................................ ................... 38 Pulsed light (PL) ................................ ................................ ......................... 39 Justification of Study ................................ ................................ ............................... 4 1 3 INACTIVATION OF AFLATOXINS B 1 B 2 IN WITH SKIN AND WITHOUT SKIN PEANUTS BY PULSED LIGHT AND THE EFFECT OF PULSED LIGHT ON PEANUT PROPERTIES ................................ ................................ ......................... 43 Materials and Methods ................................ ................................ ............................ 45 Preparation of the Samples ................................ ................................ .............. 45 Sample inoculation ................................ ................................ ..................... 45 Moisture optimization ................................ ................................ ................. 47

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7 Uniformity test ................................ ................................ ............................ 47 Pulsed UV light treatment ................................ ................................ .......... 48 Aflatoxin extraction ................................ ................................ ..................... 49 Competitive ELISA ................................ ................................ ..................... 49 High performance liquid chromatography mass spectrometry analysis (HPLC/MS/MS) ................................ ................................ ....................... 49 Temperature Measurements ................................ ................................ ............ 50 Infrared thermometer ................................ ................................ ................. 50 In situ temperature measurements ................................ ............................ 50 Chemical Analysis ................................ ................................ ............................ 51 Peroxide value ................................ ................................ ........................... 51 Free fatty acids and acid value ................................ ................................ .. 52 Results and Discussio n ................................ ................................ ........................... 53 Uniformity Test ................................ ................................ ................................ 53 Moisture Content ................................ ................................ .............................. 54 Temperature Measurements ................................ ................................ ............ 55 ELISA Results ................................ ................................ ................................ .. 56 HPLC MS/MS Analysis ................................ ................................ ..................... 57 Peroxide Value and Free Fatty Acids ................................ ............................... 60 4 OPTIMIZATION OF THE PULSED LIGHT (PL) EXPOSURE METHOD FOR AFLATOXIN DETOXIFICATION FOR WITH SKIN AND WITHOUT SKIN PEANUTS ................................ ................................ ................................ ............... 72 Materials and Methods ................................ ................................ ............................ 74 PL Exposure ................................ ................................ ................................ ..... 75 Experimental Design ................................ ................................ ........................ 76 In plate samples ................................ ................................ ......................... 76 Shaking treatment ................................ ................................ ...................... 76 Sliced peanuts ................................ ................................ ........................... 76 In ice tray treatment ................................ ................................ ................... 77 In tube treatment ................................ ................................ ........................ 77 Results and Discussion ................................ ................................ ........................... 78 ELISA and HPLC MS/MS Results ................................ ................................ .......... 79 With Skin Samples ................................ ................................ ........................... 79 In plate treatment ................................ ................................ ....................... 79 Shaking treatment ................................ ................................ ...................... 80 In ice treatment ................................ ................................ .......................... 80 In tube treatment ................................ ................................ ........................ 80 Without Skin Samples ................................ ................................ ...................... 81 In plate treatment ................................ ................................ ....................... 81 Shaking treatment ................................ ................................ ...................... 82 Slices treatment ................................ ................................ ......................... 82 In ice treatment ................................ ................................ .......................... 82 In tube treatment ................................ ................................ ........................ 83 PL ................................ ................................ ................................ ..................... 83

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8 5 INACTIVATION OF AFLATOXINS B 1 AND B 2 IN PEANUTS BY COMBINING TWO TREATMENTS: PULSED LIGHT (PL) AND CITRIC ACID ............................ 94 Material and Methods ................................ ................................ ............................. 96 Experimental Design and Statistical Analysis ................................ ................... 97 Results and Discussion ................................ ................................ ........................... 97 ELISA Results ................................ ................................ ................................ .. 97 HPLC MS/MS Results ................................ ................................ ...................... 98 Discussion ................................ ................................ ................................ ........ 98 Possible Mechanisms of AFT Degradation by PL and Citric Acid Treatments 99 6 THE EFFECT OF STORAGE TIME ON THE PEROXIDE VALUE, FREE FATTY ACID CONTENT, AND COLOR IN PL TREATED PEANUTS .............................. 104 Materials and Methods ................................ ................................ .......................... 105 Samples Preparation and Storage Condition ................................ ................. 105 Experiment Design ................................ ................................ ......................... 105 Peroxide Value (PV) ................................ ................................ ....................... 106 Free Fatty Acids and Acid Value D etermination. ................................ ............ 106 Color Evaluation ................................ ................................ ............................. 106 Results and Discussion ................................ ................................ ......................... 107 Peroxide Value and Free Fatty Acids Tests ................................ ................... 107 FFA and Acid Value ................................ ................................ ....................... 108 Color E valuation ................................ ................................ ............................. 111 7 OVERALL CONCLUSION ................................ ................................ .................... 119 APPENDIX A INOCULATION OF WITHOUT SKIN AND WITH SKIN PEANUTS WITH ASPERGILLUS FLAVUS ................................ ................................ ...................... 121 B PULSED LIGHT TREATMENT ................................ ................................ ............. 122 C COMBINE PULSED LIGHT WITH CITRI C ACID AS A HURDLE TECHNIQUE AND COMPARE THE RESULTS WITH THE PL TREATMENT ........................... 123 LIST OF REFERENCES ................................ ................................ ............................. 124 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 138

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9 LIST OF TABLES Table Pa ge 3 1 AFT uniformity test results. The conc entration of AFTs for 5, 10, and 15 g A. flavus inoculated peanuts. ................................ ................................ .............. 61 3 2 AFT concentration for peanuts with different moisture content after PL treatment for 300 s at 5 cm. ................................ ................................ ................ 61 3 3 LC MS/MS analysis of PL effect on diluted mix AFB 1 and AFB 2 100 ppb. .......... 61 3 4 AFT reduction percentage in with skin PL (in plate) treated peanu t samples using different distances and different time. ................................ ....................... 61 3 5 Correlation coefficient for in with skin PL (in plate) treated peanut samples using different distances and different time. ................................ ....................... 62 3 6 LC MS/MS analysis for 5 g peanuts treated in plate with PL for 240 s, and 7 cm distance from the strobe. ................................ ................................ ........... 62 3 7 AFT reduction per centage in w/o skin PL (in plate) treated peanut samples using different distances and different time. ................................ ....................... 62 3 8 Correlation coefficient of the reduction percentage with all other parameters in wi thout skin PL (in plate) treated peanuts with the increasing in temperature. ................................ ................................ ................................ ....... 62 3 9 Peroxide value, free fatty acids, and total acid number for peanuts oil after PL treatments of peanuts. ................................ ................................ ........................ 63 4 1 LC MS/MS analysis for 5g without skin peanuts with different PL treatments. ... 86 4 2 LC MS/MS analysis for 5 g with skin peanuts t reated with PL. .......................... 86 4 3 Peroxide value (meq of peroxide/L) of peanuts oil after PL treatments of peanuts. ................................ ................................ ................................ .............. 86 5 1 ELISA results for the comparison between citric acid + PL (shaking) treatment and citric acid treatment for without skin (w/o skin) and with skin peanuts ................................ ................................ ................................ ............. 102 5 2 LC MS/MS results for the comparison between pulse d light (PL) treatment and the pulsed UV light with the citric acid treatment without washing. ............ 102 5 3 LC MS/MS results for the comparison between citric acid treatment and citric acid followed by PL treatment for without skin peanuts with washing. ............. 102

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10 5 4 LC MS/MS results for the comparison between citric acid treatment and citric acid followed by PL treatment for with skin peanuts with washing. .................. 102 6 1 The peroxide value for 0, 1, 2, and 3 months stored PL treated with skin peanuts at 5 cm distance from the strobe. ................................ ........................ 114 6 2 The peroxide value for 0, 1, 2, and 3 months stored PL treated with skin peanuts at 7 cm distance from the strobe. ................................ ........................ 114 6 3 The peroxide value for 0, 1, 2, and 3 months stored PL treated without skin peanuts at 5 cm distance from the strobe. ................................ ........................ 114 6 4 The peroxide value for 0, 1, 2, and 3 months stored PL treated without skin peanuts at 7 cm distance from the strobe. ................................ ........................ 114 6 5 The FFA % for 0, 1, 2, and 3 months stored PL treated with skin peanuts at 5 cm distance from the strobe. ................................ ................................ ......... 114 6 6 The FFA % fo r 0, 1, 2, and 3 months stored PL treated with skin peanuts at 7 cm distance from the strobe. ................................ ................................ ......... 115 6 7 The FFA % for 0, 1, 2, and 3 months stored PL treated w/o skin peanuts at 5 cm distance fro m the strobe. ................................ ................................ ......... 115 6 8 The FFA % for 0, 1, 2, and 3 months stored PL treated w/o skin peanuts at 7 cm distance from the strobe. ................................ ................................ ......... 115 6 9 The acid value results for 0, 1, 2, and 3 months stored PL treated with skin peanuts at 5 cm distance from the strobe ................................ ......................... 115 6 10 The acid value results for 0, 1, 2, and 3 months stored PL tr eated with skin peanuts at 7 cm distance from the strobe ................................ ......................... 115 6 11 The acid value results for 0, 1, 2, and 3 months stored PL treated w/o skin peanuts at 5 cm distance from the strobe ................................ ......................... 116 6 12 The acid value results for 0, 1, 2, and 3 months stored PL treated w/o skin peanuts at 7 cm distance from the strobe ................................ ......................... 116 6 13 L*, a *, b* values for color evaluation of commercial peanuts roasted peanut, control (untreated peanut), and four different times PL treated peanut samples. ................................ ................................ ................................ ........... 116 6 14 Hue angle, Chroma, and whiteness values for color evaluation of commercial roasted peanut, control (untreated peanut), and PL treated peanuts. .............. 117

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11 LIST OF FIGURES Figure page 2 1 Ammonization of aflatoxin ................................ ................................ ................. 30 3 1 Peanuts inoculation. ................................ ................................ .......................... 64 3 2 Temperature measurements. ................................ ................................ ............. 64 3 3 Xenon pulsed light machine. Model# LHS40 LMP HSG from Xenon Corp ......... 65 3 4 The AFB 1 AFB 2 percentages of Pulsed Light tre ated pure AFB 1 AFB 2 mixture in solvent. ................................ ................................ ............................... 65 3 5 The instantaneous temperature measurements. ................................ ................ 66 3 6 The instantan eous temperature measurements. ................................ ................ 68 3 7 LC MS/MS calibration curve for the AFB 1 AFB 2 standards. ............................... 70 3 8 LC/MS/MS c hromatograms for PL treated peanuts.. ................................ .......... 71 4 1 The model structure of peanut cells: ................................ ................................ ... 86 4 2 Reduction percentages and temperat ure measurements of in plate PL treated peanuts. ................................ ................................ ................................ .. 87 4 3 Reduction percentages and temperature measurements of shaking PL treated peanuts. ................................ ................................ ................................ .. 87 4 4 Reduction percentages and temperature measurements of in ice PL treated peanuts. ................................ ................................ ................................ .............. 88 4 5 R eduction percentages and temperature measurements of in tube PL treated peanuts. ................................ ................................ ................................ ............. 88 4 6 Reduction percentages and temperature measurements of in plate PL treated peanuts. ................................ ................................ ................................ 89 4 7 Reduction percentages and temperature measurements of shaking PL treated peanuts. ................................ ................................ ................................ 89 4 8 Reduction percentages and tempera ture measurements of slices PL treated peanuts. ................................ ................................ ................................ ............. 90 4 9 Reduct ion percentages and temperature measurements of in ice PL treated peanuts. ................................ ................................ ................................ .............. 90

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12 4 10 R eduction percentages and temperature measurements of in tube PL treated peanuts. ................................ ................................ ................................ .. 91 4 11 In plate PL treatment for peanuts. ................................ ................................ ..... 91 4 12 Schematic representation of Shaking PL treatment. ................................ .......... 91 4 13 Shakin g device inserted inside the P L machine ................................ ................ 92 4 14 Shaking device for shaking treatment for PL tr eatment of peanuts. .................. 92 4 15 Double dishes for In ice PL treatment of peanuts. ................................ ............. 92 4 16 Rotating motor for In tube rotating PL treatment of peanuts. ............................. 93 5 1 LC/MS/MS chromatograms for peanuts. ................................ ......................... 103 6 1 The color vision machine images for peanuts samples. ................................ .. 118

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13 LIST OF ABBREVIATIONS AFTs Aflatoxins AFB 1 Aflatoxin B 1 AFB 2 Aflatoxin B 2 AF M 1 Aflatoxin M 1 AFG 1 Aflatoxin G 1 AFG 2 AflatoxinG 2 AOAC Association of Analytical C hemists AOCS American Oil Chemists Society ANOVA Analysis of Variance ESI Electrospray Ionization ELISA Enzyme Linked Immunosorbent A ssay FDA Food and Drugs Admini stration FFA Free Fatty Acids FAO Food and Agriculture Organization KMC Kernel Moisture Content LSD Least Significant Differences LP Low Pressure meq Milliequivalent MP Medium Pressure

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14 PV Peroxide Value WHO World Health Organization SPSS Stat istical Package for the Social Science s

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INACTIVATION OF AFLATOXIN S B 1 B 2 IN PEANUTS BY PULSED LIGHT (PL) By Manal Othman Abu agela December 2017 Chair: Paul Sarnoski Major : Food Science Aflatoxin(s) (AFTs) are secondary metabolites of Aspergillus molds These toxigenic, carcinogenic, and immunosuppressive compounds are commonly found in contaminated peanuts. Most of the methods studied to eliminate AFTs on peanuts either degraded peanut quality or were not cost effective. Pulsed light (PL) treatment has shown promising results in AFT degradation in beverages and oth er food products. Five methods were tested using with and without skin peanut kernels that were with A.falvus and PL treated using: 1) aluminum plate on a conventional conveyor, 2) shaker, 3) rotating glass tubes, or 4) on a plate with ice; additionally, sliced peanuts were tested on 5) aluminum plate on a conventional conveyor. The shaker treatment was further tested using a hurdle technique of soaking the peanuts in a citric acid solution prior to PL treatment AFB 1 and AFB 2 contents were determined usin g an enzyme linked immunosorbent assay (ELISA) and liquid chromatography mass spectrometry (LC MS/MS). Finally, the effect of storage time on PL treated peanuts peroxide value and free fatty acids were investigated. Data were analyzed by Analysis of Varian

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16 Peanuts treated on conveyer plate for 300 s at 5 cm from the strobe showed AFTs were reduced by 82% with skin and 95.3% without skin. However, the surfaces of the peanuts were burned. Surface burning was avoided by shaking or rotating the peanuts during the treatment. Shaking treatment had the significantly (p<0.05) higher AFTs reduction (91.73%) compared to other methods, presumably due to exposing all peanut sides to the light during the treatment. The hurdle techn iques achieved 100% reduction with 240s of PL treatment. Peroxide and free fatty acids values were not significantly increased after 3 months of storage. This study indicates that a cost effective PL technique can achieve significant reductions in AFTs tha t are superior to other methods without altering the quality of the peanuts

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17 C HAPTER 1 INTRODUCTION Fungi or molds have played a paramount destruction role in agriculture commodities throughout history. They can contaminate and colonize crops before harv esting and during storage for a relatively long time in a warm and high humidity condition or exposure to a stressful environment such as drought. Molds can excrete extremely harmful secondary metabolites called mycotoxins inside and over the crops. There are hundreds of mycotoxins ; few have been detected in food and are considered to have a serious impact on human healt h (Serra et al ., 2005). Mycotoxins have been studied since the early nineteen th century ( Sinha & Arora 1911) Many fungi species are r epo rted as mycotoxin producers; however, the f ilamentous fungi Aspergillu s is the dominant producer. Aspergillus currently is one of the most economic ally distinguished genera due to its ability to produc e aflatoxin ( s ) (AFTs). Aspergillus species produce AFTs during its li ving phase However, the exact function of these mycotoxins remains a mystery. It is hypothesized that mycotoxins act as a defense mechanism, protecting the fungus from plants, animals and other competing fungi (Smith and Moss, 1985). Asperg illus flavus and Aspergillus parasiticus are the main AFT producers. In addition, the gene responsible for producing these some other Aspergillu s species such as the food fermentation useful strains A. oryzae and A. sojae ( El Nagera bi et al., 2012; Hassan et al., 2017; Kusumoto et al., 1998 ). The major AFT concern to human health is from four types of aflatoxins B 1 B 2 G 1 and G 2 The a flatoxin B series, especially B 1 is the most severe AFT. It has multifactorial toxic and chronic e ffects. Particularly those which are attributed to hepatocarcinogenic, hepatotoxic, teratogenic, and mutagenic effects

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18 (Moreau et al., 2013; Moudgil et al., 2013), M ajor AFT i nfected commodities are dried crops such as oil seeds, cocoa, coffee, dried peas, spices, fruit, beans, and nuts, especially peanuts (Turner et al., 2009). Peanuts ( Arachis hypogaea ) is commonly named worldwide as groundnut and earthnut, monkey nuts and goobers. It is one of the most nutritious crops and one of ar and consumable commodity, cultivating in approximately 100 countries in all six continents. Peanuts have grown originally in South America in tropical and subtropical regions. Peanuts are consider ed one of the most produced and consumed commodities in t he United States for its high nutrient value. Peanuts contain 51.9% lipid, and 21 % to contains approximately 43% oleic acid 35% linoleic acid, and 0.1% linolenic acid. P eanut oil has a low vul nerability to oxidation because of the less double bonds present in the 6 ; Zhao 2013). In addition, peanuts are rich in Vitamin E and p hytosterols which are related to cardiovascular and c immune system fortification demonstrated the consumption of peanuts or peanut butter could reduce the threat of developing type two diabetes ( Eis enstat et al., 2007 ). Peanuts are contaminat ed with AFTs when subjected to extended periods of pre harvest heat and drought stress (Holbrook et al., 2000). Changes of chemical composition of peanut kernels due to fungal infection are inevitable. Protei ns, lipids, free and total amino acids, and free fatty acids are reported to change significantly when peanuts are infected by A. flavus (Chiou et al., 1994 ).

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19 Since AFTs have been discovered, several investigations have been conducted regarding detoxificat ion and elimination methods AFTs are notoriously difficult to remove. Most AFTs are chemically stable and heat resistant toxins. Thus, they tend to survive processing stages. AFTs tolerate the high temperature s encountered during baking, roasting, and bre akfast cereal production. Most of the chemical physical microbial and irradiation approach es th at have been tested never reached complete AFT elimination. Th is makes it essential to prevent AFT production in the first place by avoiding the conditions wh ich lead to mycotoxin formation, which may n o t always be achieved during practice (Turner et al., 2009). Additionally, a ny detoxification process to be applied practically, should be economically and technica lly feasible. Pulsed u ltra v iolet light (PL) is one o f the proposed new technologies which is presented as non thermal food treatment regarding microbial inactivation by using broad spectral wavelengths (200 to 1100 nm), including the UV spectrum. PL has proven its capability of destroying and reducing harmful compounds, bacteria, viruses, enzymes, allergens and toxins. This research proved the efficiency of PL to inactivate AFTs to significant ly lower levels in peanut kernels. Objectives. The overall purpose of this study was to investigate the effect of the pulsed light technique on AFTs in peanuts T o inactivate the AFTs totally or partially to meet the regulatory requirement s (e.g. FDA, EU, etc.) t he specific objectives that w ere investigated in this project are: Objective 1: D etermine the degrada tion percentage of aflatoxins (B 1, B 2 ) in without skin (w/o the testa ) and with skin peanuts (with the testa ), after PL treatment using different distances and times using an enzyme linked immunosorbent assay (ELISA) and liquid chromatograph y mass spectrom etry (LC MS/MS) for quantification

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20 Objective 2: O ptimize different conditions and treatments ( i n plate, in ice, in rotating tube, slices, and shaking treatment) using several distances 5, 7, and 10 cm, and different t imes of PL treatment Objective 3: To c ombine PL with citric acid and compare the results with the PL treatment alone Objective 4: M onitor the treated peanuts after storage by recording any possible chang es in peroxide value, free fatty acids, and acid value as proof of lipid oxidation, after variant storage periods (1, 2, and 3 months).

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21 CHAPTER 2 LITERATURE REVIEW and which means poison. Mycotoxins are a e Fungal m etabolites generally have low molecular weight and are produced by the primary metabolic processes of various fungi ( Hussein & Brasel, 2001 ). The specific functions of mycotoxins are still considered a mystery even after 30 years of research and investigation. Aflatoxins, ochratoxins, zearelenone, trichothecenes, tremorgenic toxins, ergot alkaloids, and fumonisin are the mycotoxins of greatest agro economic pernicious ( Hussein & Brasel, 2001 ). The se toxins are the on e of the greatest worldwide li fe threatening and food and feed contaminants. While all mycotoxins are of fungal derivation, however, not all toxins produced by fungi are under the term mycotoxins (Zain 2011). In 1962, the word mycotoxin was connected to the a ftermath of a significant crisis that happened in London, England, when a pproximately 100,000 turk eys died. The aflatoxin ( s ) ( AFT s ) were isolated and identified a nd linked with peanut consumption. The turkey feed was contaminated with Aspergillus flavus an d their secondary metabol ites, AFT s. This crisis promptly encouraged scientists to trace and examine the toxicity of all other ambiguous mold metabolites, which could be deadly (Bennett & Klich, 2003; Zain 2011.) The highly carcinogenic AFTs are produced mainly by two specific st rains: Aspergillus parasiticus and Aspergillus flavus They secrete four major types of aflatoxin: B 1 B 2 G 1 G 2 (Belc et al., 2016; Bennett & Klich 1999 ; Piermarini et al., 2007 ). They are named blue (B) or green (G) according to their fluorescence und er UV light and the differences in their chromatographic mobility in the thin layer

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22 chromatography (TLC). In addition, there are two other less toxic AFT metabolites designated M 1 and M 2 The aflatoxin B series, especially B 1 is the most toxic. The domina nt aflatoxigenic species in c r ops, Aspergillus f lavus was divided into two different morphotypes. The two types of the A. flavus are the L strain and the S strain. The L strain forms a large sclerotia >400 mm in diameter. The S strain, classified as A. pa rvisclerotigenus which basically forms several small sclerotia <400 mm (Samson et al. 2004 ). The L strain is excessively adaptable to AFT production, with isolates fluctuating from non aflatoxigenic to extremely aflatoxigenic (Horn and Dorner 1999). Gen erally, the S strain produces greater amounts of AFT s, unlike the L strain. The required infectious doses of AFTs to develop harmful health effects are varied among fungi and the different infected animals or humans. Toxicological effects on humans and an imals can be either acute or chronic Acute effect is an instant onset of a harmful impact from a single high dose exposure, whereas chronic toxicity is gradual or delay symptoms and is typically due to multiple or long term exposures. Chronic toxicant is the primary mechanism of infection, generally associated with ingestion of low doses over long periods of time creates difficulties across the body ( Hussein & Brasel, 2001 ; Makun 2013). Aflatoxins cause hepatocellular carcinoma (liver cancer) especially i n combination with the virus of hepatitis B in animals and humans. Regarding the mentioned hazards, the maximum tolerance levels of AFB 1 allowed is 4 20 ng/g in food and feed ( Beasley et al., 1981; Hussein & Brasel, 2001; Turner et al., 2009 ). As is the ca se with any contemning food agent, the amount of consumed contaminants and the infectious dose should always be considered.

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23 Aflatoxins Regulation Food and health organizations through over 100 nations throughout the world have established food safety regu lations for AFT maximum tolerable levels. These standards were to protect public health and avoid lethal crises at tributed to AFT consumption (Wu & Guclu 2012). For instance, in Kenya in 2004 125 people died from ingesting homegrown corn, which was conta minated with AFT above tolerance levels ( Lewis et al ., 2005). Aft er exposure, AFT s are bio activated in humans via the cytochrome P450 enzymes to start their toxic effects. Once activated, AFT can bind to and damage cellular targets such as DNA and proteins (Guengerich et al., 1998). The high lipophilic nature of this toxin facilitates its crossing of the placental barrier increasing fa tal exposures. These exposures have led to immunodeficiency, altered growth factor behavior, and intestinal toxicity in chil dren (Wild et al ., 1991). Due to the highly lethal nature of AFT s, enacted, food regulations are very stringent with regard to the amount allowed in foods for human consumption. Therefore, most of the nations regulate the sum of the four most exist ent types of AFTs in food: B 1 B 2 G 1 G 2 and aflatoxin M 1 in dairy products ( Van Egmond, 1989). Within the European Union ( EU ) aflatoxin B 1 content must be <2 ng/g. Total AFTs must not exceed 0.1 ng/g in 15 ng/g for other produ cts (EU Committee) .The FDA regulations stipulate that AFT Australia and Canada put a restriction for the total AFTs in nuts not to exceed 15 ng/g for all foods ( Wu & Guclu, 2012 ). The same was recommended fro m the international limit for raw peanuts by the Codex Alimentarius Commission (CAC). The European Scientific Committee for food (ESC) concluded that aflatoxin B1 is genotoxic therefore, no tolerable daily intake can be set. Similarly, aflatoxin M 1 is

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24 re gulated in at least 14 countries ; the permit levels generally are within the range of 0.05 0.5 parts per billion (Coker 2000). Although these regulatory standards are supported in the United States, the European Union (EU), and in developing countries, it is common for commodities to be contamina ted with concentration levels > 100 ng/g. In fact, the variations between the AFT standards in the most industrial countries are somehow quite large, which affect the global market. This leads to the urgent demand o f harmonization of mycotoxin regulations all around the world ( Van Egmond, 1989). In industrialized countries, the impact of food contamination with AFT s adversely affects the economy more than the public health ( Wu & Guclu, 2012 ). The health impacts of AF T in low income countries are more severe. Many individuals are exposed to high levels of AFTs which affect them chronically through the essential foods such as maize and peanuts. This usually leads to frequent deaths from live r cancer and aflatoxicosis. L ow income countries are suffering from lack of the resources infrastructure s ervices, and technology, which are important for daily food control and AFT monitoring. Aflatoxin exposures are generally highest in Asian and in sub Saharan African nations. Th rough the examination of many types of agricultural commodities, many researchers have suggested that the peanut is the most frequently contaminated with AFTs among all other crops. This has been attributed to such factors suc h as high moisture and nutrien t content, methods of harvest, and storage circumstances, which could provide favorable conditions for the growth of contaminating molds (Wogan 1966). Usually, s as a source of ch as peanut because it grow s unde rground, with the developing pods contacting directly with the soil living organisms. This ma kes peanuts one of the

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25 most susceptible seeds to be invaded by A. parasiticus and A. flavus e specially under conditions of str ess, high temperatures, and drought. Usually damaged peanut kernels contain the highest AFT concentrations (Horn and Do r ner 199 9 ; Lee & Krochta 2002 ). Furthermore, the peanut is consider ed to be one of the highest produced and consumed seeds. P eanut wor ld production is approximately 29 million metric tons yearly. The United States is the third produc er in the world. Roughly 2.4 billion pounds of peanuts are consum ed yearly, on e half of it as a peanut butter; approximately 90 million jars of peanut butter are annually sold. In addition, peanuts and its product s covered two thirds of all nut snack s consumed in the U.S (Jain and Lee 2006). I ndividual consumption is 6 pounds of peanuts or peanut products annually in the U.S. (Beyer et al., 2001). Seven state s produce 99% of all U S productio n of peanuts. The major producing state is Georgia with 41% production, then Texas with 24% production, Alabama produces approximately 10%, followed by North Carolina 9%, Florida with 6% production, Oklahoma 5%, and final ly Virginia with just 5% of the total yield ( American Peanut Council, 201 7 ) In addition, there is a noticeable increasing demand for all peanut products all around the world; for instance, peanut oil, peanut butter, salted a nd roasted peanuts, and peanut confections. U.S. peanuts fall under four categories: Runner, Virginia, Valencia, and Spanish. Each of these peanuts types are distinguished by the differences in their size and flavor. However, in the early 1970s Runner bec a me the m ost commonly grown pea nut in the U.S. Runner has an attractive kernel size range which is the reason for gaining wide acceptance through the U S. Th e majority of Runner peanuts are used for peanut butter

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26 production. Runner is now 80% of the total U.S. production mainly grown i n Georgia, Florida, Texas, Alabama and Oklahoma ( American Peanut Council 201 7 ) AFT Detoxification Methods AFTs are consider ed one of the greatest food safety concerns. AFTs are a major economic pr oblem for the peanut industr y which must spend more mo ney for monitoring procedures to confirm that all products are going to the consumers free of aflatoxin (USDA). Several methods since the 19 60s has been used for AFT de toxification The Food and Agriculture Organization (FAO) stipulate that the method shou ld: Inactivate or destroy, or eliminate aflatoxins. Not to leave nor produce toxic or carcinogenic or mutagenic residuals in the final food products. Increase the overall safety and avoid formation of harmful residues or leave any toxic substances. The n ew technique should not adversely affect or suppress the nutritional value and the desirable sensory and physical properties of the treated product. I t should be applicable technically and feasible economically. Initially, it should be potent to kill the s pores and destroy mycelium of the toxic fungi and inhabit its ability to proliferate or produce toxins under optimum conditions again ( Cole et al. 1985; Piva et al., 1995; Rustom 1997). This r eview reveal s some of the most effective chemical, physical, and biological approaches for AFT detoxification throughout the last four decades. Chemical Approach Several chemicals have shown a high potency to react with AFT s and adverse ly affect the AFT s harmful impact by changing their chemical structure to less carcinogenic and toxic substances. These chemicals involve bases, acids, bisulphites,

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27 gases, and oxidizing agents. Most experiments ended in the formation of other toxic residues and caused unfavorable degradation in sensory, functional, and nutritional p roperties of the food. However ozone and ammonium treatments have been used to destroy AFTs in peanut meal, cottonseed and maize especially for animal feed ( Rustom 1997 ). Using the ozone in AFT detoxification FDA in 1982, stated ozone as a generally r ecognized as safe substance (GRAS) for bottled water disinfection use only (FDA, 2008). It has started to be used in food processing ( Kim et al. 1999; Mahapatra et al., 2005). Ozone has gained positive reputation in the food sanitization field instead of c hlorine, as chlorine has led to environmental and occupational problems (EPA, 1999). Even though, this substance still needs to be tested to confirm its efficiency to be safe economically, and healthy substitution of many other chemicals Ozone is a gas at levels as low as 0.00002 g/m 3 which is 10 times below the WHO limit of recommended exposure for 1 h The highly oxidation potency O 3 of 2.07 volts (V) m akes it 1.5 and 1.3 times more lethal th an hydrogen peroxide and chlorine respectively against many micro pathogenic species (Fo egeding 1985; Freitas Silva & Venncio, 2010 ; Khadre et al., 2001 ). Although there are some encouraging results recorded from applications of using the ozone to in hibit the growth of some filamentous fungi or their toxins. In several studies, ozone was found to be able to destroy microorganisms and alter their metabolites toxic structure, leaving no traces of ozone in the treated crops. This fact makes the use of oz one safe in food applications.

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28 The detoxifying effects of ozone on corn flour, flaked red paper, pistachio kernel, ground pistachio s, dried figs, and corn flour w ere all tested using different ozone levels and treatme nt time s For pistachio kernel and gr ound pistachios, the concentrations of the used ozone were 5.0, 7.0, and 9.0 g /m 3 The reduction of total aflatoxins and AFB 1 in pistachio kernels was about 24 % and 23% and the reduction of total aflatoxins and AFB 1 in ground pistachio was 5 % and 93 % res pectively in 60 min (Akbas and Ozdemir, 2006). For flaked red paper gaseous O 3 concentration s 16, 33, and 66 g/m 3 were used for 60 min a nd the reduction of AFTs reached to 93% (Inan et al., 2007). For d ried figs the used concentration was 1.7 g/m 3 li quid O 3 and 13.8 g/m 3 gaseous O 3 for 7.5, 15, and 30 min The reduction of aflatoxin B 1 was more effective in the gaseous treatment (Zorlugen et al., 2008). After corn flour was treated with 15, 30, 45 and 75 mg liquid ozone for 60 min, the concentration of AFB 2 AFG 1 and AFB 1 declined from 2.42, studies, it became obvious that as the concentration of the ozone and exposing time increased the contents of AFTs decreased. Ozone either partially degrade s AFT s or causes chemical modification. Investigations revealed that the degradation by chemical alteration was found just in AFG 1 and AFB 1 ; however, AFG 2 and AFB 2 showed a greater resistance (Cullen & Newberne, 2013 ). The mechanism of ozone on the detox ification of AFB 1 and AFG 1 involves an electrophilic reaction of the C8 C9 double bond in the furan ring causing the formation of ozonized species These compounds are then rearranged into monozonide byproducts such as ketones, aldehydes, carbon dioxide, a nd acids ( Alexandre et al.,

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29 2012 ; Lou et al., 200 4 ). Since there is no C8 C9 double bond within the structure, AFB 2 and AFG 2 are more resistant to ozonization than AFB 1 and AFG 1 (Agriopoulou et al., 2016; Chen et al., 2014). Even though the efficiency of o zone as a chemical detoxifier is high, a greater concentration is required to kill fungi or contaminated surfaces, while low concentration of ozone and short fumigation time is generally considered necessary in order to preserve product properties like col or, flavor, aroma, and vitamins ( Bocci, 2010; Chen et al., 2014). The disadvantages of using the ozone for AFT detoxification : The ozone oxidizes acids to smaller mole cular fragments ; ozone degrades the cell wall envelope of unsaturated lipids resulting in leakage of cellular contents (Da s et al., 2006). Ozone treatment significantly affects the moisture content of the treated food, especially when used in high concentr ation (Lou et al ., 200 4). Ammonia NH 3 t reatment Exposing to ammonia in solution, in the gaseous phase, or with other compounds that have the ability to release it, fulfi lled optimum results in detoxification of several crops such as peanut, corn meal, and cotton. Efficiency is related to the amount of ammonia used, reaction time, levels of pressure and temperature, and being combined with formaldehyde. The mechanism of action induced by ammonia on aflatoxin AFB 1 is that the chemical structure of this toxin is irreversibly changed after the reaction with ammonia for a long time. In contrast, if the exposure the AFBs molecule structure can return to its original structure ( A llameh et al., 2005; Piva et al ., 1995; Weng et al., 199 4 ). A study conducted on yellow corn naturally contaminated with 12 ,500 ng/ g of AFB 1 and treated with ammonia hydroxide (NH 4 OH)

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30 at high temperatures, showed AFB 1 degradation greater than 99% (Weng et al., 1994). Several studies on ammonia treatment reveale d that the high temperature aqueous NH 4 OH or gaseous NH 3 can eliminate AFTs successfully from food and animal feed. It has been proposed that the resulted compound from the ammonia AFB s reaction is the less toxic than Aflatoxin D (AFD) which is a non flu orescent phenol with molecular weight (MW) 286 I t s functional group, which is consider ed the reason of the toxicity of AFBs. AFD 1 was biologically examined in order to determine the potential toxicity and m utagenic potency of the residual. Results showed that AFD 1 is as much as 450 fold less toxic than AFB 1 Figure 2 1 Ammonization of aflatoxin (Piva et al., 1995) Ammonia treatment has some disadvantages, t his chemical decomposes metals In addition, ammonia usually forms unfavorable brown color in food, which cannot be avoided. The elevation in non protein nitrogen, and in total nitrogen parallel with a significant decline in the solubility of the nitrogen, in the amino acids con tent such as methionine, cystine, and especially lysine, and protein efficiency ratios ( Dollear et al., 1968; Piva et al ., 1995).

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31 Addition of s orbents Materials such as silicas, aluminas, and aluminosilicates are being us ed to pursue specific approaches in tended to reducing the AFM 1 carryover in milk, meat, and egg. These materials have the ability to decrease the AFT absorption in the animal intestine. Several studies have been conducted on different types of animals on using hydrated sodium calcium alumin osilicates (HSCAS). This material can bind AFTs and reduce the production of AFM 1 In addition, an experiment had examined bentonite as an AFT sorbent in dairy cows and the results showed a 33% decline in the AF M 1 c arryover, while in vitro trials on trout feed accomplished adsorption of 70% AFB 1 in the feed ( V eldman 1992 ). Another in vitro test confirmed the efficiency of the Myco B ond which is a commercial product made of improved chemical phylloalumino silicate accompanied with multiple layer montmorillon ite and formed an inert stable complex potent to prevent the absorption of mycotoxins in the intestine (Piva et al. 1995 ; Winfree, 1992 ). Citric acid treatment It was hypothesized that acid treatments have the ability to alter the chemical structure of B series AFTs The converted substance has mutagenicity 1000 times lower than the original toxin ( Rustom 1997). Ciegler & Peterson ( 1968 ) had tested the effect of c itric acid on AFB 1 by adding 500 AFB 1 to 1 liter of 0.1 N citric acid. The solution was agi tated at 28 C for 24 48 h He noticed the production of a new compound which was more polar and differ ed in terms of fluorescent intensity. For all it s physical properties the researcher suggested that this compound could be hy droxydihydro aflatoxin ; howe ver this reaction is reversible. Chemical inactivation of AFB 1 and AFB 2 in maize grain, duckling feed, and extruded sorghum was tested by using 0.1 N aqueous citric

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32 acid. Conversion was measured by the AFLATEST TM immunoaffinity column method, high perform ance liquid chromatography (HPLC), and the toxicity were tested using the improved Ames test. Results revealed that treatment with aqueous citric acid achieved 96.7%, 86%, and 92% degradation in AFT s concentration in those foods respectively. In addition, the AF T B series fluorescence intensity of th e acid treated samples was far lighter than the non treated samples as appeared in the chromatograms of the HPLC. On the other side, the results from the Ames test showed a great reduction of the mutagenic abil ity of the acidified maize ( Ciegler & Peterson, 1968 ; Mndez Albores et al., 2005). In 2010 rice was treated with 1 N aqueous citric acid to destroy AFT. The AFTs in rice samples were 97.22% degraded. However, the best degradation occurred in rice contami 30 ppb (Safara et al., 2010). Biological A pproach One of the most familiar methods for the mycotoxins quality control in foods is using microorganisms to biologically decontaminate mycotoxin s Saccharomyces cerevisiae and lactic acid bacteria two possible decontaminating microorganisms, have proven that their detoxification abilities are significant amongst the other microorganisms. Yeast and lactic acid bacteria ( LAB ) are best known to bind variant molecules. For example, structures on the c ell wall surface could bind some toxins or metal ions. In addition, the wall structure of both yeast and LAB is completely different, which results in the different mechanism of binding molecules in both cells ( Juodeikiene et al., 2012) According to some studies reported, they confirm that the binding process, with AFTs is by adhesion to the cell wall components rather than by metabolism or by binding covalently ( Shetty & Jespersen 2006).

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33 Adsorption of mycotoxins by y easts In the 1980s, when there was so me information regarding the usage of zearalenone (a mycotoxin) contaminated maize of ethanol production a fter the fermentation process, some unusual action was discovered. The included toxins disappeared in the residual substances, which garb l ed the hypo thesis that the toxin by some means might be bound to yeast cells. A recent study tested isolates of yeasts from several species, including Candida krusei and S. cerevisiae for AFT binding. Toxin binding was found to be highly strain specific. Specific is olates from maize from West Africa was found to have the ability to bind with 60% of the added toxins in phosphate buffer saline (PBS). Most of the yeast strains could bind with 15% of aflatoxin B 1 Although there are numerous reports of animal feeding exp eriments on yeasts and their cell wall components, resulting in different levels of degradation of AFT toxicity, induced strain of S. cerevisiae that could bind AFTs un der laboratory circumstances ( Shetty & Jespersen 2006) The c ell wall is a greatly dynamic structure responding rapidly to changes in the environment and stress. Regarding the physical nature and chemical composition of the S. cerevisiae cell wall, it is rational to think that the cell surface presents numerous sites on its surface for physical adsorption of molecules. Some studies revealed that mannan components of the S. cerevisiae cell wall has a role in AFT binding. The cell wall of the S. cerevisiae i s a bi layered structure and considers 30% of the cell total weight and its content of up to 1, 3 glucan and 1, 6 glucan. Mannoproteins proteins are the major protein in the cell wall. They have a covalent link to 1, 3 glucans through 1, 6 glucan chains. They contain an actual hete rogeneous type of glycoproteins; 70 of them have

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34 been ident ified. Another important piece is the phosphodiester bridges in the mannosyl side chains, which provide many negative charges at the cell surface, which could be the possible sight of mycotoxin binding. Adsorption of mycotoxins by lactic acid b acteria (LA B) Many scientists are s up porting the idea that the best technique for AFT decontamination should be by using specific microorganisms, giving an advantage of eliminating the toxins under mild conditions, avoiding the use of harmful chemicals and without a substantial loss in nutrit ive value, in addition to the provided safety to the food and feed. Using l actic acid bacteria in fermenting food is practice f or centuries. Early studies revealed that several strains of LAB are AFT biosynthesis inhibiters but n ot sufficient factors in AFT detoxification. W hen five isolate s of LAB, including Lactobacillus rhamnosus strains GG and LC 705, L. casei Shirota L. gasseri and L. acidophilus were screened for AFT binding (LAB cell wall components, peptidoglycan, inclu ding lipoteichoic and teichoic acid, neutral polysaccharides, and proteinatious S layer ) a ll played diverse functions in macromolecular binding and adhesion to AFT. Both the probiotic L rhamnosus strains effectively removed aflatoxin B 1 from inoculated cu lture media. The destruction was through a fast process immediately eliminating around 80% of AFTs. L. rhamnosus strains GG and LC705 which was found effectively binding to aflatoxin B 1 better than any other AFT ( El Nezami et al., 2002 ). Furthermore simil ar AFB 1 binding strategy had reported from these two strains, even though they had some differences in respect to other toxins ( Shetty & Jespersen 2006).

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35 E nzymes for detoxification There have been numerous reports of using microbial enzymes such as AFO (a flatoxin oxidase) for AFT detoxification. One of the most known enzymes is isolated from the mushroom of Armillariella tabescens ( Liu et al., 1998) This enzyme demonstrated high AFB 1 degradation ability and has been named aflatoxin detoxifizyme (ADTZ). A study in 2015 used AFO in a concentration as small as 0.2 mg/ml with a specific enzyme activity of 180 /mg revealed that this negligible amount can decline 1 mol of AFB 1 per minute at 30 C (Wu et al., 2015). AFO is a selective enzyme for AFB 1 or similar structures, such as sterigmatocystin (ST), versicolorin A, or any chemical that has a furan or p yran ring s The proposed site for AFO to act with AFB 1 is the bisfuran structure. The production of hydrogen peroxide is important to induce the AFO e ffectivene ss Physical A pproach F ungi contaminated seeds can be eradicated by hand selecting or using photoelectric detecting machines such as X ray images and Computing Tomography (XCT) to detect and separate the infected kernels (Kotwaliwale et al., 2014 ; Pearson & Wicklow, 2006 ). This method is time and money consuming. However, other physical aspects are more logical and successful in terms of fungi infec tion elimination and AFT i nactivation. These methods includ e: solvent extraction, adsorption, and inact ivation by heat and irradiation ( Rustom 1997). Extraction Organic solvents such as chloroform, methanol, hexane and acetone have been used to extract AFTs from agronomic crops.

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36 E xtraction by chemical solvents was used to eliminate AFT s from the cottonseed and o ilseeds peanut. Scientists suggested that materials treated by these chemicals are only appropriate for animal feed. The typical used solvents are: 80% isopropanol, 90% diluted acetone, 95% ethanol, methanol water, hexane methanol, a cetonitrile water, and acetone hexane water The ratio of solvent: sample is considered critical for the recovery of the AFTs ( Cole & Dorner, 1994 ). The e xtraction method can eliminate all traces of AFTs from oil seed with no harmful residual formation or protein reduction in tre ated food. However, this technique is very expensive in large scale applications compared with other detoxification techniques, in addition to the crucial problems related to the difficult ies in the disposal of the left over toxic extracts ( Rustom 1997). Heat Aflatoxins are consider ed heat resistance substances. They have high breakdown temperatures varying from 237C to 306C. AFB 1 is relatively st able under dry heating at temperatures below its thermal decomposition temperature of 267C. Using temperatu re to eliminate AFTs from contaminated food and food product s is a logical technique to be tested. However, heating using the regular home cooking conditions like frying or boiling at approximately 150C have failed to remove AFB 1 and AFG 1 completely Many variables have been connected directly to the extent of AFT destruction; the type of the food, the initial level of contamination, the heating temperature, cooking time, pH, and ionic strength of the food. However, the most crucial factor affecting the de gradation of AFT was found to be the moisture content. Heat easily inactivates AFTs in contaminated foods that have high moisture content. Mann et al. (1967) have noticed that when cottonseed meal containing 30% moisture was heated to 100C for 1 h, 74.8% of AFB 1 and AFB 2 w as destroyed, w hile only 32.7% of

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37 the AFTs were inactivated after heating the same meal at the same temperature when it has 6.6% moisture content. Several studies have been conducted on AFT inactivation in peanuts by high temperature, roa sting or boiling in different means; direct roasting at 204 C for 20 min showed 52% reduction. The dry heating treatment at 150 C for 30 min for AFT contaminated peanuts reduced AFB 1 to 4 5 % ( Pluyer et al., 1987 ) AFB 1 inoculated peanuts treated with autocl ave moist temperature 116C, under pressure 0.7 bar for 30 min, and in 5% NaCl brine, recorded an outstanding reduction reaching to 80 % 100% (Farah et al., 1983). Even though the direct and indirect high heat treatments could be e ffective in terms of AFT d etoxification in peanuts, the final quality and produce unfavorable compounds. Irradiation Radiation is divided into two classifications: ionizing and non ionizing. Ioni zing radiation includ es gamma ray, X ray, and ultraviolet ray s The irradiated molecules possibly change when exposed to small increase in temperature. On the contrary, non ionizing radiation such as radio wave, microwave, infrared wave, and visible light in high intensity may increase the temperature significantly, which leads to chang es in food structure with no known hazard to human health. Despite the doubts about the safety of irradiated food, it has become a technique for commercial scale food product sterilization. Microwave Microwave treatment showed sufficient results in terms of AFT destruction in pre contaminated peanuts. Reduction was depend ent on the level of the power and the treatment time. For example, roasting peanuts artificially contamina ted meal in a

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38 microwave at 6 kW for 4 min eliminated 95% of the meal AFT content ( Yazdanpanah et al., 2005 ) Likewise, microwavi ng decreased the AFT level s in peanut kernels at low energ y at 0.7 kW for 8.5 min resulting in 48 % 61% of AFB 1 reduction in the peanut kernels which were artificially contaminated. However, the same treatment resulted in only 30 % 45% reduction of AFB 1 in the peanut kernels which were naturally contaminated (Pluyer et al., 1987). Ultraviolet (UV) l ight Several methods since the 196 0 s were investigated for AFT destruction or elimination purposes. UV irradiation with its four regions of wavelength s : vacuum UV 100 200 nm, UV C 200 280 nm, UV B 280 315 nm, and UV A 315 400 nm, was the most tested method because AFTs showed sensitivity t o UV radiation AFTs absorb UV light at 222, 265 and 362 nm, with maximum absorption at 362 nm, which leads to the production of up to 12 photo d estruction compounds which are less toxic (Krishnamurthy et al., 2008 ; Samarajeewa et al., 1990 ). AFB 1 and AFG 1 were s ubject to multiple photochemical chang es when exposed to UV light 365 nm for 1 h our Protein bound AFTs showed less susceptibility to photo destruction than the free toxins ( Shantha & Murthy, 1977). Standard vials of AFT with differ ent solution con centrations of 1000 1 200 2 1000 1 200 2 and 2400 irradiati on at 366 nm wave length for 10 min. Degradation was by 98, 99.5, 99.8, 100 and 99.1% respectively ( Sharareh et al., 2015 ). Pea nut samples were treated by UV 260 nm, intensity ( 108 J / cm 2 ) for 45 min at room temperature and t he reduction in AFT content was 87.76 96.49 % (Jubeen et al., 2012) From previous studies UVA, which is consider ed the highest penetration region and the lowe st energy, has shown to be less effective than UVC, which has been concluded as

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39 the most effec tive region in terms of detoxified AFT s (Diao et al. 2014). Another recent study also used the UV radiation intensity of 6.4 mW cm 2 to treat peanut oil with a th ickness of <3 mm. Results verified that (UV) radiation reduced the AFB 1 in peanut oil kg 1 1 in 10 min and reduced by 86.08% (Diao et al ., 2015). Raw milk was initially inoculated with AFM 1 and exposed to UV light for 20 min at 25 C. The AFM 1 declined by 60.7%, and the destruction mechanism was attributed to the double bond opening in AFM 1 terminal furan ring ( Yousef & Marth 1986). In addition, artificially AFB 1 co ntaminated (250 ppb) dried figs had exposure to UV irradiation for 30 min. The treatment caused 45.7% reduction in the toxin level ( Altug et al. 1990). Sixty min utes of UV and enzymatic exposure for AFB 1 contaminated red chili powder showed a reduction by 87.8% (Tripathi & Mishra, 2010). Another study stated that the exposu re to short waves (254 nm) and long wave s (362 nm) of UV for 30 min destroy ed AFB 1 completely in wheat grains (Atalla et al., 2004) These studies support that UV irradiation is an effective technique in AFT termination, although the efficiency of detoxifi cation is varied with the irradiation condition differences Pulsed l ight (PL) Pulsed UV light is a relatively new technique modified and developed from continuous wave UV light (CW UV), which was discovered in the 1930s. This broad spectrum electromagneti c energy (100 1200) ranges from UV to the infrared region. Pulsed light contains approximately 54% UV, 25% visible, and 20% infrared light (Yang et al., 2011). T his light is rich in UV C (200 to 280 nm), which is often used to kill microorganisms. The diff erence between UV and PL is that the UV technique works continuously while the PL emits discontinuous pulses. In addition, both have different lamp type s ; the most used lamps in continuous UV light techniques are low pressure

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40 (LP) and medium pressure (MP) mercury vapor lamps known as CW UV (Diffey, 2012) T he LP mercury lamp is the oldes t ; it discharges monochroma tic wavelength at 254.7 nm, and this wavelength is close to the 260 nm, which is a germicidal effective wavelength. UV C is usually used to prese rve food and surfaces. A more developed technique is MP UV C lamps, which release polychromatic light at wavelengths between 200 and 300 nm. The wavelength between 254 and 264 nm has the highest germicidal properties. UV light absorbed by microorganisms in duces photo chemical and photo thermal effects. Therefore, in order to improve the absorbance of light by microorganisms, innovative lamp design is essential ( Sandeep, 2 001). Consequently, the PL works with xenon or krypton lamps, which can emit a number o f flashes per second ( 2007). The PL energy is magnified multiple folds by accumulating in a capacitor in a fraction of a second then releasing it as short duration discontinuous pulses using a lamp full of ionized inert gas, such as xenon. These pulses form an intensive light within a short time (nanoseconds to milliseconds with a negligible amount of additional energy consumption ( et al., 2007; Oms Oliu et al. 2010). This provides an amplification for the UV intensity to approximately 20 ,000 tim es more than the conventional, continuous mercury UV light and the sunshine at sea level (Dunn et al., 1995; Krishnamurthy et al., 2007). PL is four to six ti mes more efficient than continuous UV light in terms of p athogen inactivation ( Krishnamurthy et a l., 2008 ). PL has not been sufficiently studied in terms of AFT illumination. Studies such as mycotoxins degradation in solvent (Moreau et al., 2013) food allergen reduction (Yang et al., 2010) and enzym e inactivation studies have been conducted Th e P L spectrums have three main effects on materials: photo chemical, photo thermal, and

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41 photo physical (Shriver et al., 2011). For instance, Moreau et al. (20 1 3 ) recorded that eight flashes of pulsed light can eliminate 72.5 1.1, 84.5 1.9, 92.7 0.8 and 98.1 0.2% of deoxynivalenol, zearalenone, aflatoxin B 1 and ochratoxin in solution respectively. Their work showed that this type of irradiation can eliminate some mycotoxins. Although most PL detoxification methods for AFTs were effective at some lev el, the method could not achieve a sufficient level of AFT degradation accompanied with maintaining food quality. However, many successful experiments were conducted using PL to eliminate or destruct several types of microorganisms and allerg en ic compounds Therefore thi s study will contin ue the required investigation on the ability of the PL to destroy specific types of aflatoxin (AFB 1 a nd AFB 2 ) in inoculated peanuts, which is consider ed an important worldwide crop. Justification of Study Aflatoxins grab bed the world attention beca use of the massive economic loss due to the noxious influence they have on human health and animal livestock industries in both local and global markets. The annual estimated losses from this progressive dil emma in Canada and the USA were U S $5 billion (Lyn et al., 2009) In developing countries where basic foodstuffs such as maize and peanuts are susceptible to be contaminated, premature death and morbidity is associated with the consumption of AFTs. Several methods have bee n used to detoxify or destroy AFTs in different crops (Coker 2000). However, AFTs are still considered an elevated risk contaminating substance because in terms of thermal methods, the AF T B series is relatively high ly resistant to thermal degradation A c omplete destruction is hard to achieve even at temperatures of around 250 C in dry grains (Magan & Olsen 2004 ; Torres et al., 2001 ).

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42 Given AF T s resistance to thermal degradation, this study will use PL as a semi thermal AFT degradation method.

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43 CHAPTER 3 INACTIVATION OF AFLATOXINS B 1 B 2 IN WITH SKIN AND WITHOUT SKIN PEANUTS BY PULSED LIGHT AND THE EFFECT OF PULSED LIGHT ON PEANUT PROPERTIES The natural incidence of aflatoxin ( s ) (AFT s ) in feed and food is unavoidable and unpredictable creat ing a unique challenge for scientists that study food safety. These toxicants are highly abundant in tropical and subtropical areas, where the weather is warmer and humid more than other places. These conditions are optimum for the growth of fungi and their products. T he growth of AFT producer fungi in some grains naturally is influenc ed by several factors, including the type of the grain temperature, moisture content, fungal species, existence of minerals, relat ive moisture of the surrounding air, and the physical con dition of the kernels (Viquez et al., 1994). Peanuts as one of the most consumed commodities around the world have grabbed attention for high susceptibility to f ung al growth, such as A. flavus and ultimately AFT production. Th e AFT dilemma need s to be stu died from all aspects starting from the cause to the elimination and destruction effects. A. flavus is the dominant AFT producer in peanuts. Broken, immature, undersized, de shelled, discolored, and rancid peanut kernels are most sus ceptible to fungi conta mination ( Beu chat, 1987 ; Chiou et al., 19 8 4; Rucker et al., 1994). Most of the food safety commun ity has emphasized that farms, manufactures, and markets should take steps to prevent peanuts fungi contamination starting from pre harvest stage. An effectiv e treatment is urgently needed in food processing and safety fields. Multiple chemical treatments (ammonia sodium bisulfite, hydrogen peroxide, ozone, chlorine, acids, and alkali ), and physical treatments (heat gamma irradiation, microwave radiation, vis ible light, and ultraviolet) have been studied to destroy AFT s

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44 and fungal sustainability in several types of food. However, few methods have been tested on peanuts because most of these methods have major drawbacks in terms of lacking the expediency for u s e in solid food, limited AFT elimination, ret ention of some toxic residual compounds chang e of some nutrients in the food, or the formation of unfavorable by products (Wang et al. 2016) Many factors influence the effectiveness of any detoxification meth od, including the chemical persistence of the AFT the conditions of the method, the interaction and the matrix of the food. Practically, a good and efficient mycotoxin detoxification method should destroy, detoxify, or eliminate the AFT ; treatment must n ot produce any toxic residual in the food, keep the high nutrient value of the food, and retain or improve the consumer acceptability of the product (Park 2002). Several irradiation methods such as microwave and PL have been investigated for AFT detoxifi cation purpose in peanuts. AFB 1 contaminated peanuts were treated in a microwave for 0.7 kW for 8.5 min resulting in just reduction of about 48 61% of AFB 1 level in peanut, but the same treatment caused only 30 45% AFB 1 decline in the naturally contaminate d peanut (Pluyer et al., 1987). Pulsed light treatment is a relatively novel food processing and preservation technology. This technique uses pulses of intensive broad spectra, including 54% UV, 20% infrared and 26% visible light (Shriver et al. 2011). D uring PL radiation, the inert gas inside the PL lamp, such as Xenon, is excit ed by high voltage, and when these Xenon molecules want to come back to the ground state, energy is released as photons and absorbed by the food particles, leading to photo physic al, photo thermal, and photo chemical effects on foods ( Choi et al.,2010; Shriver et al., 2011).

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45 The former three photo effects are responsible for the assumed mechan istic effect of PL on food or any targeted molecules (Krishnamurthy et al., 2007). The te chnique of PL was originally accepted as a non thermal technology used for microorganism elimination. Nevertheless, recent study has clarified that PL may also have a photo thermal effect in addition to its non thermal nature. The prolonged treatment (e.g. minutes) has led to temperature elevation and moisture evaporation inside the food matrix (Yang et al., 2012) Although PL is known as a non thermal technique, with a relatively long time process, about 30 s or more depending on a PL strobe distance from food, heat will be generated (Faidhalla, 2013). In addition, this technology showed promising results in terms of AFT reduction. Moreau et al. ( 20 1 3 ) recorded that eight flashes of PL can eliminate 84.5 1.9, 72.5 1.1, 92.7 0.8 and 98.1 0.2% of d eoxynivalenol, zearalenone, ochratoxin and aflatoxin B 1 in solvents respectively. Their work showed that one or several flashes of pulsed light on zearalenone and deoxynivelenol ended with a negligible decline in the toxicity of the mycotoxins. However, the same treatment for AFB 1 showed a complete elimination of the mutagenic ability of this AFT Therefore, the presented study was conducted to investigate the effect of PL on the aflatoxin B 1 and B 2 degradation in with skin and without skin peanuts. Mat erials and Methods Preparation of the Samples Sample inoculation Raw with skin (with testa) and without skin (w/o testa) peanuts were purchased from a domestic market. Peanuts were autoclaved for 15 min at 115 C to eliminate any pre contaminated fungi or AFTs. Peanuts were inoculated with A. flavus which was

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46 purchased from the American Type Culture Collection (ATCC) ( Manassas, VA, US ) This specific strain anamorph (ATCC 16875 TM ) has the highest ability of producing AFB 1 AFB 2 together, among all other strains (Wei and Jong 1986). Fungus was proliferated on PDA media (potato starch 4.0 % dextrose 20%, 2% agar), which was distributed by a hockey glass stick for 1 min, and incubated in a lab incubator for 5 7 d (Doyle et al., 1982; Nam et al., 2009). Inoc ulum was incubated at 30 C Fungi spores were harvested from the Petri dished with a spatula, filtered using cheesecl oth, and a suspension of a sterile water spore roughly 100,000 conidia per milliliter w as prepared. The conidia were counted using a h emo cytometer 500 g of peanuts was placed in Erlenmeyer flasks (1000 ml), and 50 mL of distilled water was added and autoclaved at 121 C for 15 min and allowed to stand overnight. To minimize the moisture loss from the grain, flasks were sealed with thin poly ethylene film. The film was punctured by ma king a few holes using a pin to avoid carbon dioxide production from respiration which could accumulate in the head space of the flask. After the inoculation process with A flavus conidia, flasks were inoculated with 25 mL of the spore suspension, incubated at 30 C in dark/night of 12/12 h for 21 d Flask s were shak en daily for moisture and fungi distribution. After this long period of incubation, flasks were boiled in water for 1 h to stop AFT production. Moistu re was optimized to 16% as in previous studies ( Mndez Albores et al., 2005 ; Wei and Jong, 1986 ) Hemocyt ometer calculation used E quation 3 1, 3 2, 3 3. % of viable cells = Number of viable cells / total # of cells x 100 (3 1) Dilution factor = F inal volu me / volume of cells (3 2) Concentration (viab le cells/ml) = Average number [number of cells / number of squares] x dilution factor x 10 4 (3 3)

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47 Moisture o ptimization production of AFT is influenced by the competition of natural microflora on peanu t pods. Factors such as moisture content are crucial for AFT accumulation in peanut kernels and the development of A. flavus over other microorganisms (Chiou et al., 1984). The minimum moisture content of peanuts for A. flavus growth is 8 10% at a relative humidity of approximately 82%. T he optimum moisture content for AFT production on peanuts is 15 35% (Doyle et al., 1982). Thus, moisture was controlled and adjusted to 16% The peanuts were kept in sealed plastic bags and stored in 18 C freezer for treat ment and further analysis. Peanut kernel moisture content was determined in triplicate, using the Association of Official Analytical Chemists (AOAC) number 925.40 method (AOAC, 1990) Using a mechanical convection oven (Precision Sci entific, Winchester, VA ), 5 g samples of peanuts were taken directly from the inoculum. Samples were gr ound to a fine powder to accelerate the moisture loss inside the oven. The following modification was required because of the high moisture cont ent of the samples. After weigh i ng the samples in ceramic crucible, peanuts inside the crucibles were put in the oven at 103C for 12 h The samples were then cooled in a desiccator and weighed. Equation 3 4 was used to calculate the moisture content: % m oisture (wt/wt) 100 (3 4) Uniformity t est This test was conducted in triplicate to confirm the uniformity of the peanut inocul um A. flavus inoculated peanuts with and without skin samples 5, 10, and 15 g, in triplicate were used AF Ts were extracted from all samples using the same extraction

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48 method using 70 % aqua methanol Then a competitive ELISA test w as used to determine the differences in AFT concentration in all samples. Data were analyzed by A nalysis of V ariance (ANOVA) using S PSS at 0.05, and to evaluate the significance of the differences between samples. Pulsed UV l ight t reatment Pulsed light treatment was conducted using a Xenon PL applicator of Model # LHS40 LMP HSG from Xenon Corp (Wilmington, MA ) This system consists of an RC 747 power/control module, a treatment chamber that contains two xenon lamps (mercury free ) and two blowers ( air cooling system ) at the top of the lamp housing chamber, and one treatment chamber with a conveyor belt The xenon lamp used has an e lectrical efficiency of 10 % 30 % and UV intensity of 30.000 w/cm (Koutchma 2009). The PL system generates a broadband spectrum between 100 and 1100 nm. Approximately 20%, 26%, and 54% from the energy are infrared, visible light, and ultraviolet regions, respectivel y. This system provide s high intensity PL at a pulse rate of three a specification. Treatment delivers 1.27 J per centimeter squared (J/cm 2 ) for an input of 3800 V at 1.9 cm below the c entral axis of the quartz window of the PL xenon lamp ( Wang et al., 2016) The treatments were conducted in triplicate. Each time 5 g samples of with skin and without skin raw runner type peanuts were arranged in small aluminum dishes (7.2 cm diameter, a nd 1 cm high) obtained from Fisher Scientific, In c. (Allentown, PA ). A combination of diverse illumina tion durations and distances were tested to explore the effe ctive conditions for PL treatment of whole peanuts. The samples were treated by the PL for sev eral duration time s 30 60, 90 120, 180, 210, 240 and 300 s in three different

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49 distances 5, 7, and 10 cm from the PL strobe. The treatment times were selected based upon the degree of visual roasting that occurred and AFTs reduction for the peanut sample s, ranging from lightly roasted to burnt, during preliminary tests. Aflatoxin e xtraction After the PL treatment, 5 g of peanuts were well milled in a grinder (Model LXC 150 50 /60Hz 180W Keunex, Korea). Then the peanuts were blended (31BL91, Waring, Dyn amics Corporation of America, Hartford, C T ) with 25 mL of 70% methanol (aqueous) for 2 min to extract AFTs Then the extract was transferred to conical flasks and was shaken (MTS 2/4 D S1, IKA, Wilmington, NC ) for 30 min at 300 rpm. The extract was allowed to settle, then filtered through a Whatman Number 1 filter paper and moved to autoclaved containers and stored in a freezer ( 18 C) until analysis for AFB 1 AFB 2 content. Extractions after storage were shaken before ELISA analysis by using an orbital shak er (Heidolph, Schwabach, Germany) for 3 min similar to the method of Zheng et al. (2005) and Nyirahakizimana et al. (2013) Competitive ELISA An AgraQuant ELISA Aflatoxin Kit (Romer Labs, Getzersdorf, Austria) was used to determine the concentration of A FB1 and AFB2. All procedures for ELISA analysis were performed according to manufacturer specifications. The kit is able to quantitate AFTs in the range of 4 40 ppb. High performance liquid chromatography mass spectrometry analysis (HPLC/MS/MS ) Chromatogra phic analyses were performed on s amples that had the highest reduction percentage to confirm the E LI SA results by using HPLC/MS/MS on an A gilent (Palo Alto, CA) 1100 series A thermo Scientific Hyper Sil Gold aQ (2.1 x 150 mm; 3

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50 m; + guard column ) was use d for compound separation. Elution was performed using mobile phase A (H 2 O, 0.2% ac etic acid) and mobile phase B (m ethanol, 0.2% acetic acid) The flow rate was 0.2 mL/min, and the linear gradient used was: 0 min 90% A; 13 min 50% B; 45 55 min 100% A, 65 7 5 min 90% B. Electrospray mass spectrometry was performed (ESI MS/MS) of the [M+H ] + ions of the AFB 1 and AFB 2 LCQ DECA quadrupole ion trap mass spectrometer was operated in the positive electrospray ionization (ESI) mode operating with XCALIBUR 2.0.7. SP 1 ( Thermo Fisher Scientific; San Jose, CA). The spray voltage was 4.0 kV; heated capillary voltage was +32 V; tube lens offset was 4 V. Nitrogen was used as the spray gas. Source and desolation temperatures were set at 250 C. The ESI Duration was 60 min, a nd the number of s can e vents was five A series of standards of AFB 1 and AFB 2 were also analyzed to create an external calibration curve Standards were Sigma products: Aflatoxin B 1 (Sigma A6636 1 mg; Lot # 025M4092V ) ; Aflatoxin B 2 ( Sigma A9887 1 mg; Lot # 016M4012V). Temperature Measurements I nfrared thermometer An Omega OS423 LS ( Omega Engineering, Inc, S tanford, CT) infrared thermom eter was used for temperature measurements of treated peanuts befor e and right after PL treatment at the exact moment when the conveyor br ought the sample s out of the chamber. In situ temperature m easurements Temperature was measured i nstantaneous by inserting tiny thermal sensors in to three locations of the peanut kernel. Each sensor was inserted in distinctive locations (t op, middle, and bottom) of the three different peanut kernels (Fig ure 3 2 A). The kernels were attached to the aluminum plate, using a lab made flour glue, to avoid any

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51 movement during the PL treatment. Samples were treated in situ for 120 and 240 s at two distances 5 cm and 7 cm ( Figure 3 2 C). Temperature was me asured by a K type thermocouple (Omegaette HH306, Omega Engineering Inc., Stamford, C T) using a Pi co 8 channel thermocouple data logging interface (PC 08) attached to a computer running Pico softwa re ( Figure 3 2 B). Data w as recorded at 1 s interval s Chemical A nalysis Peroxide v alue Peroxides are the initial products of lipid oxidation (number of millimoles of peroxide in 1 kg of oil). The effect of PL on lipid oxidation of peanuts was investigat ed by comparing the peroxide value (PV) of the raw peanuts (control) with 240 s shaking treatment, 300 s shaking treatment, and 240 s in plate treatment ( t he highest PL treatments). The peanut seeds were milled with a coffee grinder (LXC 15 AC220 240V, 180 W) and dried to final weight in a thermostatically controlled oven at 105C. An oil extr action was carried out using a Soxhlet apparatus following the Soxhlet extraction official method of AOAC ( 199 5 ) A 250 mL round bottom flask was washed and dried in a n oven at 103C for 25 min and left to cool at room temperature before it was weighed. Five grams of the dr ied sample were weighed into an extraction thimble (Advantec N08425X100MM) made of cellulose This thimble was inserted into the extraction column wit h the condenser connected. Two hundred milliliter of the extracting solvent (chloroform, boiling point 60C) was poured into the 250 mL flask and fixed to the condenser under the extraction unit. The flask was then heated by electro thermal heater at 60C for at least 8 h (Eshun et al. 2013). Extracting solvent was evaporated leaving the concentrated peanut oil sample for analysis.

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52 Peroxide values were measured using the Official American Oil Chemists' Society ( Hortwitz, 2002 ) method 965.33 for peroxide va lue of oils and fats A 2.0 mL amount of peanut oil was placed in a 250 mL flask, and 20 mL of the appropriate solvent mixture (glacial acetic acid:chloroform 3:2 ) was transferred into a flask and gently shook. Saturated potassium iodide solution 1.0 mL wa s added and stirred at a slow speed for 1 min Then 100 mL distilled water, and 1 mL of starch solution was added to the solution, mixed with high speed stirring, and immediately titrated with 0.01 N or 0.1 N sodium thiosulfate. Solution was turned from a purple to a slight yellow or colorless, and this was considered the endpoint (t hese colors may be affected by the initial color of the tested oil). Blank determination was carried out under the same conditions. A n expir ed sample of commercial peanut oil ( 2013 ) was examined for comparison. Peroxide value results were reported as mill equivalents peroxide per killogram oil using Equation 3 5 (Eshun et al. 2013 ; Silva 2010 ). PV [meq/kg] = ((V 1 V 2 ) C 1000) / m (3 5) Where: V 1 = volume of sodium thiosulfat e for titrate the oil, V 2 = volume of sodium thiosulfate for titrate a blank C = molar concentration of sodium thiosulfate, m = weight of samples in grams. Free fatty acids and acid v alue Free Fatty Acid (FFA) is a good indicator of oil quality because it measures the concentration of fatty acids that are released from triacylglycerol s due to hydrolysis, lipase action, or even oxidation. FFA were measured according to the official method Ca 5a 40 of American Oil Chemists' Society (AOCS, 2009). Oil was extra cted from

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53 untreate d (control) and PL treated with skin and without skin peanuts using Soxhlet apparatuses. Five grams of peanut oil were weigh ed into a 125 mL Erlenmeyer flask. In the fume hood 50 mL of ethanol was added to dissolve the oil. B lank s which contain just 50 mL of ethanol were prepared for the same purpose. Five drops of phenolphthalein indicator were added to all samples. Then a titration to the endpoint (faint pink) with a standard solution of Na O H was controlled, and the volume titrated for blank and samples all were carried out for the final calculation using E quation 3 6 for FFA determination. % FFA (as oleic acid) = (3 6 ) Where: V = mL of NaOH required for sample, b lank = mL of NaOH for the blank, M = Molarity of the NaOH in mol/L, F = molar mass of the oleic acid (28.2 g/mole), and SW = S ample weight (lipid) in gram s In addition acid value for u ntreated peanuts and treated peanuts was determined. It is defined as the number of milligrams of KOH needed to neutralize 1 g of sample. To convert percentage free fatty acids (as oleic) to acid value, the free acid percentages were multiplied by 1.99. Re sults and D iscussion Uniformity Test The uniformity test results showed that there were no significant differences among all 5 g, 10 g, and 15 g of the inocula ted peanuts regarding AFT concentration (Table 3 1) F igure 3 1 shows the difference in fungi gr owth distribution of the inoculum after 5 d of inoc ul ation at the same conditions. These results were very helpful in terms of avoiding the misinterpretation of the results As Food and Agriculture Organization of U nited N ation FAO and the World Health Org anization and WHO indicated in the codex

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54 alimentarius of general standard for contaminants and toxins in food and feed (Codex Standard 193 1995) that AFT dis tribution is usually highly non homogeneous in peanut samples, which were laboratory inoculated. Th erefore, samples were prepared and homogenized extremely carefully. Moisture Content Moisture distribution is one of many factors affect ing AFT distribution in any crop. Previous study results indicated that the elevation in moisture content enhanced nut quality and PL efficiency ( Nkama & Muller 1988; zdemir and Devres 1999). In this study AFT reduction achieved in pean uts with moisture content of 16 % was much higher than the peanuts with moisture content of 10% (Table 3 2) by a difference of about 30 7 %. Nkama & Muller ( 1988 ) tested several primary moisture content s in mill ed rice containing 1100 g AFTs /kg rice, exposed UV light with high intensity of about 64 mW/cm 2 and at 36C. Results clarified that increasing the moisture to 24% led to AFT conten t to d ecline to 351 g /kg, and 14.1 % moisture resulted in reduction to 413 g /kg, which agreed with the former hypothesize. In the present study, moisture content was optimized to 16%. For future research, higher moisture levels, their effect on PL efficie ncy, and the correlation between moisture levels, temperature, and treatment times should be investigated from all aspects, including the sensory quality. exposing time o n AFT reduction percentages, which agreed with Demir (2004) who emphasized that both temperature and time are main parameters in any peanut application.

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55 A study conducted by Kita et al. (200 7 ) indicated that decline in moisture can directly decrease the me chanical characteristics such as hardness of roasted peanuts. Zhao ( 2013 ) has pointed out that traditional roasting process for dry peanuts gave a moisture loss of about 3% with consistent heating at 131 165 C for 15 min However, PL treated peanuts at 10 cm distance sho wed a moisture loss around 1.4% 5.6% for 5 9.5 min PL expo sure and samples at 7 cm distance from the lamp showed a moisture loss of about 2.0% 8.8% for 5 7 min PL treatment. The moisture loss during the PL treatment was like ly due to the hig her instantaneous temperatures generated inside the peanut kernels (Zhao 2013 ) Therefore, elevating the moisture content before the PL treatment was mandatory to enhance the photo thermal and photo chemical effect in addition to keeping the sensory quali ty of the roast or semi roast peanuts. Furthermore, the moist ure heat sensitivity of AFB 1 was confirmed in foods treated with irradiation. It has been proposed that water molecules help in opening the lactone ring in AFB 1 to form a carboxylic acid in the t erminal part of the ring. Temperature M easurements r ose parallel with expos ure time (Tab le 3 1, 3 2) in both with skin and without skin samples and opposite to increasing distance. The highe st temperature recorded by the infrared thermometer was 181 C for without skin and 150 C with skin peanuts treated for 300 s at 5 cm from the strobe The relatively high fat content (~ 50%) likely functioned as an efficient heat transfer medium leading to a rapid temperature rise in the peanuts. Furthermore, according perature measurements of peanut samples w ith skin samples recorded lower temperature compared with without skin samples probably due to the skin whic h may work as a barrier especially

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56 for light reflection The in situ temperatures ( Figure 3 5 A, B, C, D ; Figure 3 6 A, B, C, D) were recorded using sensors, which were connected to a computer that has a special software to measure the temperature every s econd in situ. Measurements showed that the homogeneity of temperature distribution was the best at 7 cm. The recorded temperature from all three sensors ( Figure 3 2 A, B, C) were close at the 7 cm distance ELISA Results According to ELISA AFB 1 and AFB 2 c oncentration inside PL treated peanuts and compared with a control of non treated samples. Results were calculated using equations brought on from running a series of AFB 1 AFB 2 mix diluted standards with a calibration curve R 2 = 0.99. The AFB 1 and AFB 2 i n solvent were treated with PL for several periods at 7 cm High reduction percentages were achieved in 240 s at 7 cm PL treatment ( Figure 3 4) The highest reduction of AFB 1 AFB 2 in peanuts achieved in this study was around 95.3 % 3.47 % for without skin peanuts treated for 300 s at 5 cm (Table 3 7 equally. For with skin, the AFT reduction reached to 82.0 % 16.1 % (Table 3 4 ) when treated for 240 s at 7 cm. This specific treatment was chosen regarding keeping the peanuts quality in addition to the significant AFT reduction comparing with other treatments of shorter time and closer or further distances from the strobe Comparably, a UV light treatment using 43 mW/cm 2 for 24 h reduced aflatoxin B 1 in milled rice from 1100 ppb to 135 ppb, which is 88% AFT reduction, while a higher intensity of 64 mW/cm 2 produced the same reduction of AFTs after 12 h of treatment (Nkama and Muller, 1988). Similar results for Wang et al. (2016) revealed th at PL treatment with intensity of about 0.52 J/cm 2 /pulse for different times. He found out that

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57 at 9 cm distance to the lamp, PL treatment for 80 s declined AFB 1 and AFB 2 in rough rice by 75.0% and 39.2%. Furthermore, a treatment of 15 s reduced AFB 1 and A FB 2 in rice bran by 90.3% and 86.7%, respectively (Wang et al., 2016). Reduction of AFTs had a strong positive correlation coefficient ( Table 3 5, 3 8 ) with the increasing of expos ure time (0.79, 0.9 1 ) and strong positive correlation coefficient with the temperature elevation (0.87 and 0.95) for with skin and without skin samples respectively. However, it had low negative correlation with the increasing in di stances, ( 0.4 and 0.31) for with skin and without skin samples respectively In addition, accor ding to the s e results the effect of the elevating in temperat ure on the reduction percentage was the highest attributed factor to AFT reduction percentage HPLC MS/MS Analysis Table 3 3 show s that the LC MS/MS results agreed with the ELISA results T he re duction of pure AFB 1 AFB 2 in solvent reached 100% and 97.9% respectively when treated with PL for 180 s at 7 cm. Table 3 6 showed AFB 1 AFB 2 reduction in AFT concentrations inside peanut kernels after PL treatment, which reached to 90.3 and 95.2 resp ectively for without skin peanuts treated for 240 s at 7 cm, and reduction for AFB 1 AFB 2 reached to 87.2 and 84.7 for with skin peanuts treated the same way. Results of AFB 1 AFB 2 mixture treatment led to a complete reduction for AFB 1 and semi complete reduction for AFB 2 ( Table 3 3). Aflatoxin B 1 and B 2 exhibited a good ESI ionization efficiency in the positive ion m ode for molecular ion m /z 313 ( fragments m/z 270.3, 285.2 and 298) for AFB 1 at RT = 22.67 min. W hile for AFB 2 the molecular ion was m/z 315 ( fragments m/z 259.2, 287.2, and 297.1) and eluted at RT = 21.79 min MS/ MS fragmentation pathway of AFB S

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58 revealed that ions were formed by loss of carbon monoxide, oxygen, hydrogen, and methyl group (Iram et al., 2016). LC MS/MS chromatograms (Figure 3 8 ) showed the obvious decrease in AFB 1 AFB 2 never showed a complete reduction even though the initial concentration was lower than the AFB 1 These results agreed with the previous work of Basaran (2009), which recorded that the UV light of 254 nm did not degrade AFG 2 and AFB 2 but significantly reduced AFG 1 and AFB 1 AFTs have different sensitivity to UV light. Thus, to improve the UV detoxification efficiency, the type of contaminating AFTs should first be identified, and then specific UV wavelength where AFTs have the maximum absorption should be chosen before irradiation. In this study obviously the wave spectrum used 220 1200 had a better effect on AFB 1 than on AFB 2 In general, results showed that after 240 s PL treatment for AFB 1 AFB 2 ; AFB 1 could not be d etected and AFB 2 was barely discernible. Factorial statistical analysis t w o way ANOVA was conducted to determine the significant effect s of the differences in times and distances during PL treatment on the AFT reduction percentages, and on temperature elevation (Table 3 4, 3 7). The treatments were significantly different at = 0.05 confidence interval. Fisher Least Significant Differences test (LSD) was conducted to determine the mean differences f or both with skin and without skin samples. For with skin samples (Table 3 4 ), at 5 cm distance to the lamp, significant differences in the reduction percentage 64.6% was observed at the 240 s exposing time when temperature reached 136 C. At 7 cm, significant reduction differences among treatments at 240 s w ere recorded 62.4% at temperature 105C. At 10 cm distance to

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59 the lamp, there were no significant differences among treatments in terms of reduction percentage. For without skin samples (Table 3 7 ) at 5 cm distance to the lamp, significant differences star ted at 90 s exposing time when temperature reached 90 2C. At 7 cm the significant reduction 44.6% was achieved at 120 s w hen temperature reached 86.2 C. At 10 cm distance to the lamp, the significant reduction 45.2% was at 210 s when the temperature reac hed 130 C. The LSD analysis showed increasing in the LSD between means equivalent to the increasing on distances. The same temperature with different distance resulted in different reduction levels. The effect of temperature was greater when the distances were closer. These results led towards the conclusion that the elevation in temperature is not the main factor of AFT reduction. Aflatoxins particularly are stable against irradiation when in a solid state (Aibara & Yamagishi, 1970). Therefore, one of the most crucial conclusions from this study is that increase in peanut moisture content enhanced AFT degradation. Lower m oisture content showed lower red uction The possible interpretation of this is that UV absorption was increased at higher moisture conten t since water is highly UV absorbent. F or opaque foods such as peanuts, the turbidity of the color reduces the penetration capability of the UV light which likely occurred for the peanut samples (Guerrero Beltran & Barbosa Canovas, 2004). When the matrix i s transparent, it can penetrate perfectly, permitting a final decontamination of samples as was achieved when AFB 1 and AFB 2 in solvent was treated by PL (Feuilloley et al., 2006). However, in general, the effect of PL penetrates 2 mm into the samples (Wall en & Fraenkel, 2001).

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60 Peroxide Value a nd Free Fatty Acids Lipid oxidation occurs in three stage s: initiation, propagation, and termination. This dynamic process is controlled by several factors including temperature, light, oxygen, and storage time. The l evels of peroxides chang e over time. At the beginning of shelf life, the peroxide value (PV) is low. However, during the food's life the PV increases to the top point and then declines down again. P eroxide value (PV) and free fatty acid test s are both con sidered shelf life indicator s for peanuts In this experiment t he PV was iodometrically determined and expressed as meq/kg Results (Table 3 9) showed that after 240 s 7 cm treatment PV was 1.03 for without skin and 0.89 for with skin samples The free f atty acids were 0.56 % for with skin and 0.55 % for without skin samples. The acid value was 1.08 for with skin and 0.88 for without skin samples. The acid value was alkalimetric ally deter mined and expressed in mg KOH/g Comparably, Pokorny et al. ( 2003 ) fou nd that Virginia raw peanuts extracted oil contains free fatty acids arou nd 0.72 %, the acid value was 1.48, and the PV was 1.46 Thus the p eroxide values of PL treated peanuts were fully acceptable according to the USDA Foreign Agricultural Service ( 2004 ) regulations for quality indexes of peanut oil products : PV mmol/kg 7.5 ( 1 meq/kg = 0.5 mmol/kg) and acid value (KOH) mg/g are within specification P eanuts are consider ed rancid when the PV > 30 meq/kg according to the Chemistry a nd Technology o f Oils a nd Fats (Chakrabarty 2003). Recently, s everal studies have de termined the PV of freshly roasted peanuts to be found between 1.0 and 10.0 meq peroxides O 2 /kg oil and the acid value was 0.6 0.99 mg KOH/G oil ( Akhtar et al., 2014; zcan 20 03 ). According to PP12 USDA commodity requirements ( 2010 ) peroxide value should meg/kg for roasted peanut s

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61 Table 3 1. AFT u niformity test results. The concentration of AFTs for 5, 10, and 15 g A. flavus inoculated peanuts. Peanut s amples (g) W ith skin (ppb) w/o skin (ppb) 5 75.7 8.13 a 69.7 11.9 a 10 75.8 4.86 a 74.6 7.24 a 15 78.8 4.32 a 77.0 6.05 a Different alphabetic means significantly different at = 0.05 using one way ANOVA and LSD system for mean differences analysis Table 3 2. AFT concentration for peanuts with different moisture content aft er PL treatment for 300 s at 5 cm. Peanut samples (g) Moisture content % AFT R % 1 2 4 2 00 10 1.13 1 2. 6 2 .13 a 64.7 11.9 b 3 16 2.32 95.3 3.47 c R% = Reduction percentage. Different alphabetic means significantly different at = 0.05 using one way ANOVA and LSD system for mean differences analysis Table 3 3. LC MS/MS analysis of PL effect on diluted mix AFB 1 and AFB 2 100 ppb. Samples AFB 1 R% AFB 2 R% Non treated AFB 1 AFB 2 in solvent (control) 0 a 0 a PL 180 s treated AFB 1 AFB 2 i n solvent 100b 97.9b PL 240 s treated AFB 1 AFB 2 in solvent 100b 97b Different alphabetic means significantly different at = 0.05 using one way ANOVA and LSD for mean differences analysis Table 3 4. AFT reduction percentage in with skin PL (in plate) treated peanut samples using different distances and different time. Time(S) 5 cm (C) 5 cm R % 7 cm (C) 7 cm R% 10 cm (C) 10 cm R% 0 23 2.35 0a 23 2.35 0a 23 2.35 0 a 30 58.5 2.43 0a 55.6 0.89 0 a 53.1 2.82 0 a 60 62.8 4.26 26.9 4.2a,b 60.3 0.25 20.9 6.32a,b 59.1 1.5 1.2 0 1.40a 90 83.0 1.64 33. 9 4a,b 67.2 1.83 23.1 8.42a,b 70.2 4.10 3.63 4.62a 120 105 8.65 39.3 4.9a,b 81.9 7.21 34.5 5.97a,b 71.1 13.8 4.16 0.00a 180 123 4.85 40.1 7a,b 96.8 2.62 39.3 7.05a,b 74.8 15.4 4.39 3.15a 210 130 12.2 43.4 17a,b 103 3.25 40.3 9.5ba,b 92.0 8.40 15.1 5.05a 240 1 36 1.11 64.6 19.4b 105 7.35 62.4 15.0b 98.3 4.29 25.1 15.0 a 300 150 17.9 82.0 16.1b,c 114 11.2 64.8 14.5 b 103 5.57 32.8 16.2a Mean standard deviation (n = 3) R% = Reduction percentage. Control was 128.993 36.540 ppb.

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62 T able 3 5. Correlation coefficient for in with sk in PL (in plate) treated peanut samples using different distances and different time. Time (s) Distance % R C Time (s) 1 Distance 3.2E 17 1 % R 0.79 0.49 1 C 0.88 0.30 0.87 1 R% = Reduction percentage Table 3 6. LC MS/MS analysis for 5 g peanuts treated in plate with PL for 240 s and 7 cm distance from the strobe Treatments AFB 1 R% AFB 2 R% With skin 87.2a 84.7a W/O skin 90.3a 95.2a Control 0 0 Different alphabetic means significantly different at = 0.05 using one way ANOVA and LSD for mean differences analysis. Table 3 7 AFT reduction percentage in w/o skin PL (in plate) treated peanut samples using different distances and different time. Mean standard deviation (n = 3) R% = Reduction percen tage. Control was 171 .87 22.2 ppb. Table 3 8. Correlation coefficient of the reduction percentage with all other parameters in without skin PL (in plate) treated peanuts with the increasing in temperature. Time Distance %R C Time (s) 1 Dist ance 3.2E 17 1 %R 0.91 0.31 1 C 0.92 0.29 0.95 1 R% = Reduction percentage. Time (S) 5 cm (C) 5 cm R % 7 cm (C) 7 cm R% 10 cm (C) 10 cm R % 0 22.1 0.67 0 a 22.2 2.27 0 a 22.2 1.45 0 a 30 58.7 6.88 0 a 50.6 0.29 0 a 45.1 14.9 0 a 60 69.1 4.41 32.6 2.40 a 65.2 0.98 23.5 0.46 a,b 53.2 3.69 13.0 1.16 a,b 90 90.2 5.03 36.2 2.65 b 79.3 2.26 28.8 1.10 a, b 57.7 1.20 3.00 4.78 a,b 120 110 6.47 48.6 0.46 b,c 86.2 0.73 44.6 6.09 b 77.0 0.81 30.4 1.60 a,b 180 128 3.97 68.6 5.65 b,c 99.4 0.82 60.2 16.9 b,c 79.3 1.10 41.3 0.31 a,b 210 131 3.66 76.3 3.30 c 104 2.56 70.3 4.13 b,c 82.0 2.34 45.2 0.67 b 240 148 3.02 86.9 1.99 c,d 137 0.01 77.9 2.95 b,c 120 1.59 50.1 4.53 b 300 180 12.9 95.3 3.47 c,d 164 2.54 80.1 1.49 c 130 1.61 55.1 1.19 b

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63 Table 3 9 Peroxide value, free fatty acids, and total acid number for peanuts oil after PL treatments of peanuts. Treatment FFA % Total Acid Mg KOH/g PV Control w/o sk in 0.58 0.15cb 0.93a 2.13 0.25c Control with skin 0.65 0.03b 0.90a 1.32 0.02c 240 s / 7 cm w/o skin 0.55 0.09 b 0.88a 1.03 0.10c 240 s / 7 cm with skin 0.56 0.21 b 1.08a 0.89 0.06c Mean standard deviation (n = 3) Different alphabet ic means significantly different at = 0.05 using one way ANOVA and LSD for mean differences analysis.

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64 Figure 3 1. Peanuts inoculation. A) Without skin peanuts, B ) With skin peanuts, C) A. flavus 5 d ay inoculated without skin peanuts, D ) A. flavus 5 d a y inoculated with skin peanuts ( Photo courtesy of author). A B C Figure 3 2. Temperature measurements. A) S of the thermo sensors inside the peanut kernels, B) K type th ermocouple and laptop running Pico software, C) S chematic representation of PUV treatment and t emperature data acquisition ( Photo courtesy of author).

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65 Fig ure 3 3. Xe non pulsed light machine. Model# LHS40 LMP HSG from Xenon Corp (Wilmington, MA ) ( Pho to courtesy of author). Figure 3 4. The AFB 1 AFB 2 percentages of Pulsed Light treated pure AFB 1 AFB 2 mixture in solvent 100 22 3 3 3 0 0 Control 60 s 90s 120s 140s 180s 240s Aflatoxin B1,B2 (%) Treatments PL treatment for AFBs in solvent

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66 Figure 3 5. Th e instantaneous temperature measurements. A), B), C), and D ) Temperature measurement during PL running: for with skin and without skin peanuts each second : A and B for 120 s; C and D for 240 s Sensors were inserted at the top, in the center, and at the bottom of the grain a t 5 cm distance from the stro be 0 30 60 90 120 150 0 20 40 60 80 100 120 140 Temperature ( C ) Time(s) A: In situ temp of with skin peanuts 120 s, 5 cm Surface C Middle C Bottome C 0 30 60 90 120 150 0 20 40 60 80 100 120 140 Temperature ( C ) Time(s) B: In situ temp of without skin 120 s, 5 cm Surface C Middle C Bottome C

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67 Figure 3 5. Continued. 0 50 100 150 200 250 300 0 50 100 150 200 250 300 Temperature ( C ) Time(s) C:In situ temp of with skin peanuts 240s, 5cm surface middle bottom 0 50 100 150 200 250 300 0 50 100 150 200 250 300 Temperature ( C ) Time(s) D: In situ temp of without skin 240 s, 5 cm Surface C Middle C Bottome C

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68 F igure 3 6. Th e instantaneous temperature measurements. A), B), C), and D ) Temperature measurement during PL running: for with skin and without skin peanuts each second : A and B for 120 s; C and D for 240 s Sensors were inserted at the top, in the center, and at the bottom of the grain a t 7 cm distance from the strobe 0 30 60 90 120 150 0 20 40 60 80 100 120 140 Temperature ( C ) Time (s) A: In situ temp of with skin peanuts 120 s, 7 cm Surface C Middle C Bottome C 0 30 60 90 120 150 0 20 40 60 80 100 120 140 Temperature ( C ) Time (s) B: In situ temp of without skin peanuts 120 s, 7 cm Surface C Middle C Bottome C

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69 Figure 3 6. Continued. 0 30 60 90 120 150 0 50 100 150 200 250 Temperature ( C ) Time (s) C: In situ temp of with skin peanuts 240 s, 7 cm Surface C Middle C Bottome C 0 30 60 90 120 150 0 50 100 150 200 250 Temperature (C) Time (s) D: Without skin 240 s 7 cm Surface C Middle C Bottome C

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70 Figure 3 7 LC MS/MS calibration curve for the AFB 1 AFB 2 standards.

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71 A B Figure 3 8 LC/MS/MS chromatograms for peanuts (A) before PL treatment ( control) (B) after PL treatment for 240 s at 7 cm

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72 CHAPTER 4 OPTIMIZATION OF THE PULSED LIGHT (PL) EXPOS URE METHOD FOR AFLATOXIN DETOXIFICATION FOR WITH SKIN AND WITHOUT SKIN PEANUTS Pulsed light is a powerful technique that appli es intensive flash es of white light, including broad spectrum with wavelengths ranged from 200 nm in the ultraviolet region to 1100 nm near infrared region. The power of each pulse is high er ( about 20 000 times ) than the power of the sunlight at sea level. The pulse continu es for a few hundred millionths of a second and can produce a high power p ulse in this negligible time. Thus, PL has been successfully used to sterilize and eliminate fungi and bacteria in food. The killing ability of PL is four to six times greater than t hat of the continuous UV light at equal energy levels. The advantages of using PL is that PL light can increase the quality and the shelf life of foods without carrying significant sensory changes ( Chung et al., 2008; Dunn et al., 1995 ; Gmez Lpez et al., 200 7 catalytic activity is known to be responsible for breaking down many and various pollutants. The specific PL decontamination mechanisms have not been fully elucidated yet, but the most recent hypothesis is that the intensive power o f PL is due to the rich broad spectr um used, and its extreme high power peak s in a very short time, particularly the UV part of th e spectrum and its photo chemical and photo thermal effects. This photo effect is attributed to the alteration of UV photons i nto energy, triggering local, sharp, and short temperature elevation at the surface of the treated object Although the PL has a low penetration level (2 mm from the surface of solid food), this technique effectively chang es the structure of the molecules which absorbe d the UV light; suggesting that this technique cou ld also be effective in detoxification and

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73 changing the structure of mycotoxins in addition to being active on micro organisms e specially mycotoxins concentrated at the surface of plants ( More au et al., 2013 ). Aflatoxin ( s ) (AFT s ) are the secondary metabolites of some Aspergillus species such as Aspergillus flavus and Aspergillus parasiticus Since the f atal effects of AFTs have been discovered, 8 000 pub lished research studies have investigate d their exposing acute and chronic toxic, carcinogenic, and mutagenic e ffects. In addition, at the end of the 1960 s, the toxigenic and the carcinogenic potency of AFTs bec ame an emergency matter demanding more rese arch, since the acute hepatotoxi c disease (X diseases) struck 100, (Eaton & Groopman 2013). S everal other outbreaks initiated intensive investigations all around the world about AFT s and their potency related to diseases in animals and humans (Eaton & Groopman 2013). Aflatox ins appeared to have existed for a long time before these outbreaks. However, those dramatic outbreaks grabbed the world attention to the seriousness of many other diseases that could be attributed to AFT consumption. Chemically, to understand AFT behavi or as a chemical compound, AFT is a lipophilic compound. In the human body, AFT can pass any barrier inside the body and be bio activated in the organs. Generally, the primary AFT target organ is the liver. This fact has been proven by thousands of studies on AFT toxicity conducted on rodents, poultry, fish, and monkey livers. These animals were fed a diet containing AFB 1 Their livers were analyzed to discover that the damage occurred in the liver after AFT consumption for a specific period. However, not al l results were identical since there are fundamental differences in the susceptibility of each species. Furthermore, within the tested species, the level of response was influenced by various other factors such as

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74 their diet, weight, age, sex, exposure to infectious sources, the presence of pharmacological active compounds, and other mycotoxins ( Cullen & Newberne, 201 3 ; Zain 2011). Pulsed light has been tested on AFB 1 in solution and achieved complete AFT elimination ( Moreau et al., 2013 ). However, PL trea tment is known to be affected by the transparency of the treated food. As the transparency increases the P L penetration increase s and the efficiency increase s too. As UV absorbance increases, the intensity throughout the product in the reactor decreases a nd results in a reduction of UV dose delivery (Koutchma 2009). The mechanisms involved in the decontaminating effects of PL a re not clearly known yet, but is probably due to the rich broad spectrum UV content, short duration and high peak power delivered to the treated food. PL started to be examined on several types of food after its effic acy was approved in terms of AFT reduction ( Mndez Albores et al., 2005 ). Therefore, this experiment was conducted to evaluate the efficiency of PL at the ra nge between 100 and 2200 wavelength spectrum on the AFB 1 a nd AFB 2 detoxification for peanuts which is consider ed to h ave big kernels compared with other previously PL treated commodities or foods. The big challenge is treating peanuts practically, given that AFB 1 and AFB 2 are lipophilic compounds. The p eanut cell is composed of a 1.2 m diameter oil field cavity surrounded by a double membrane. The cell membrane is not permeable for oil but is able to pass water. Thus, the toxins which are inside this cavity will be h ard to treat by PL or any other treatment ( Figure 4 1). Materials and Methods Locally purchased (Publix, Gainesville, FL) with skin and without skin runner raw peanuts prepared to be exposed to the PL light. All used tools, materials, and labware

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75 were san immediately followed by rinsing with sterilized water as previously done by Potrebko & Resurreccion ( 2009). Peanuts were autoclaved and inoculated with a specific strain of A. flavus (ATCC 16875 TM ) as me ntioned in Chapter 3. This strain produc es AFB 1 AFB 2 in satisfactory levels (Wei & Jong, 1986) Peanuts were then in cubat ed at optimum conditions for fungi growth and AFT production for a sufficient time (21 d ) m oisture was optimized to 16% and peanuts k ept in sealed plastic bags and stored in a 18C freezer until analys is PL Exp osure The P L system consists of three major components: the power supply, the pulse configuration and the lamps. Energy is stored in a high power capacitor for a comparatively long time (a fraction of a second), then released to a xenon lamp within a shorter time (nanoseconds to milliseconds). The X enon lamp was specially designed for this technique. The intensive energy the lamps deli ver produces powerful pulses of light that lasts a few hundred mic roseconds. These pulses focus on the treatment area. C hapter 3), a major challenge was to achieve a homogenized treatment for peanut samples and increase both the uniformity i n treatment and the max imum AFT reduction at the same time. Thus, peanuts need to be rotate d or move d during PL treatment, especially at short distances. Therefore, five PL exposing methods (in plate, sliced peanuts, in ice tray, rotating in a test tube, and shaking) were examin ed to achieve best color uniformity besides greatest AFT reduction in shorter time.

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76 Experimental D esign The inoculated peanuts were prepared as mentioned in C hapter 3. However, this time five exposing methods were used for without skin samples and four m ethods for with skin samples In plate samples Samples of 5 g of with skin and without skin peanuts were placed in an aluminum plate. To fulfil better results, the appro priate amount of whole peanut kernels was placed in a plate hav ing a relatively small s ize (7.2 cm radi us ) given that the PL machine used for this experiment has the highest power intensity directly under the center of the Xenon lamp. Shaking treatment A shaker device was built in the lab by connecting an appropriate s ize iron tray to a lab shaker ( MTS 2/4 D S1, IKA, Wilmington, NC ) by two long metal rods to provide flexibility and fixing the tray to the shaker body tightly at the same time ( Figure 4 1 4 ). Peanuts were placed in the middle of this tray and the tray was inserted inside the P L m achine to be right under the quartz window Activating the shaker made the peanuts flip and rotate and shake during expos ure to the light ( Figure 4 1 2 4 1 3 ). In this treatment the only distances used were 7 10 cm due to the non suitable height of the ma chine chamber to house the shaker at 5 cm Sliced peanuts Peanut kernels were cut in slices to improve the UV penetration, given that the penetration of PL is very limited to a few millimeters, and the peanut kernels are relatively thicker than the sugges ted penetration distance. Slices were then placed in al uminum plates and trea ted by PL

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77 In ice tray treatment To understand the effective power of PL treatment, and whether the destruction is the action of the photo chemical of the UV photons or from the h eat due to UV photo thermal and i nfrared region, peanut plates (6 cm diameter) w ere placed in larger plates (7. 5 cm diameter) t hat contained ice surrounding all the plate's sides except the upper side. These samples were treated for 30, 60, 90, 120, 180, 1 20, and 240 s and at distances of 5, 7, and 10 cm ( Figure 4 1 5 ). In tube treatment Zhao ( 2013 ) in her research made a motor, which was buil t in the Processing Lab of the University of Florida. The motor was fixed to steel plate with a control panel, metal rod, and wires ( Figure 4 1 6 ) The steel rod rotates carrying a regular glass tube ( Fisher Scientific Rockford, IL ) full of peanut s Once the motor is turned on, the device flips the peanuts inside the test tube while exposing to PL. The same method was u sed in this experiment. Five grams of peanuts were placed in a transparent glass test tube. A hole was made at the bottom of the test tube to release the vapor pressure to prevent the tube from exp lod ing This glass tube was capped by a suitable rubber cap tighten ed to the edge of the metal ro d which was belt and adjusted to make sure that the test tube was located directly under the center of the light source ( Figure 4 1 6 ) (Zhao 2013). The distances used her e we re 7 10 cm since the d Treatments used a 7x3 factorial design; the treatment factors were PL exposure times of 30, 60, 90, 120, 180, 120, and 240 min and distances of 5, 7, and 10 cm.

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78 Samples were treated in triplicate. Each treatment used three 5 g samples treated separately. After PL treatment, s amples were ground in a coffee grinder (Model LXC 150 50 /60Hz 180W Keunex, Korea ), then extracted by mixing and blending in a lab blender (31BL91, Waring, Dynamics Corporation of America, Hartford, C T ) for 2 min with m ethanol 70% and filtered using Whatman n umber 1 filter paper as in Chapter 3 The extrac ted samples were analyzed using ELISA ( Agra Quant ELISA A flatoxin Kit Romer Labs, Getzersdorf, Austria). The kit included five different AFT concentration standards 0, 4, 10, 20, and 40 ppb to create a standard calibration curve to calculate AFB 1 and AFB 2 concentrations for all PL treated samples and compare th e treated samples AFT concentration with the positive control (fungi inoculated peanuts without PL treatment). Chromatographic analyses were performed on samples that h ad the highest reduction percentage to confirm the ELISA results by using HPLC/MS/MS ins trument (the same as C hapter 3). The highest treatment of all method were statistically compared using 2016 M icrosoft Excel software ; f actorial analysis one way ANOVA. Means and standard divisions were calculated. The mean significant differences were obt ained using the least significant differences (LSD) procedure Results and D iscussion P ulsed light treatment is strongly affected by the transparency of the treated food. The colored or the opaque foods have UV light penetration capacity less than transpa rent food such as water and some juices (Guerrero Beltran & Barbosa Canovas, 2004). When the transparency increases the UV penetrate s deeply inside the food

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79 matrix permitting a complete decontamination for the contaminant (Feuilloley et al., 2006). Wallen & Fraenkel ( 2001 ) implied that the effect of PL is limited to the first few millimeters from the surface of the solid food Thus, it is necessary to control the thickness of irradiated foods due to lower UV light penetration ( to several millimeters depend ing upon their optical properties ). Pulsed light easily penetrates water but difficultly passes through milk and other turbid foods. Granular or opaque foods such as peanut need to be presented as a thin layer or constant shaking or moving during the UV li ght detoxification (D i ao et al., 2015). In addition, expanding the penetration capacity of UV light is an essential step to improve its detoxification efficiency. Therefore, many PL exposing methods were investigated in this chapter to fulfil the best AFT reduction with respect to peanut nutrient and sensory properties ELISA and HPLC MS/MS Results With Skin Samples In plate treatment After PL treatment, AFT concentrations in peanut kernels showed reduction depending on the treatment method, expos ure time and the distance between samples to the Xenon lamp. The presented study results revealed that PL treatment can reduce AFTs. This reduction had a strong positive correlation coefficient to the temperature elevation and treatment time ( Table 3 5, 3 8 ). Howe ver, it had a negative correlation coefficient to the distance for both with skin and without skin peanuts in all treatment methods ( Figure 4 2). The highest reduction (82%) was achieved after treatment for 300 s ith increasing the treatment time the reduction would increase. However, increasing the treatment time above 300 s accompanied with decreasing the distance led to some burn to the upper surface of the

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80 peanut kernels during in plate treatment causing a degr adation in peanut quality. LC MS/MS results agreed with the ELISA results. The reduction reported for AFB 1 AFB 2 for treatment duration 240 s at 7 cm were 87.2 % and 84.7 %, respectively. Shaking treatment Shaking treatment was the b est in terms of achieved a high reduction percentage reach to 86% after 240 s treatment ( Figure 4 3) of PL with maintaining good peanut quality T here is a possibility of having bet t er reduction with keeping the peanut sensory quality by increasing treatment time with respect to t he amount of light absorbed by the food. LC MS/MS results agreed with the ELISA results. The reduction reported for AFB 1 AFB 2 for treatment duration 240 s at 7 cm were 91.3% and 88.5 % respectively. In ice treatment This treatment conducted to avoid the temperature elevation which could affect peanut quality. At the same time this treatment examined the photo effect solo, without the temperature effects. The highest temperature recorded in this treatment was 107C in the 300 s treatment ( Figure 4 4) Resu lts showed that reduction achieved in this treatment was much lower than all other treatment s. This demonstrates that the PL treatment led to mostly photo thermal destruction Strictly photo reduction (temperature controlled treatment) caused a low level o f AFTs (AFB 1 AFB 2 ) reduction w hich at maximum was 50% reduction The LC MS/MS results showed lower reduction than the ELISA results. The reduction reported for AFB 1 AFB 2 for treatment duration 300 s at 7 cm were 28% and 65% respectively. In tube treatme nt Peanut s were treated by placing in a glass test tube and fixed to a rotating motor to provide treatment from all sides and the provide uniformity in the treatment and avoid

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81 e former treatments (using the same distance and time) in terms of AFTs (AFB 1 AFB 2 ) reduction, which reached to 52.4% when treated for 300 s ( Figure 4 5). LC MS/MS results were higher from ELISA results. The reduction reported for AFB 1 AFB 2 for treatment duration 400 s at 7 cm were 86.5% and 93.2% respectively. Factorial statistical analysis one way ANOVA was conducted to determine the significant effect s of the differences in the four exposing method s during PL treatment on the AFT reduction percentages and on temperature elevation ( Figure 4 2, 4 3, 4 4, 4 5 ) The highest treatment of all the four exposing method of the with skin samples were statistically analyzed Results were significantly different at = 0.05 confidence interval between the in plate treatment, in ice treatment and in tube treatment. However, the in plate treatment was Without Skin S amples In plate treatment Peanuts without skin w ere more sensitive to PL treatment. All methods recorded best reduction percentage for without skin peanuts due to the s kin which acted as a barrier The skin likely shaded the kernels and decreased the effect of PL. In addition, the dark color of the ski n will increase the light absorbance on the surface not allowing it to penetrate sufficiently. Therefore, the highest reduction achieved (90.9 % ) was in without skin peanuts treated by PL treatment without reducing the quality of the kernels for 240 s at 5 cm. Increasing the treatment time above 300 s ( Figure 4 6) accompanied with decreasing the distance led to some burning and uniform dark spots at the top of the peanut surface. LC MS/MS results agreed with the ELISA results. The reduction

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82 reported for AF B 1 AFB 2 for treatment duration 240 s at 7 cm were 90.3 % and 95.2 %, respectively. Shaking treatment Shaking treatment was the b est in terms of achieving a high reduction percentage reach ing to 91.7% after 240 s of PL treatment at 7 cm to the quartz window accompanied with saving the quality of peanuts and resulted in semi roasted peanuts. T here is a possibility of having better reduction with keeping the peanut sensory quality by increasing treatment time with respect to the irradiation absorbed by food. L C MS/MS results agreed with the ELISA results. The reduction reported for AFB 1 AFB 2 for treatment duration 240 s at 7 cm ( Figure 4 7) were 94.1% and 93.9 %, respectively. Slices treatment Peanut s were s liced to get better UV penetration treatment. R esults were good and the reduction reached to 82% in 210 s and the temperature reached 170 C ( Figure 4 consumers This treatment ended with fast burning of the edges of the peanut sli ces In ice treatment This treatment was conducted to avoid the temperature elevation which could affect the peanut s quality and a t the same time examined the photo effect solo without the temperature effects. The highest t emperature reached was 126 C ( Figure 4 9). Results showed that reduction achieved in this treatment was much lower than all other treatments by mean s that the PL treatment has photo thermal destruction ; thus the photon without increasing in temperature caused a low level of AFTs ( AFB 1 AFB 2 ) reduction which was 49.1%. LC MS/MS results were lower than ELISA results. The

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83 reduction reported for AFB 1 AFB 2 for treatment duration of 300 s at 7 cm was 28% and 65% respectively. In tube treatment Peanut s were treated by placing in a glass test tube and fixed to a rotating motor to treat all the peanuts sides and provide uniformity in the treatment and color in addition to avoiding a high increase in temperature during treatment. Results were n o t as good as the former treatments without r otation (using the same distance and time) in terms of AFTs (AFB 1 AFB 2 ) reduction, which reached to 63.16% when treated for 300 s ( Figure 4 10). LC MS/MS results were higher than ELISA results. The reduction reported by LC MS/MS for AFB 1 AFB 2 for treatme nt duration 400 s at 7 cm were 75.2 % and 78.6 % respectively for the without skin treatments Factorial statistical analysis one way ANOVA was conducted to determine the significant effect s of the differences in the exposing methods during PL treatment on the AFT reduction percentages, and on temperature elevation ( Figure 4 6 4 7 4 8 4 9 4 10 ) The highest treatment of all the f ive exposing method s for the with out skin samples were statistically analyzed Results were significantly different at = 0.05 confidence interval between the in plate treatment, in ice treatment and in tube treatment. However, the in and the slice treatment PL Pulsed light originate s from the sa me principle as UV decontamination PL is quite close to the UV treatment but PL has higher intensity and greater efficiency than UV decontamination techniques because PL amplifie s the photo catalytic activity of light which is naturally responsible for extirpating a variety of pollutants. Various

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84 mechanisms have been proposed to explain the lethal effects of PL all of them related to the UV part of the spectrum and its photo chemical and photo thermal effects ( Elmna sser et al., 2007; Gmez Lpez et al., 2007). The photo chemical mechanism involves the UV part. However, this study results indicated that the AFT degradation was the result of two companied PL effects : the photo thermal and the photo chemical effect. Infrared heat can also contribute to th e AFT destruction effect (Yang et al ., 2012) Therefore, PL is consider ed a fast method to transfer a large amount of photo thermal An infrared thermometer was used to measure the increasing of temperature after all treatme nt s and compare d with the initial temperature (room temperature 23 25C). The highest temperature reached among all treatment s was 180 C for the i n plate treatment. However, the AFT s have high decomposition temperatures ranging fro m 237C to 306C, and sol id AFB 1 is quite stable to dry heating at temperatures below its thermal decomposition temperature of 267C ( Rustom 1997). Thus, in the present study the heat e levation should not have been high enough to destroy AFTs thermally. Generally, PL treatment accompanied with manipulating time and distance and shaking the samples fulfilled a high reduction at a relatively low temperature. Surrounding the samples with ice was conducted to examine the effect of the photo chemical effect in solo on the AFT degra dation. However, all the results showed lower reduction percentages compared with other treatm ents. On the other hand, treating the peanuts (the whole kernels ) inside rotating test tube ( Figure 4 18) showed a low reduction percentage because of the layer o f glass which worked as a barrier to prevent part of the light from reach ing the samples. However, the quality of the

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85 produced samples was perfect in terms of the distribution of the roasting color. Thus, this treatment could be used for a longer time with respect to the increasing of the power expenses and time which will be the main disadvantages of this method. Zhao ( 2013 ) used the rotating tube method to provide uniform PL treatment to inhibit the allergen ic effects of peanuts b y destroy ing proteins re sponsible for the s e allergic symptoms in human results were promising s i nce using this device increases the PL treatment time allowing the peanuts to absorb as much as available of the effective spectrum, avoiding the quality deterioration of pea nuts, and allowing longer treatment than in plate treatment (Zhao 2 01 3).

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86 Table 4 1 LC MS /MS analysis for 5g without skin peanuts with different PL treatments. Treatments R % AFB 2 R % AFB 1 In Plate 95.2a 90.3a Shaking 93.9a 94.1a Tube 78.6 b 75.2 b Con trol 0 0 R%: reduction percentage, PL treatment: 240 s at 7 cm distance from the strobe Different alphabetic means significantly different at = 0.05 using one way ANOVA and LSD for mean differences analysis Table 4 2 LC MS /MS analysis for 5 g with s kin peanuts treated with PL Treatments R % AFB 2 R % AFB 1 In Plate 84.7a 87.2a Shaking 88.5a 91.3a Tube 93.2a 86.5a In ice 65.0b 28.0b Control 0 0 R%: reduction percentage, PL treatment : 240 s and 7 cm distance from the strobe Different alphabetic mea ns significantly different at = 0.05 using one way ANOVA and LSD for mean differences analysis Table 4 3. Peroxide value (meq of peroxide/L) of peanuts oil after PL treatments of peanuts Treatment Control 240 s Shaking 240 s Plate 300 s Shaking 300 s Plate 240 s / 5 cm 1.30 0.14 a 0.43 0.05c 0.96 0.05c 0.67 0.06bc 0.83 0.03c 240 s / 7 cm 1.10 0.14a 0.50 0.04c 0.89 0.04c 0.40 0.07b 1.20 0.26c Mean standard deviation (n = 3). Different alphabetic means significantly different at = 0.05 using one way ANOVA an d LSD for mean differences analysis Figure 4 1 The model structure of peanut cells: I = cell cavity; II = amorphous matrix; III = external membrane film; IV = internal membrane film (Zakhartchenko et al. 1998)

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87 Figure 4 2. Reduction percentag es and temperature measurements of in plate PL treat ed peanuts. Three different distances ( 5, 7, and 10 cm ) were used for with skin peanuts. Figure 4 3. Reduction percentages and temperature measurements of shaking PL treat ed peanuts. Two different distances ( 7, 10 cm ) we re used for with skin peanuts. 0 50 100 150 200 250 300 350 0 20 40 60 80 100 120 0 20 40 60 80 100 120 140 160 180 30 60 90 120 180 210 240 300 AFT Reduction% Temperature Time (s) Plate treatment with skin 5cm-T 7cm-T 10cm-T 10cm-R% 5cm-R % 7cm-R% 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 160 180 200 0 30 60 90 120 180 210 240 300 AFT reduction% Temperature Time (s) Shaking treatment with skin peanuts 7cm-T 10cm-T 7cm-R% 10cm-R%

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8 8 Figure 4 4. Reduction percentages and temperature measurements of in ice PL treat ed peanuts. Three different distances ( 5, 7, and 10 cm ) were used for with skin peanuts. Figure 4 5. Reduction percentages and temperature mea surements of in tube PL treat ed peanuts. Two different distances ( 7, 10 cm ) were used for with skin peanuts. 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 160 180 200 0 30 60 90 120 180 210 240 300 AFT reduction% Temperature Time(s) Ice treatment with skin samples 5cm T 7cm T 10cm T 5cm R% 7cm R% 10cm R% 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 180 210 240 300 400 AFT reduction % Time (s) Tube treatment with skin 7cm-R% 10cm-R%

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89 Figure 4 6. Reduction percentages and temperature measurements of in plate PL treat ed peanuts. Three different distances ( 5, 7, and 10 cm ) were used for w /o pean uts. Figure 4 7. Reduction percentages and temperature measurements of shaking PL treat ed peanuts. Two different ( 7, 10 cm ) were used for w/o skin peanuts. 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 160 180 200 AFTs Reduction % Temperature 30 60 90 120 180 210 240 300 Time (s) Plate treatment w/o skin (5,7,and 10 cm) 5cm-T 7cm-T 10cm-T 5cm-R % 7cm-R% 10cm-R% 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 160 180 200 0 30 60 90 120 180 210 240 300 AFT reduction% Temperature Time (s) Shaking treatment w/o skin peanuts 7cm-T 10cm-T 7cm-R% 10cm-R%

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90 Figure 4 8. Reduction percentages and temperature measurements of slices PL treat ed peanuts. Two different ( 7, 10 cm ) were used for w/o skin peanuts. Figure 4 9. Reduction percentages and temperature measurements of in ice PL treat ed peanuts. Three different distances ( 5, 7, and 10 cm ) were used for w/o skin peanuts. 0 20 40 60 80 100 0 50 100 150 200 0 30 60 90 120 180 210 AFT reduction% Temperature Time(s) Peanuts slices treatment (5,7 cm) 5cm T 7cm T 5cm R% 7cm R% 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 160 180 200 AFT reduction% Temperature 0 30 60 90 120 180 210 240 300 Time (s) Ice treatment w/o skin 5cm T 7cm T 10cm T 5cm R% 7cm R% 10cm R%

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91 Figure 4 10. Reduction percentages and temperature measurements of in tube PL treat ed peanuts. T wo different distances ( 7, 10 cm ) PL treatment for w/o skin peanuts. Figure 4 1 1 In plate PL treatment for peanuts. ( Photo courtesy of author). Figure 4 1 2 Schemat ic representation of Shakin g P L treatment ( Photo courtesy of author). 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 180 210 240 300 400 AFT reduction % Time (s ) Tube treatment without skin 7cm-R% 10cm-R%

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92 Figure 4 1 3 Shakin g device inserted inside the P L machine ( Photo courtesy of author). Figure 4 1 4 Shaking device for shaking treatment for PL treatment of peanuts. ( Photo courtesy of author). F igure 4 1 5 Double dishes for In ice PL treatment of peanuts. ( Photo courtesy of author).

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93 Figure 4 1 6 Rotating motor for In tube rotating PL treatment of peanuts. ( Photo courtesy of author).

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94 CHAPTER 5 INACTIVATION OF AFLATOXINS B 1 AND B 2 IN PEAN UTS BY COM BINING TWO TREATMENTS: PULSED LIGHT (PL) AND CITRIC ACID The AFTs are secondary metabolites of Aspergillus flavus and Aspergillus parasiticus There are 18 different identified types of AFTs Nevertheless, the major members are AFT B 1 B 2 G 1 and G 2 AFT s are coumarin derivatives with a fused dihydrofurofuran moiety (Gupta 201 2 ; Iram et al., 2016 ). They are potent toxigenic, carcinogenic, and immunosuppressive compounds commonly found in peanut and associated products. Chronic exposure to low levels of A FB 1 with the presence of hepatitis B virus could cause a serious development of hepatocellular carcinoma in the human body. In addition, the International Agency for Research on Cancer (IARC) announced aflatoxin B 1 as a group 1 carcinogen to humans i n the Sixth Annual Report on Carcinogens in 1991 ( National Toxicology Program, 2011) Since then AFTs have been confirmed as dangerous contaminants causing acute and chronic effects. The health and science society intensified their effort to fin d a way to g et rid of this risk which has threaten ed Numerous methods for reducing AFTs have been investigated since the 1960s: c hemical physical, and biological approach es Most of these methods have some disadvantages over the fav orable attributes in human edible substances. Some points should be taken into consideration as a nominal requirement: the safety of the food after treatment, the used method should eliminate AFTs to at least the FDA requirement 20 ng/g) (FDA 1994 ; Mob een toxic residual, and finally it should maintain food quality The c hemical approach for AFT detoxification in food in mild con dition s was one of the scientist favor ed methods, since the treatment will not lead to nutrient value degradation because of temperature

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95 elevation. Therefore, several chemicals have been tested through the last four decades for AFT destruction in several types of commodities. Most of them achieved a remarkable reduction. However most of them carried out the concerns of the possible toxic residual production and sensory damaging attributes Recently, ozone and ammonium treatments have been used widely to destroy AFTs in water, peanut meal, cottonseed and maize especially for an imal feed ( Rustom 1997 ). Pulsed ultra v iolet L ight (PL) is one o f the proposed new technologies which is presented as non thermal food treatment regarding microbial inactivation by using broad spectral wavelengths (200 to 1100 nm), including UV spectrum. P ulsed light has proven its capability of destroying and reducing several types of harmful compounds, including bacteria, viruses, enzymes, allergens and toxins. In high intensity continuous UV treatment, the photo degradation rate of AFB 1 is strongly aff ected by UV intensity. Only few literatures showed the same UV intensity (Liu et al., 2011). AFB 1 (2 mg/kg) in peanut oil can be destroyed completely when exposed to UV irradiation for 30 min at an intensity of 8000 J/cm 2 while it was degraded by about 85 % and 79% at the lower intensity of 4000 and 2000 J/cm 2 respectively (Diao et al., 2015 ) However, great irradiation intensity may cause critical degradation in pea n ut quality. There fore, the intensity and exposure time of the UV irradiation must be contr olled to keep the safety and quality of irradiated foods (Diao et al., 2015 ). The p ulsed intensive light is 20,000 times mo re than the continu ous light In the presented experiment, PL was used to degrade AFTs in peanuts. The expos ure times and distances w ere investigated from all aspects to provide an optimum treatment for the best AFB 1 AFB 2 reduction and keeping good quality to the product. The used pulsed

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96 light delivered 1.27 J/cm 2 to the peanuts at 1.9 from the strobe Results were promising as shown i n C hapter 3 and 4 been achieved. Therefore, soaking the peanuts in citric acid before PL treatment was investigated in this chapter as earlier literature declared that low pH enhances the UV efficiency (Diao et al., 201 5). Citric acid has been tested before alone for AFT detoxification in duckling feed and rice ( Mndez Albores et al., 2007 ). Many physical, chemical, biological, and irradiation methods have been explored to decrease or remove AFT s in foods. Pulsed light ( PL) and citric acid treatments are two of these techniques. Individually, each treatment showed good results in the degradation of AFT s for some commodities. However, a complete elimination of AFTs was not achieved by either method. Many current studies co ncluded that UV wavelength, types of AFT s, irradiation intensity, moisture contents of foods, exposure time, thickness and pH of irradiated foods drastically affect UV elimination efficacy. AFB 1 is reported to be highly sensitive to UV irradiation at a pH of less than 3 or greater than 10 (Diao et al., 2015). Thus, adding c itric acid to PL treatment was tested in this study. Material and M ethods Sample preparation was the same as Chapter 3. With skin and without skin peanuts were inoculated with Aspergillu s flavus (dominant AFB 1 AFB 2 producer) A fter a proper incubation period 5 g of p eanuts were soaked in 0.1 N citric acid at a concentration of 2 m L /g for 1 h as i s similar to the method used by Mndez Albrose (2004) Samples were either washed with dist illed water or citric acid solution. Moisture was optimized to 16% as in Chapter 3 Then PL treatment was conducted (Model# LHS40 LMP HSG from Xenon Corp, Wilmington, MA ) in a shaking tray for 210 s or 240 s

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97 at 7 cm AFTs were extracted using methanol/wate r (70/30) S amples were analyzed using ELISA and HPLC MS/MS to determine AFB 1 AFB 2 reduction Experimental Design and Statistical Analysis This experiment was conducted in triplicate. After the preparation step as in C hapter 3 and the citric acid treatme nt for specific samples, e ach sample was placed in the tray which was fixed to a shaker ( MTS 2/4 D S1, IKA, Wilmington, NC) to be exposed to the light from all sides at the same time. Samples were extracted and AFTs were measured by using ELISA and HPLC MS /MS. Data were analyzed statistically usin g t wo way ANOVA test at 0.05 level of confidence (Microsoft E xcel 2016) followed by Fisher Least Significance Differences ( LSD ) method for means differences to evaluate the significance in differences among differe nt treatments and the control ( untreated peanuts) at this confidence interval. Results and D iscussion ELISA Results ELISA results showed a reduction percentage of AFB 1 AFB 2 in with skin and without skin peanuts with citric acid of about 88.29 % and 98.12 % respectively (Table 5 1 5 2 ) When citric acid treatment was applied individually t he reduction percentages were 20 6 and 2 9 3 respectively for with skin and with out skin peanuts, and the PL treatment individually resulted in 78.9 and 87.1 for with skin and without skin respectively. It was obvious that the hurdle technique and soaking the peanuts in 0.1N citric acid preceding t he PL treatments achieved the best results for AF B 1 B 2 reduction (Table 5 1 )

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98 HPLC MS / MS Results The LC MS results reveale d that treatment with citric acid individually cause d an AFB 1 AFB 2 reduction with washing reached to 12.6% and 51.1% respectively. However treating the peanuts with the PL alone led to AFB 1 AFB 2 reduction reaching to 90.3 % and 95.2 % respectively. Howe ver the hurdle technique (citric acid) and PL led to A FB 1 AFB 2 reduction reaching to 98.9% and 98.1% respectively when treatment was applied without washing the citric acid from the surface of the peanuts (Table 5 2, 5 3) Acid treatments have the abil ity to alter the chemical structure of AFB specifically alteration of AFB 1 to AFB 2 The converted substance has mutagenicity 1000 times lower than the original toxin ( Rustom 1997). Discussion Aflatoxin B 1 is an invisible food threat AFT s residue conta mination has been reported in peanuts and peanut products. Therefore, a practice to obtain peanuts free of AFTs is important since peanut consumption has increased in the last century due to its distribution all around the world. However, any detoxificat ion method in tended to be use d should not cause any undesirable alterations to the nutritional and organoleptic qualities of the peanu ts (Samarajeewa et al. 1990). Previous studies revealed that t here was an obvious change in the chemical composition of f ood components and some product quality de gradation occurred when the UV light treatment is applied at a high dose on some foods (Kolakowsk a 2002 ). However, as shown from other stud y results, UV irradiation may not cause any alteration effects if UV ir ra diation is applied in reasonable amounts (Caminiti et al., 2012; Taze et al., 20 15 ; Tripathi & Mishra, 2010).

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99 Therefore, a hurdle technique or combining two methods is essential to decrease the UV expos ure time and enhance the output of both treatments. Th e two techniques we used in this experiment, individually each one of them achieved good but not sufficient results in terms of AFT degradation, and quality retention of the peanuts. Soaking the peanuts in 0. 1 N citric acid for 30 min at a lower concentrat ion (2 mL /g) from what Mndez Albores et al. ( 2005 ) used (15 min in 3 m L /g, 1 N citric acid) was more effective than the shaking treatment at 7 cm for 240 s (Chapter 4) Thus, these parameters efficiency were enhanced by the soaking step in 0.1 N citric ac id for 1 h Comparably, in terms of using chemicals to enhance irradiation treatment Farah et al. (1983) used an autoclave at 116 C, 0.7 bar to cook raw with skin peanuts after soaked it in a 5% NaCl solution for 30 min. This treatment showed a good redu ction for total content of AFB 1 AFG 1 AFB 2 and AFG 2 by 80 100%. The removal of the toxins was attributed to the addition of NaCl, as compared to unsalted controls (Masimango et al., 1978; Rustom 1997). In addition, Rastegar (2017) showed that r oasting A FB 1 contaminated pistachios with 30 ml water, 15 ml lemon juice and 2.25 g of citric acid at 120 C for 1 h reduced the level of AFB1 in 49.2 3.5% from the initial level (268 and 383 ng/g) without a noticeable change in desired attributes of pistachios ( R astegar et al., 2017). Possible Mechanisms of AFT Degradation by PL and Citric Acid T reatment s During peanut PL expo sure three possible degradation mechanisms could have happened to the AFTs: one is the effect of light, since the range of spectrum used in this experiment is included UVA and UVC, which have been proven their ability for the destruction of AFT s due to their photosensitivity. Diao et al. ( 2015 ) in his review showed

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100 that exp osing to UV at wavelength s 220 400 to several types of food (peanut, p eanut oil, coconut oil, red chili powder, walnut, almond, pistachio, and dried figs) resulted in AFT reduction from 45.7 % to 100% during different expos ure time from 10 to 60 min and light intensity ranged from 64 J /cm 2 to 8000 J /cm 2 (Diao et al ., 2015). L illard & Lantin ( 1970 ) indicated that the best degradation of AFB 1 by irradiation is at 362 nm. Atalla et al. (2004) and Jubeen et al. (2012) reported that the C8 9 double bond in the terminal furan ring of AFB 1 can be degraded easily by UV light at 362 nm However, AFB 2 and AF G 2 9 double bond in their terminal furan ring. Thus, they were easily degraded by UVC (especially 254 nm). However, Basaran (2009) reported that the UV light of 254 nm did not affect AFG 2 and AFB 2 but significantly r educed AFG 1 and AFB 1 The second possible degradation mechanism is that th e effect of citric acids on AFTs AFB 1 is reported to be highly sensitive to UV irradiation at a pH of less than 3 or greater than 10 (Lillard & Lantin, 1970; Rustom 1997 ). Thus, t o decrease the pH, 0.1 N citric acid ( pH 2.9) for 1 h (3 m L /g contaminated peanuts) was added to peanuts before PL treatment to enhance the UV efficiency. Mndez Albores et al. ( 2007 ) study of treating duckling feed with 1 N (pH 2.4) aqueous citric acid fo r 15 min (3 m L /g of contaminated feed) showed that feed with initial concentration 110 ng/g was partially detoxified to 86% by citric acid treatment. In addition, acidified AFB 1 molecules from acid treated feed, exhibit neither toxigenic activity nor carci nogenic and mutagenic activity compared with molecules of untreated feed. Additionally AFB 1 is reported to be highly sensitive to UV irradiation at a pH of less than 3 or greater than 10 (Lillard & Lantin, 1970). Rustom ( 1997 ) mentioned that heat treatmen t at pH 8.0 w as not effective

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101 to reduce AFT s mutagenic activity. However, the treatment at pH 10.2 for 121 C for 15 min reduced the mutagenicity to 88%. The third possible degradation mechanism is the effect of the high temperature generated from infrared wave length. ELISA and HPLC MS / MS results showed a lmost complete AFT reduction in without skin peanuts ( Table 5 1 5 2; Fig ure 5 1) which was soaked in citric acids and not washed before the PL treatment. These results showed that the citric acid enhanc es the PL efficiency in terms of AFT destruction.

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102 Table 5 1 ELISA results for the c omparison between c itric acid + PL (shaking) treatment and citric acid treatment for without skin (w/o skin) and with skin peanuts Samples Time(s) Temperature ( C ) Reduc tion% C with skin 0 21.7 1 .02 0 C w/o skin 0 21.75 1.02 0 PL, w/o skin/sh 210 125 10.6 73.9 3.13b PL, with skin/sh 210 130 2.05 40.9 14.6a PL, with skin/sh 240 140 9.19 78.9 0.4b c PL, w/o skin/sh 240 168 9.19 87.1 10b Ci with ski n 3600 20.1 0.0 20.6 0.08 a Ci w/o skin 3600 20.1 0.0 29.3 3.45 a Ci+ PL w/o skin 240 178 4.7 98.1 1.13 c Ci+ PL with skin 240 168 2.4 88.2 1.9 8 c Different alphabetic means significantly different at = 0.05 using one way ANOVA and LSD system for mean differences analysis. C = control, Ci = citric acid treatment, PL = pulsed light treatment, Ci+PL = Citric acid+ pulsed light 7 cm sh = shaking treatment. Table 5 2 LC MS /MS results for the comparison between pulsed light (PL) treatment and the p ulsed UV light with the citric acid treatment without washing. Treatments AFB 1 R% AFB 2 R% 240 s PL, 7 cm + Ci 240 s PL, 7 cm Control 98.9 a 90.3 a 0 98.1 a 95.2 a 0 Different alphabetic means significantly differ ent at = 0.05 using one way ANOVA and LSD system for mean differences analysis. C = control, Ci = citric acid treatment, PL = pulsed light treatment, R%= reduction percentage. Table 5 3 LC MS/MS results for the comparison between citric acid treatment and cit ric acid followed by PL treatment for without skin peanuts with washing. Treatment AFB 1 R % AFB 2 R % Citric acid (1h) 12.6 a 51.1 a 240 s shaking PL 7 cm + Citric acid, 91.3 b 88.9 b Control 0 c 0 c Different alphabetic means significantly different a t = 0.05 using one way ANOVA Table 5 4 LC MS/MS results for the comparison between citric acid treatment and citric acid fo llowed by PL treatment for with skin peanuts with washing. Treatment AFB 1 R % AFB 2 R % Citric acid (1h) 19.1 a 24.2 a 240 s shakin g PL 7 cm + Citric acid, 79.1 b 80.1b Control 0 c 0 c Different alphabetic means significantly different at = 0.05 using one way ANOVA.

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103 A B C Figure 5 1. LC/MS/MS ch romatograms for peanuts A) Without treatment (control), B) A fter PL treatment C) After PL + citric acid treatment

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104 CHAPTER 6 THE EFFECT OF STORAGE TIME ON THE PEROXIDE VALUE FREE FATTY ACID CONTENT AND COLOR IN PL TREATED PEANUTS Raw peanuts and its products are important foods all over the world and are part of many famous re cipes. C onsumers typically us e peanuts in food products in different way s (Camargo et al., 2012). Peanuts recently have been identified as contributing to lower occurrence of coronary heart disease (CHD). Peanuts contain many vitamins, and are consider ed a n excellent vitamin E source. Peanut butter alone provides 2.3% of the vitamin E needed in the diet of the U.S. (Chun et al., 2005). Lipids are found in a great percentage in peanuts, almost 47 52% of the dry weight. The majority of the lipid composition i s around 80% unsaturated fatty acids. However, greater amounts of unsaturated fatty acid s accelerate rancidity, which affects the sensory quality, and flavor attributes in peanuts. Lipid oxidation is the main reason behind the off flavor of rancid peanuts. Oxidation at la ter stage s form the directly off flavor contributed compounds such as alcohols, ketones, and aliphatic aldehydes. For peanuts, these chemicals are related to painty, oxidized, and cardboardy flavors connected to peanut flavor fade (Powell 2004). Oxidation is mainly affected by peanut composition, processing method, treatment temperature, and storage conditions, including temperature, time, light and oxygen (Riveros 2010). Peroxide value (PV) is an indicator of the initial stages of oxidat ive changes. This method evaluate s the total hydroperoxide content, which is one of the most common quality indicators of lipids through storage periods (Shahidi and Zhong 2010). UV irradiation initiates free radical oxidation and forms lipid radicals, su peroxide radical (SOR), and H 2 O 2 and then can lead to cross linking of carbohydrate and protein Therefore, there are some changes in the chemical composition of food components

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105 and possibl y some product quality de gradation that could occur when UV light treatment is used especially when large fl u e nce s are applied (Kolakowska 2002 ). However, from other stud y results, UV irradiation may not cause any components alter ed if UV irradiation is used in small to moderate amounts ( Diao et al., 2015) Materials a nd Methods Samples Preparation and Storage Condition Raw with skin (with testa) and without skin (w/o testa) peanuts were purchased from a local market. Five grams of peanuts samples were treated by Pulsed Light at 5 and 7 cm for several time periods and different methods ( 240 s shaking treatment, 240 s in plate treatment, 300 s shaking treatment, and 300 s in plate treatment ) Then 5 g of each treatment was placed in small plastic bag, sealed thermally, and stored at ambient temperature inside a cardboard box to prevent excess light. Experiment Design Each month samples were ana lyzed for their peroxide value, free fatty acids and acid value content. Factorial two way ANOVA statistical analyses were conducted. Means and standard deviation were determined w ith = 0 .05 confidence interval. Then the least significant differences (LSD) for mean differences analysis to determine the effect of stored time and different treatment on the shelf life of the PL stored peanuts was conducted In this experiment, the ef fect of PL and storage time on lipid oxidation in the peanut kernels was dete rmined by compar ing the peroxide value (PV), free fatty acids and the acid value of the raw peanuts (control) with 1, 2, and 3 months stored peanuts ( previously PL treated peanut s ) Treatments assessed w ere the most effective PL

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106 treatm ents in terms of AFT reduction: 240 s shaking 7 cm 300 s shaking 7 cm and 240 s in plate 7 cm 300 s in plate 7 cm treatments Peroxide Value ( PV ) T he International Dairy Federation standard metho d 74A (International Dairy Federation, 1991, Brussels, Belgium) was used to determine PV Free Fatty Acid s and Acid Value Determination FFA and acid value were determined according to the official method Ca 5a 40 of American Oil Chemists' Society (AOCS, 1 990 ) the same as in Chapter 3. Color Evaluation To quantify the color changes, a machine vision system consisting of a Nikon D 200 digital camera housed inside a light box [42.5 cm (W) x 61.0 cm (L) x 78.1 cm (H)] was used (Luzuriaga et al., 1997). The c amera was used to measure the color of peanut kernels with a D65 (daylight) lamp and 10observer angle. Each image includes traditional roasted peanuts (control) and PL peanuts as measured samples. Each image was calibrated against an orange yellow color r eference tile (L=71.4, a=12.2, b=70.14) that was obtained from the ColorChecker Classic X Rite Company (MI, USA). The software LensEyeSK v10.0.0 from Engineering and Cyber Solutions Inc. (Gainesville, FL, USA) was used to analyze the images. The Hunter L, a, and b color space is organized in a cube form. All black color was surrounding the references orange and yellow cube s were erased to maintain a precise calibration without interference from the dark border. Later to obtain correct images the samples im ages were subjected to background corrections and the final images were calibrated with a standard s orange and yellow color s The maximum L is 100 (white) and the minimum L is 0 (black). A positive a value denotes red, a negative a

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107 value for green; a posi tive b value for yellow, and a negative b value for blue. The coordinates of three color parameters were expressed using C* (chroma), h (hue angle), and total color difference Th e color analysis w as triplicated for each sample. A ll samples L, B, and A va lues were analyzed statistically using one way ANOVA, and the LSD mean difference s method Results and Discussion Peroxide Value and Free Fatty Acids Tests The PV test was used as a shelf life indicator by detecting the early stage s of the autoxidation pro cess. This study illustrated the bigger picture of the effect of PL treatment on the lipid oxidation in light exposed peanut kernels was the tendency of the PL to accelerate lipid oxidation Thus in this experiment, a long storage period was conducted to monitor the levels of lipid oxidation of PL treated peanut kernels by determining both peroxide value and FFA percentage directly after PL treatment and after each month of a three month storage period According to previous s tudies conducted on roasted pe anuts to evaluate quality the rate of PV increasing for roasted peanuts was significantly faster than that of raw peanuts under both air and vacuum storage conditions, which indicated the low oxidative stability of roast ed peanuts. Chun et al. ( 2005 ) reco rded that the shelf life of dry roasted peanuts is about 2 weeks when stored at 21 C under air. However, under vacuum, the shelf life is extended beyond 38 weeks. Evranuz (1993) reported the shelf lives of salted roasted peanuts of 28 d at 15 C 10 d at 25 C, and 11 d at 35 C under the assumption that the products remain acceptable until the PV reaches 25 meq/kg oil. Results ( Table 6 1, 6 2 ; Fig ure 6 1, 6 3) showed that the peroxide values of stored PL treated peanuts were of good qualit y, because the sampl es were below the specified

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108 PV of 10 meq/kg for foods containing fats (AOAC, 1995) for both peanut s with skin and peanuts without skin, and for the distances 5,7 cm. However the 7 cm results were better after three months of storage T he highes t PV value was for the 300 s i n plate treatment. For the 5 cm treatment, the PV started at zer o time with 0.89 meq/kg peanut oil, and reached 12.05 and 9.89 for without skin and with skin peanuts respectively af ter 3 months of storage ( Table 6 3, 6 5 ). T he AOAC has declared that peanuts are consider ed ra n cid when the PV reach es 30 meq/ kg. Peroxide value increased gradually during the storage time due to the activity of enzymes such as lipoxygenase which acts as a catalyst to unsaturated fatty acid oxidat ion reaction to produce hydroperoxides and the secondary products respectively (Fennema 1996) FFA and Acid Value The FFA and the acid value were determined for with skin peanuts exposed to PL using four different treatments: 240 s shaking PL, 240 s in plate PL, 300 s in plate, at 5 cm from the strobe Results were analyzed using tow way ANOVA statistical test, to determine the significant variances among treatments and storage periods at = 0.05. The P value of between treatments of FFA and the acid value was 0.42 (> 0.05). Thus However, there were significant differences among samples FFA and acids value before and after storage. showed no significant differences at the first two months neither in the control nor in the 240 s 300 s in plate samples with FFA rang ing from 0.43 to 0.57. However, after storage for three months, the LSD results showed that all treatments are significantly different. The l east FFA for 3 months of storage w ere found in the 240 s PL shaking treat ment (Table 6 5). T he acid value had no significant differences among trea tments

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109 in the zero time and after 1 mean were statically di fferent = 0.05 at the second and the third months ( Table 6 2, 6 8 ). R esults of FFA (Table 6 10 ) show the FFA percentages and the acid value of with skin peanut s exposed to PL using four different treatments: 240 s shaking PL, 240 s in plate PL, 300 s in plate, at 7 cm from the strobe Results were analyzed using t w o way ANOVA statistical test, to determine the significant differences among treatments and storage periods at confidence intervals = 0.05. The P value of between treatments was 0.27 (> = show significant differences among treatments. FFA and the acid value percentages was evaluated for without skin peanuts samples, which exposed to PL using four different treatments: 240 s shaking PL, 240 s in plate PL, 300 s in plate, at 5 cm then 7 cm from the xenon lamp. Results were statistically analyzed using tow way ANOVA test, to determine the significant differences among treatme nts and storage periods at confidence intervals = 0.05. The p value of between treatments was 0.002, (< = 0.05) and 0.29 (> = 0.05) for 5 cm and 7 cm respectively. Thus, the distances were a crucial factor in all treatment since the results were significantly differen t among treatment s at 5 cm but not at 7 cm from the strobe On the other hand, storage time effect the FFA and acid value measurements. The P value of the storage time was 0.0002 and 0.047 (< = 0.05) for 5 cm and 7 cm respectively, which means there were significant differences among the Least Significant Differences (LSD), test analysis showed no significant differences at the first two months between treatments. However, it showed differences among a ll

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110 the storage periods times. Especially after three month storage, all treated samples were varied in their F FA and acid values. The least FFA and acid value content was a result of 240 shaking PL treatment again for with skin peanuts ( Table 6 5 6 6 ; Tab le 6 9, 6 10 ). To summarize the results, treatments at 7 cm had lower FFA than the 5 cm distan ce from the X enon lamp, and treatment without skin had more homogeneity in their results. The rate of FFA increase for PL treated peanuts was significantly faster than that of raw peanuts under the same conditions. Chun ( 2005 ) s tated that peanuts treated with high temperature such as roasting showed high susceptibility to oxidation due to the high content of unsaturated fatty acids. UV light has been found to hav e the ability to stimulate specific enzymes, which are responsible for the biosynthesis of the flavonoids, these enzymes may produce compounds that act as UV screens preventing UV induced damage in the genetic material of pl ant cells (Cantos et al., 2000). To compare the presented study results with what had published before, results emphasized that the maximum FFA measurement (3.2 6% FFA ), which was recorded during this study for without skin plate at 5 cm from the strobe and treate d for 300 s and after a 3 month storage period. These results were much lower than what Makeri ( 2011 ) had from roasting peanuts using a conventional air oven at temperature of 80, 100, 120, 140, and 160 C for 20 min and stored for 0 3, 6, 9, and 12 weeks Makeri (2011) FFA values ranged from 0.45 to 1.80 at time zero and from 0.90 to 2.20 after storage for 3 weeks, 6.30 to 8.20 after storage for 6 weeks, 7.80 to 9.10 after storage of 9 weeks, and 8.10 to 9.55 after the 12 week storage period. The FFA for the control untreated peanuts samples ranged between 0.40 and 0.49, which agreed with other references Pokorny et al. (2003) recorded FFA for raw peanuts

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111 between 0.4 and 0.72 and acid value between 0.79 1.48, and Makeri ( 2011 ) found that the FFA for raw p eanuts was 0.45. Moreover, peroxide value and FFA percentage and acid value results correlat ed The peroxide value increase d as the storage time increasing as well as the FFA and the acid value. With both methods the shaking treatment for 240 s had the l owest oxidation values Especially for the with skin samples, all PV and FFA w ere lower than the without skin, which could be due to the composition of the skin which included some polyphenols and antioxidant such as tocopherols compounds which may helped to delay the auto oxida tion of the lipids (Sales & Resurreccion, 2010). Although, Sobolev & Cole (2004) stated that regarding the general differences between with skin and without skin peanut n on significant differences were found in relation to the gener al composition of the peanut samples ( p < 0.05). Chun (2005) has demonstrated that tocopherol losses were highly correlated with lipid oxidation based on PV and the c onjugated diene values ( CDV ) for all stored peanuts, indicating the antioxidant function o f vitamin E during lipid oxidation. Similar ly, the results in the present study showed that the existence of the skin could have a quit e significant effect on the PV and the FFA and acid value to extend the storage time (Table 6 5, 6 6, 6 7, 6 8). Color Evaluation Zhao (2013) mentioned that some parts of peanut samples that were treated with PL had darker spots if the peanut kernel was not spun. However, using the shaking treatment succeeded to reduce AFTs without any loss in the color quality of the pea nut s In this study L value, b value, and a value of three different PL treatment s (180 s / 7 cm, 210 s / 7 cm, and 240 s / 7 cm) were compared with commercial ( Publix

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112 roasted peanuts) and a control ( non treated peanuts ) to find out the effect of PL treatm ent on the peanuts degree of roasting and the uniformity of color. All samples were evaluated using machine vision Samples *L, *a, *b levels revealed th at all samples tend to give greyi s h yellow color, since the *a value give s negative numbers and the *b value was low positive number (Table 6 13 ) Typically, roast color is the most important quality control parameter in commercial processes. Roast color in peanuts is generally measured by light reflectance in a colorimeter, giving an L value in a range fro m 80 (very light or no roast) to 30 (very dark roasted). The Hunter L value of roasted peanuts used in high quality dry roasted peanuts and peanut butter falls in the range of 50 51 (Sanders et al., 1989 ; Baker et al. 200 3 ). In the present study the L val ue, which described the whitening of the tested samples ranged from 70.86 for the commercial roasted samples to 76.38 for control peanuts (T able 6 13 ) A one way ANOVA statistical analysis followed by LSD means differences analysis revealed that neither be tween samples nor within each sample had significant difference s at = 0.05 except the commercial sample in its a level (describe the red intensity of the red color) which was significantly higher than the *a level for the control and all other PL trea ted samples The *b Value described the greenish yellow of the samples revealed that the commercial sample was much higher than all the PL treated sample, and the control with the b value range d from 24.5 to 25.21. Measurements of the same samples showed a negative a value ranged from 3.61 to 5.07 for the PL treated samples and ranged from 5.37 to 5.53 for the control non treated peanuts. However, for commercial sample ranged from 1.33 to 1.61. A ll other samples ha d no significant differences in their Chroma values

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113 with the control and the different PL treatments Which clarified that the color of the PL treated samples until the treatment of 240 s was unifor m and it was ntly differ ent from the control ( Table 6 14) Color evaluation r esults showed no significant differences between the three different treatments and the co ntrol but there were significant differences between the treate d samples and the commercial sample T he level of roasting after all the 180 s, 210 s, and 240 s PL treatments fell into the roast category of light Means comparison by LSD analysis showed no significant differences at = 0.05 neither between samples nor within samples for color (T able 6 13 ; Figure 6 1 ) T he shaking treatment provided good uniformity and resulted in peanuts with a homogen eous color.

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114 Table 6 1. The peroxide value for 0 1, 2 and 3 months stored PL t reated with skin peanuts at 5 cm distance from the stro be Time (month) Control 240 s shaking 240 s in plate 300 s shaking 300 s in plate 0 2.1 a 1.67 a 2.46 a,b 1.33 a 2.77 a,b 1 3.13 a 1.57 a 2.23 a,b 1.1 a 3.1 a,b 2 3 a 3.33 a,b 3.53 b 4.33 a,b 6.67 b 3 2.03 a 6.03 b 6.9 b 6.6 b 9.89 c Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 2. The peroxide value for 0 1, 2 and 3 months stored PL treated with skin peanuts at 7 cm distance from the strobe Time (m onth ) C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate 0 1.1 a 1.63 a 1.91 a,b 1.76 a 2.17 a,b 1 1.13 a 1.23 a 2.17 a,b 1.5 a 2.77 a,b 2 2 a,b 2.86 a,b 3.4 a,b 3.26 a,b 4.66 b 3 2.03 a,b 4.73 b 6.67 b ,c 6.67 b,c 7.7 c Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 3. The peroxide value for 0 1 2 and 3 months stored PL treated without skin peanuts at 5 cm distance from the strobe Time (month) C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate 0 2.8 a 2.67 a 2.96 a 2.83 a 2.77 a 1 3.03 a 2.57 a 2.23 a 3.1 a,b 3.1 a 2 3.05 a 6.78 a 8.53 a ,b 5.33 a,b 8.86 a,b 3 4.13 a 8.13 a,b 10.9 a,b 7.6 a,b 12.89 b Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 4. The peroxide value for 0 1, 2 and 3 months stored PL treated without skin peanuts at 7 cm distance from the strobe Time (month) C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate 0 2.8 a 2.67 a 2. 96 a 2.83 a 2.77 a 1 2.13 a 2.23 a 2.97 a 1.5 a 3.77 a,b 2 3 a 4.86 a,b 5.4 b 3.26 a,b 6.66 b,c 3 3.13 a 6.73 b,c 7.67 b,c 7.69 b,c 9.74 c Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 5. The FFA % for 0 1, 2 and 3 mont hs stored PL treated with skin peanuts at 5 cm distance from the strobe Storage time C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate Month 0 0.45a 0.43a 0.51a 0.45a 0.46a Month 1 0.54a, a 0.57a 0 .57a 0.56a 0.53a Month 2 0.69 a 0.65 a 0.7 8 b 0.61 a 0.78 b Month 3 0.61 a 1 b 1.34 c 2.01d 2.87 e Different alphabetic means significantly different at = 0.05 using one way ANOVA

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115 Table 6 6. The FFA % for 0 1, 2 and 3 months stored PL treated with skin peanuts at 7 cm distance from the strobe Storage time C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate Month 0 0.43a 0.43 a 0.51 a 0 .49 a 0.49 a Month 1 0.58 a 0.59 a 0.63 a 0.6 a 0.61 a Month 2 0.59 a 0.73 b 0.78 b 0.69 a 0 .93b Month 3 0.6 a 1.05 c 1.17 c 2.23 d 2 d Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 7. The FFA % for 0 1, 2 and 3 mo nths stored PL treated w/o skin peanuts at 5 cm distance from the strobe Storage time C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate Month 0 0.49a 0.54 a 0.59 a 0.59a 0.7 a Month 1 0.53a 0.75 a 0.76 a 1.5 a,b 1 .77 b ,c Month 2 0.64 a 0.56 a 0 .78 a 1.04 a ,b 2.02 c Month 3 1.02 a,b 1.68 c 1.98 c 2 c 3.26 d Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 8. The FFA % for 0 1, 2 and 3 months stored PL treated w/o skin peanuts at 7 cm distance from the strobe Storage time C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate Month 0 0.44 a 0.54 a 0.65 a 0. 65 a 0.59 a Month 1 0.51 a 0.54 a 0.72 a 0.63 a 0.79 a Month 2 0.56 a 0.59 a 2.53 c 0.65 a 1.94 b Month 3 0.60 a 1.58 b 1.88 b 1.87 b 2.45 c Different alphabetic means significantly different at = 0.05 using one way ANOVA T able 6 9. The acid value results for 0 1 2 and 3 months stored PL treated with skin peanuts at 5 cm distance from the strobe Storage time C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate Month 0 0.90 a 0.86 a 1.02 a 0.90 a 0.92 a Month 1 1.08 a 1 .13 a 1.13a 1.11a 1.06a Month 2 1. 37a 1 .29 a 1.55b 1.21a 1.55b Month 3 1.21a 1.99 b 2.67c 4.00 d 5.71e Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 10. The acid value results for 0 1, 2 and 3 months stored PL treated with skin peanuts at 7 cm distance from the strobe Storage time C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate Month 0 0.86 a 0 .86 a 1.02 a 0.98 a 0.98 a Month 1 1.15 a 1.17 a 1.25 a 1.19a 1.21 a Month 2 1.17 a 1.45a,b 1.55a,b 1.37a 1.85 b Month 3 1.19 a 2.09 b 2.33 b 4.44c 3.98 d Different alphabetic means significantly different at = 0.05 using one way ANOVA

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116 Table 6 11. The acid val ue results for 0 1, 2 and 3 months stored PL treated w/o skin peanuts at 5 cm distance from the strobe Storage time C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate Month 0 0.98 a 1.08 a 1.17 a 1.17 a 1.39 a Month 1 1.06 a 1.49 a 1.51 a 2 .99 b 3.5 2 c,d Month 2 1.27 a 1.11 a 1.55 a,b 2.07b 4.02 d Month 3 2.03 a,b 3.34 c 3.94 d 3.98 d 6.49 e Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 12. The acid value results for 0 1, 2 and 3 months stored PL treated w/o skin peanuts at 7 cm distance from the strobe Storage time C ontrol 240 s shaking 240 s in plate 300 s shaking 300 s in plate Month 0 0.88 a 1. 08 a 1.29 a 1.29 a 1.17 a Month 1 1.05 a 1.08 a 1.43 a 1.25 a 1.57 a Month 2 1.11 a 1.17 a 7.02 d 1.29 a 3.86 b Month 3 1.19 a 3.14 b 3.74 b 3.72 b 4.88 c Different alphabetic means significantly different at = 0.05 using one way ANOVA Table 6 13. L*, a*, b* values f or color evaluation of c ommercial peanuts roasted peanut, control (untreated peanut), and four di fferent times PL treated peanut samples. Commercial 0 s 180 s 210 s 240 s L* value 70.86 6.21a 74.33 4.49 a 73.85 4.76 a 75.23 3.96 a 74.1 4.76 a 73.49 6.38a 76.38 3.93 a 75 4.53 a 75.49 4.95 a 76.79 4.22 a 71.87 5.65a 24.5 6.22 b 75.11 4.93 a 18.56 5.31 b 75.12 4.6 a 18.5 4.38b 76.31 3.85 a 19.6 4.49b 74.39 4.72a 19.94 4.5b b* value 25.95 6.79 b 20.21 4.22 b 19.79 4.32b 20.98 5.57b 19.2 3.99b 25.21 5.88 b 19.01 4.02b 19.07 4.1b 17.88 3.77b 19.74 3.83b a* value 1.61 3.84 c 5.53 1.66 d 5.07 2.04 d 4.87 1.9 d 4.05 2.37 d 1.32 4.02 c 5.37 1.53 d 4.66 2.03 d 3.52 2.8 c, d 4.35 1.77 d 1.33 3.39 c 5.44 2.28 d 4.85 1.89 d 4.88 1.62 d 3.61 2.36 d Average of three samples within the same sample. Different alphabetic means significantly different at = 0.05 using one way ANOVA

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117 Table 6 14. Hue angle, Chroma, and whiteness values for color evaluation of c ommercial roasted peanut, control ( un trea ted peanut), and PL treated peanuts Commercial 0 s 180 s 210 s 240 s Hue Angle 73.5 145 a 139 39.9 b 134 59.3 b 142 47.8 b 127 87.6 b 63.3 151 a 144 37 b 138 62.9 b 113 107 b 141 57.4 b 66.5 151 a 136 52.7 b 141 48.5 b 142 41.4 b 134 80.8 b Chroma 24.9 6.07 a 19.5 4.95 a 19.4 3.96 a 20.4 4.05 a 20.6 4.1 3 a 26.3 6.66 a 21.0 3.9 a 20.5 3.94 a 21.6 4.18 a 19.8 3.63 a 25.5 5.69 a 20 .0 3.61 a 19.8 3.75 a 18.7 3.42 a 20.3 3.56 a 72.7 5.14a 76.0 5.43a 75.4 3.59a 75.0 6.49a 74.6 5.67a Average of three samples within the same sample Different alphabetic means significantly different at = 0.05 using one way ANOVA

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118 Figure 6 1 The color v ision machine images for peanuts samples A) Control ( untreated peanuts), B) Publix Commercial roasted peanuts C) 180 s per 7 cm PL treatment D) 240 s per 7 cm PL treatment E) 210 s per 7 cm PL treatment ( Photo courtesy of author)

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119 CHAPTER 7 OVERALL CONCLUSION Pulsed light ( PL ) has been considered a non thermal technique and has been used successfully to eliminate bacteria and viruses usin g very short expos ure times. Short duration of PL treatment di temperature. Howeve r, in this study since the AFT s are extremely stable compounds, especially in solid food such as peanuts, relatively long exposing times were used to reach a sufficient level of AFT degradation PL treatment achieved complete AFT reduction when treated in solvent. Overall, this study suggested that there is a good potential to optimize peanut quality by simply adjusting the time/temperature profiles during PL treatment Treatment s 240 s at 5 cm or 7 cm almost achieved complete AFT detoxification Among the five different treatments which were investigated in this study the s haking treatment was the best treatment in terms of uniformity of the treatment which led to higher AFB 1 AFB 2 reduction a long with homogeneous color distribution PL irradiation is a successful technique in terms of AFT degradation. However, all other parameters especially the moisture content have a robust influence on AFT degradation. With PL technique samples should be placed accurately right under the quartz window of the Xenon lamp. The sensitivity of the without skin samples to fungi contamination, PL treatment, and experiment parameter s changing was higher than the with skin samples. The hurdle te chnique by adding citric acid treatment enhanced the PL treatment and the AFTs were dramatically reduced in most treatments especially after 240 s of PL treatment. Peroxide value FFA and acid value for PL treated samples revealed that PL treatment didn have a detrimental effect on peanut quality even after 3 months of

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120 storage However, UV light has been known for its ability to accelerate the lipid oxidation ; t hus, the low peroxide value could be due to the fast production of the final product of the unsaturated fatty acids oxidation. Therefore, f or future work, gas chromatography analysis should be conducted to profile the secondary oxidation products originating from the oxidized lipids

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121 A PP ENDIX A INOCULATION OF WITHOUT SKIN AND WITH SKIN PEANUTS WITH ASPERGILLUS FLAVUS A. flavus strains should be prepared by growing on dextrose agar for 5 7 d with After incubation at 30 C, spores will be harvested then filtered using cheesecloth, then counted using Hemocytometer. Aflatoxin extraction CH 3 OH/H 2 O Aflatoxin determination by ELISA and HPLC MS/MS 500 g of peanuts will be placed in Erlenmeyer flasks (150 mL), 50 mL of distilled water will be added and autoclaved at 121 C f or 1 h and will be allowed to stand overnight. Each flask will be inoculated with 25 mL of the spore suspension, incubated at 29 1 C in a dark place for 21 d and shaken once or twice daily.

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122 A PP ENDIX B PULSED LIGHT TREATMENT Pulsed light treatment for 30, 60, 90, 120, 180, 210, 240 ,300 s and different distances 5, 7, and 10 cm. Temperature meas urement Without skin and with graded levels of aflatoxins. ELISA HPLC MS/MS Aflatoxin extracti on CH 3 OH/H 2 O Control

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123 A PPENDIX C C OMBINE PULSED LIGHT WITH CITRIC ACID AS A HURDLE TECHNIQUE AND COMPARE THE RESULTS WITH THE P L TREATMENT ELISA Result comparison for objectives two and three: Statistical analysis. HPLC MS/MS Aflatoxin extraction: CH 3 OH/H 2 O Without skin and with with different levels of af latoxins. Pulsed UV light treatment for 210 and 240 ,300 s shaking treatment and at 7 cm. The previously contaminated peanuts will be treated with 0.1 N aqueous citric acid for 1 h (2 mL per gram of contaminated peanuts).

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124 LIST OF REFERENCES Agriopoulou, S., K oliadima, A., Karaiskakis, G., Kapolos, J. (2016). Kinetic study of aflatoxins' degradation in the presence of ozone. Food Control, 61, 221 226. Aibara, K., Yamagishi, S. (1970). Effect of ultraviolet irradiation on the destruction of aflatoxin B 1 In Proceedings of the first US Japan conference on toxic microorganisms. (pp. 211 221). US Department of the Interior, Washington, DC. Akbas, M. Y., Ozdemir, M. (2006). Effec t of different ozone treatments on aflatoxin degradation and physicochemical properties of pistachios. Journal of the Science of Food and Agriculture, 86(13), 2099 2104. Akhtar, S., Khali d, N., Ahmed, I., Shahzad, A., Suleria, H. A. R. (2014). Physicochemi cal characteristics, functional properties, and nutritional benefits of peanut oil: a review. Critical Reviews in F o od Science and N utrition, 54(12), 1562 1575. Ale xandre, E. M., Brando, T. R., Silva, C. L. (2012). Emerging technologies to improve the saf ety and quality of f ruits and vegetables. In Novel Technologies in Food S cience (pp. 261 297). Springer New York. Alla meh, A., Safamehr, A., Mirhadi, S. A., Shiva zad, M., Razzaghi Abyaneh, M., Afshar Naderi, A. (2005). Evaluation of biochemical and produ ction parameters of broiler chicks fed ammonia treated aflatoxin con taminated maize grains. Animal Feed S cience an d T echnology, 122(3), 289 301. Altug, T. O. M. R. I. S., Yousef, A. E., Marth, E. H. (1990). Degradation of aflatoxin B1 in dried figs by sodi um bisulfite with or without heat, ultraviolet energy or hydrogen peroxide. Journal of Food Protection 53(7), 581 582. Atalla, M. M., Has sanein, N. M., El Beih, A. A., Youssef, Y. A. (2004). Effect of fluorescent and UV light on mycotoxin production under different relative humidities in wheat grains. International Journal of Agriculture and Biology 6 (6), 1006. American Peanut Council (201 7 ). The Pea nut Industry Peanuts: a brief histor y AOAC. (1990). Official methods of analysis. 15 th edition. Association of Official Analytical Chemists, AOAC Pub., Virginia, U.S.A. pp.69 84, 951 979. AOAC. (1995). Official Methods of Analysis, 16th Edition. Assoc iation of Official Analytical Chemists, Washington, DC. Baker, G. L., Cornell, J. A., Gorbet, D. W., O'keefe, S. F., Sim s, C. A., Talcott, S. T. (2003). Determination of pyrazine and flavor variations in peanut genotypes during roasting. Journal of Food S cience 68 (1), 394 400.

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125 Basaran, P. (2009). Reduction of Aspergillus parasiticus on hazelnut surface by UV C treatment. International Journal of Food Science and T echnology, 44(9), 1857 1863. Battacone, G., Nudda, A., Cannas, A ., Borlino, A. C., Bomboi, G. Pulina, G. (2003). Excretion of aflatoxin M 1 in milk of dairy ewes treated with different doses of aflat oxin B 1 Journal of Dairy S cience, 86(8), 2667 2675. Beasley, R. P., Lin, C. C., Hwang, L. Y., Chien, C. S. (1981). Hepatocellular carcinoma and hepat itis B virus: a prospective study of 22 707 men in Taiwan. The Lancet, 318(8256), 1129 1133. Teixeira, P. (2016). Food Safety Aspects Concerning Traditional Foods. In Emerging and Traditional Technologies for Safe, Healthy and Quality Food (pp. 33 54). Bennett, J. W., Klich, M. A. (1999). Aspergillus Encyclopedia of Bioprocess Technology. Bennett, J., Klich, M. (2003). Chotoxins Clinical Microbiology Reviews 16, 497 516. Beuchat, L. R. (Ed.). (1987). Food and Beverage Mycology Springer Science and Business Media Beyer, K. Morrowa, E., Li, X. M., Bardina, L. Bannon, G. A., Burks, A. W., Sampson, H. A. (2001). Effects of cooking methods on peanut allergenicity. Journal of Allergy and Clinical Immunology, 107(6), 1077 1081. Bocci, V. (2010). Physical Chemical Properties of Ozone Natural Production of Ozone: The Toxicology of Ozone. In Ozone (pp. 1 4). Springer Netherlands. Braddock, J. C., Sims, C. A., O'keefe, S. F. (1995). Flavor and oxidative stability of roasted high oleic acid peanuts. Journal of Food Science, 60(3), 4 89 493. Camargo, A. C., Vieira, T. M. F. D. S., Regitano M., Calori Domingues, M. A., Canniatti Brazaca, S. G. (2012). Gamma radiation induced oxidation and tocopherols decrease in in shell, peeled and blanched peanuts. Int ernational Journal of Molecular Sciences 13(3), 2827 2845. Caminiti, I. M., Palgan, I., Muoz, A., Noci, F., Whyte, P., Morgan, D. J ., Cronin, D. A., Lyng, J. G. (2012). The effect of ultraviolet light on microbial inactivation and quality attributes of a pple juice. Food and Bioprocess Technology 5 (2), 680 686. Cmmerer, B., Kroh, L. W. (2009). Shelf life of linseeds and peanuts in relation to roasting. LWT Food Science and Technology, 42(2), 545 549.

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138 BIOGRAPHICAL SKETCH Manal Othman Abuagela was born in Tripoli Libya. She received S cience, Colle ge of Agriculture, University of Tripoli (Al Fateh University) in 1993 and 2006 respectively. After her degree she was a teacher for different school levels from elementary to high school. Generally teaching Chemistry S he s tarted her s stud ies at the University of Tripoli in 2000. After her master degree she worked as an assistant professor at the U niversity of So u k Al A had. Al Zay to o na h University ( Nasser ) s ince 2006 2 013. She started her Ph.D. studies at Department of Food Science and Human Nutrition at the University of Florid a in 2013 under Dr. Wade Yang. She t hen transferred to be under Dr. Paul Sarnoski.