Effect of pulsed light on allergenic proteins of shelled whole peanuts


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Effect of pulsed light on allergenic proteins of shelled whole peanuts
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1 online resource (162 p.)
Zhao, Xingyu
University of Florida
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Gainesville, Fla.
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Doctorate ( Ph.D.)
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University of Florida
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Food Science and Human Nutrition
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Subjects / Keywords:
allergens -- ige -- immunoreactivity -- peanut -- pl -- quality
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Food Science and Human Nutrition thesis, Ph.D.
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Most peanuts are consumed in dry roasted form in the U.S., but roasting is known as increasing peanut allergenic reaction. In recent years, many processing technologies are applied to inactive allergens in peanut seeds, such as high hydrostatic pressure (HHP), pulsed electric field (PEF), Gamma-irradiation, and pulsed light (PL). This study focused on the PL since it had been shown to reduce allergen levels on peanut, soybean, almond and shrimp protein extracts as a novel technology. This study has explored if this technology was validated for hypoallergenic peanuts production, which had similar quality as conventional roasted peanuts. The initial data was proved by sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAGE), western blot, indirect enzyme-linked immunosorbent assay (ELISA). The results indicated that soluble allergens were notably mitigated by PL technology with pooled human plasma. To have a deeper understand of this technology, insoluble fractions that were formed during the PL processing process caused by maillard reaction have been investigate by Novex gel system, which is specially designed for large protein molecures. The results have pointed at PL was effective on mitigation of the insoluble protein allergenic reaction. To optimize the major two parameters of PL equipment, time and distance were examined by factorial analysis and multi-analysis of variance (ANOVA) with color values and indirect ELISA readings. The interaction between these two factors was significant (p < 0.05) and 12 min treatment at 10 cm distance was the best treatment due to proper color and texture development and low immunoreactivity readings. In vitro digestion study was conducted after this optimization by using pepsin, trypsin, and a-chymotrypsin. The mimic digestion fluids were prepared for this digestion study with proper pH value, gastric digestion at pH 2.0, intestinal digestion at pH 8.3. The results revealed that peanut allergens Ara h 1- Ara h 11 were completely removed in this study. The quality test was conducted in the last chapter to evaluate the nutrient, texture, color changes after PL illumination. This part has provided evidence that PL could produce similar texture and color with increased antioxidant capacities in processed peanuts.
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by Xingyu Zhao.
Thesis (Ph.D.)--University of Florida, 2013.
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INGEST IEID EKQXTY127_TR2F4L INGEST_TIME 2014-05-13T21:28:51Z PACKAGE UFE0046099_00001




2 2013 Xingyu Zhao


3 To my husban d Jiaqing Zhou who supports me the most


4 ACKNOWLEDGMENTS I would like to express my gratitude to my major advisor Dr. Wade Yang for providing me the opportunity to study in his lab. Without his guidance and support this work would not have be en completed. I would also like to extend my gratitude to my committee members, Drs. Charles Sims, Steven Otw ell, and Steven Bruner for their guidance and support. I am truly appreciative of Dr. Susan Percival for providing s pectrophotometer and Dr. Jesse Gregory for pro viding nitrogen gas and direction for my research. Furthermore, I would like to deeply thank Dr s .Yagiz Yavuz and Ma urice Marshall for providing lab resources and support with the lipid oxidation portion of my dissertation. My labmates San dra Shriver, Chelsey li, Abbas S yed, Senem Guner, Bhaskar Janve ga ve me a lot s of assistance during my study, and I will forever be in their debt. Finally, I would like to express my heart felt gratitude to my parents and my husband. My parents provided me with the best condition s for my education and constant support throughout my life. My husband Jiaqin g Zhou has been my greatest supporter, filling my life with immense happiness. The unconditional assistance he provided while earning this degree is unmeasurable. Without his encouragement and love, I coul d not finish my doctoral studies and sta rt this new chapter in my life.


5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIA TIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 17 Justification ................................ ................................ ................................ ............. 18 Objective ................................ ................................ ................................ ................. 19 2 LITERATURE REVIEW ................................ ................................ .......................... 21 Current Situation of Food Allergy ................................ ................................ ............ 21 Food Allergy and Anaphylaxis ................................ ................................ ................ 21 The Mechanism of Interaction between Antigens and Antibodies ........................... 23 History of Peanuts ................................ ................................ ................................ .. 26 The Nutrients and Health Benefits of Peanut ................................ .......................... 28 Peanut Allergy ................................ ................................ ................................ ........ 30 Peanut Allergens ................................ ................................ ................................ .... 31 Processing Methods for Peanut Allergen Reduction ................................ ............... 37 Effect of Thermal Treatment on Peanut Allergens ................................ ............ 37 Thermal processing ................................ ................................ ................... 37 Roasting ................................ ................................ ................................ ..... 39 Boiling ................................ ................................ ................................ ........ 41 Frying ................................ ................................ ................................ ......... 42 Autoclaving ................................ ................................ ................................ 43 Non Thermal Treatment on Peanut Allergens ................................ .................. 44 Pulsed light ................................ ................................ ................................ 44 High pressure microf luidisation ................................ ................................ .. 46 irradiation ................................ ................................ ................................ 47 Pulsed electric field ................................ ................................ .................... 48 Chemical and biological methods ................................ .............................. 49 3 INVESTIGATION OF THE EFFECTIVENESS OF PULSED LIGHT ON PEANUT ALLERGENS ................................ ................................ ........................... 53 Introduction ................................ ................................ ................................ ............. 53


6 Material and Methods ................................ ................................ ............................. 54 Materials ................................ ................................ ................................ ........... 54 Pulsed Light Treatment of Peanut Kernels ................................ ....................... 55 Preparation of Peanut Extract after PL Treatment ................................ ............ 56 SDS PGAE of Peanut Extracts ................................ ................................ ......... 57 Western Blot of Peanut Extracts ................................ ................................ ....... 57 Dot Blot of Peanut Extracts ................................ ................................ .............. 58 Indirect ELISA of Peanut Extracts ................................ ................................ .... 59 Results and Discussion ................................ ................................ ........................... 59 SDS PAGE of PL Treated Peanut Kernels ................................ ....................... 59 Western Blot of PL Treated Peanut Kernels ................................ ..................... 62 Dot Blot of PL Treated Peanut Kernels ................................ ............................ 63 Indirect ELISA ................................ ................................ ................................ .. 64 Influence of PL Duration and Distance on IgE Binding Reactivity to W hole Peanut Allergens ................................ ................................ ........................... 65 Summary and Conclusions ................................ ................................ ..................... 66 4 EXAMINATION OF IGE IMMUNOREACTIVITY OF INSOLUBLE FRACTIONS OF PULSED LIGHT TREATED PEANUTS BY NOVEX NUPAGE ELECTROPHORESIS SYSTEM ................................ ................................ ............. 71 Introduction ................................ ................................ ................................ ............. 71 Materials and Methods ................................ ................................ ............................ 72 Sample Pre paration ................................ ................................ .......................... 72 Protein Extraction ................................ ................................ ............................. 73 Denaturing Electrophoresis ................................ ................................ .............. 73 Western Blot after Denaturing Electrophoresis ................................ ................. 74 Non Denaturing Electrophoresis ................................ ................................ ...... 75 Western Blot after Non Denaturing Electrophoresis ................................ ......... 75 Indirect ELISA ................................ ................................ ................................ .. 76 Results and Discussion ................................ ................................ ........................... 76 Denaturing Electrophoresis ................................ ................................ .............. 76 Western Blot ................................ ................................ ................................ ..... 77 Non Denaturing System ................................ ................................ ................... 82 SDS PAGE ................................ ................................ ................................ ....... 82 Western Blot ................................ ................................ ................................ ..... 83 Indirect ELISA ................................ ................................ ................................ .. 83 5 THE OPTIMIZATION OF PL EQUIPMENT AND THE INTERACTION EFFECT OF MAJOR PARAMETERS: TIME AND DISTANCE ................................ ............. 90 Introduction ................................ ................................ ................................ ............. 90 Material and Methods ................................ ................................ ............................. 91 Color Analysis ................................ ................................ ................................ .. 91 Indirect ELISA ................................ ................................ ................................ .. 92 Experimental Desig n ................................ ................................ ........................ 92 Results and Discussion ................................ ................................ ........................... 93


7 Motor Design ................................ ................................ ................................ .... 93 Factorial Analysis ................................ ................................ ............................. 94 Color analysis ................................ ................................ ............................ 94 Indirect ELISA ................................ ................................ ............................ 95 Multivariate ANOVA Analysis ................................ ................................ ........... 97 Color analysis ................................ ................................ ............................ 97 Indirect ELISA ana lysis ................................ ................................ .............. 97 Summary and Conclusions ................................ ................................ ..................... 98 6 IN VITRO SIMULATED GASTRIC AND INTESTINAL DIGESTIONS OF PL PROCESSED PEANUTS ................................ ................................ ..................... 104 Introduction ................................ ................................ ................................ ........... 104 Material and Methods ................................ ................................ ........................... 105 Simulated Peptic Digestion ................................ ................................ ............. 105 Simulated Intestinal Digestion ................................ ................................ ........ 106 Results and Discussion ................................ ................................ ......................... 107 Simulated Pepsin Digestion ................................ ................................ ............ 107 Raw peanut ................................ ................................ .............................. 107 Roasted peanut ................................ ................................ ........................ 108 Pulsed light sam ple preparation ................................ ............................... 109 Chymotrypsin Digestion ................................ .......... 110 Raw and roasted peanut ................................ ................................ .......... 110 Western blot ................................ ................................ ............................. 110 Pulsed light treat ed peanut ................................ ................................ ...... 111 Discussion ................................ ................................ ................................ ...... 112 Summary and conclusions ................................ ................................ .................... 115 7 EXAMINATION OF QUALITY CHANGES (TEXTURE, COLOR, ANTIOXIDANT CAPACITY, LIPID OXIDATION) OF THE WHOLE PEANUTS BEFORE AND AFTER PL TREATMENT ................................ ................................ ...................... 122 Material and Method ................................ ................................ ............................. 123 Texture Analysis ................................ ................................ ............................. 123 Color Analysis ................................ ................................ ................................ 124 Total Antioxidants Capacity Evaluation ................................ .......................... 125 Extraction of total antioxidants ................................ ................................ 125 Hydrophilic oxygen radical absorbance capacity (H ORAC) assay ......... 126 Lipophilic oxygen radical absorbance capacity (L ORAC) assay ............. 127 Lipid Oxidation ................................ ................................ ................................ 127 Peroxide value ................................ ................................ ......................... 128 Thiobarbituric a cid reactive substances (TBARS) assay ......................... 128 Results and Discussion ................................ ................................ ......................... 129 Texture Analysis ................................ ................................ ............................. 129 Temperature and Moisture Changes During PL Illumination .......................... 130 Color Analysis ................................ ................................ ................................ 132 Antioxidant Capa city ................................ ................................ ....................... 134


8 Lipid Oxidation ................................ ................................ ................................ 135 8 OVERALL CONCLUSIONS ................................ ................................ .................. 143 LIST OF RE FERENCES ................................ ................................ ............................. 146 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 162


9 LIST OF TABLES Table page 5 1 F test of distance and time interaction based on the L value .............................. 99 5 2 Tukey HSD results describing 5 min, 6 min, and 7min treatment groups of L value ................................ ................................ ................................ ................... 99 5 3 HSD of interactions between combination groups of L value ............................. 99 5 4 F test out put of indirect ELISA readings ................................ ............................. 99 5 5 F test of distance and time factors based on Indirect ELISA readings ............... 99 5 6 Means separated by treatment time: 5min, 6min, and 7min of Indirect ELISA readings ................................ ................................ ................................ ............ 100 5 7 Tukey HSD of interactions between combination groups (p <0.05). ................. 100 5 8 F test of distance and time interaction based on the color readings (p<0.05). 100 5 9 Means separated by treatment time of color analysis L value (p<0.05) ............ 100 5 10 F test of time and distance interaction based on Indirect E LISA (p<0.05). ....... 100 5 11 Means separated by Tukey HSD of indirect ELISA readings. .......................... 101 7 1 Texture comparison between the dry roasted and PL treated peanuts ............ 138 7 2 Surface temperature and moisture loss of dry roasted and PL treated peanuts. ................................ ................................ ................................ ............ 138 7 3 Comparison of color between dry roasted and PL treated peanuts. ................. 139


10 LIST OF FIGURES Figure page 2 1 Thermal effect on the protein structure changes. ................................ ............... 52 3 1 SDS PAGE profiles of raw and PL treated peanut extracts at diff erent durations of illumination ................................ ................................ ..................... 67 3 2 Antigenic protein bandin g patterns in raw and PL treated peanuts via western blot in vitro an alysis with pooled human plasma ................................ ................. 68 3 3 Dot blot analysis of raw (control) and PL treated peanuts ................................ .. 69 3 4 Indirect ELISA of PL treated peanuts at 10 cm and 7 cm distance to lamp with pooled human plasma containing IgE at different illumination durations ..... 70 4 1 Protein profiles of insoluble fractions of PL processed peanuts we re analyzed by Novex denaturing electrophoresis. ................................ ................................ 85 4 2 Western blot of insoluble fractions of PL processed peanuts samples were c ompared to roasted peanut ................................ ................................ ............... 86 4 3 Protein profiles of insoluble fractions of PL processed peanuts were analyzed b y Novex non denat uring electrophoresis ................................ ......................... 87 4 4 Insoluble antigenic protein fractions were analyzed by western blot on non denaturing gel ................................ ................................ ................................ ..... 88 4 5 Effects of PL treatment on total protein of peanut kernels with different duration at 10cm were determined by Indirect ELISA with pooled human plasma containing IgE antibodies ................................ ................................ ....... 89 5 1 A model and a picture of a motor designed for uniformly roasting for PL treatment. ................................ ................................ ................................ ......... 102 5 2 Factorial analysis: Tukey HSD of interactions between combination groups of color analysis values (p <0.05). ................................ ................................ ........ 103 5 3 Factorial analysis: Tukey HSD of interactions between combination groups of Indirect ELISA readings (p <0.05). ................................ ................................ ... 103 6 1 Protein profiles and Western blot of pep sin digested raw peanut sample s ....... 117 6 2 Pepsin digested roasted peanuts were determined by SDS PAGE and Western blot ................................ ................................ ................................ ..... 118 6 3 Pepsin dig estion of PL treated peanut at 12 min ................................ .............. 119


11 6 4 SDS PAGE and Western blot of mimic intestinal digestion of raw and roasted peanuts ................................ ................................ ................................ ............. 120 6 5 SDS PAGE and Western blot of mimic intestinal digestion of PL treated peanut at 12 min ................................ ................................ ............................... 121 7 1 H ORAC of PL treated peanuts. Roasted and raw peanuts were considered as controls. Tukey HSD was applied to analyze the means separation. .......... 140 7 2 L ORAC of PL treated peanuts. Roasted and raw peanuts were considered as controls. Tukey HSD was applied to analyze the means separation. .......... 141 7 3 Peroxide value of raw peanut, PL treated peanuts at 9 min, 12 min, 15 min on 0 day, 5 days, and 10 days shelf life at room temperature .......................... 142 7 4 TBARS of raw peanut, PL treated peanuts at 9 min, 12 min, 15 min on 0 day, 5 days, and 10 days shelf life at room temperature ................................ .......... 142


12 LIST OF ABBREVIATION S Atmosphere Atm AAPH 2, 20 azobis (2 amidino propane) dihydrochloride AGE S Advanced glycation end products ANOVA Analysis of variance A RA H Arachis hypogaea BCA Bicinochoninic protein assay BSA Bovine serum albumin DPPH 1, 1, diphenyl, 2 picrylhydrazyl DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol tetraacetic acid EIA Enzyme immunoassay ELISA Enzyme linked immunosorbent assay EM Electromagnetic FIR Far infrared HHP High hydrostatic pressure H ORAC HPLC Hydrophilic oxygen radical absorbance capacity High performance liquid chromatography HRP Horseradish peroxidase HSD Honestly significant difference IgE Immunoglobulin E IgG Immunoglobulin G IR Infrared JMP


13 L ORAC Lipophilic oxygen radical absorbance capacity LSD Least significant difference MIR Mid infrared MRPs Maillard reaction products NIR NMR Near infrared Nuclear magnetic resonance OPD O phenylenediamine dihydrochloride ORAC Oxygen radical scavenging activity PBS Phosphate buffered saline PEF Pulsed electric field POD Peroxidase PL Pulsed light PVDF Polyvinylidene Difluoride RMCD Randomly methylated beta cyclodextrin RNA Ribonucleic acid RT Room temperature SAS Statistical analysis system SGF Simulated gastric fluid SIF Simulated intestinal fluid SDS PAGE Sodium dodecyl sulfate polyacrylamide electrophoresis TA Texture analyzer TBARS Thiobarbituric acid reactive substances TBS Tris buffered saline TBST 1X TBS with 0.05% Tween 20 TE Trolox equivalent


14 UV Ultraviolet radiation WHO World health organization UNEP United nations environment program


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 EFFECT OF PULSED LIGHT ON ALLERGENIC PROTEINS OF SHELLED WHOLE PEANUTS By Xingyu Zhao December 2013 Chair: Wade Yang Major: Food Science and Human Nutrition Most peanuts are consumed i n the dry roasted form in the United States However, roasting has been affiliated with an increasing peanut allergenic reaction. In recent years, many processing technologies were applied to inactivate allergens in peanut seeds, which include the following: high hydrostatic pressure (HHP), pulsed irradiation, and pulsed light (PL). This study focused on PL since it has been shown to reduce allergen levels in peanut, soybean, almond and shrimp protein extracts During this research project, this technology was used to identify a hypoallergenic peanut production method th at retained similar qualities as conventional ly roasted peanuts. The data was validated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), western blot, and indirect enzyme linked immunosorbent assay (ELISA). The results indicated tha t soluble allergens were notably reduce d b y PL technology in pooled human plasma. I nsoluble fractions formed during the PL process as a result of the Maillard reaction were analyzed by the Novex gel system which is specially designed for large protein molecules. The results have illustrated that PL was ef fective in the reduction of all insolu ble allergenic protein s To optimize the two major parameters, time and distance the difference in the means were


16 separated b y factorial analysis and multi analysis of vari ance (ANOVA) based on effects of color values and indirect ELISA readings. The interaction between these two factors was significant ( p < 0.05) The best treatme nt, determined by proper color/ texture developme nt, and low immunoreactivity was a 12 min treatment at a 10 cm distance An i n vitro chymotrypsin. To mimic digestion, fluids were prepared with the appropriate pH value for gastri c digestion at pH 2.0 and intestinal digestion at pH 8.3. The results revealed that peanut allergens Ara h 1 thru 11 were com pletely removed in this study via pulsed light The quality test s performed after PL illumination has proven that PL can produce si milar texture and color as compared to conventional ly roasted peanuts The hydrophilic and lipophillic antioxidant capacities were significantly high er in 12 min PL treated peanut s than in the raw peanuts (p < 0.05).


17 CHAPTER 1 INTRODUCTION Peanut allergies are among the most common food allergies affecting children and adults. It is estimate d to affect between 0.5% and 1.1% of adult s in Western and European countries and is the third most common allergy in children with 4% affection ( Sicherer and others 2003 ) Most peanuts are consumed in a dry roasted form in the United States which is known to trigger and increas e peanut allergenic reaction s ( Maleki and others 2000a ; Poms and others 2004 ) If peanut allergens can be masked during roasting, the peanut allergy problems can be alleviated for peanut allergy patients In recent years, s everal technologies such as HHP PEF and PL have been developed that show potential s for reducing food allergens ( Johnson and others 2010 ; Li and others 2013 ) The focus of this study is on the effect on allergen reduction of PL treated peanut kernels Previous studies have demonstrated that PL can reduce allergen levels in shrimp extract, soybean extract, and peanut extract ( Chung and others 2008 ; Yang and others 2010b ; Shriver and others 2011 ) It was shown that PL at prolonged exposure s ranging from 6 min to 9.5 min has an effect on protein solubility reduction To further validate this technology, the optimization of PL applicator was conducted and In vitro tests, such as Western Blot, ELISA, simulated gastric and intesti nal digestion with pooled human serum were conducted and validated for the IgE reactivity reduction to allergenic proteins in the P L treated peanuts. A recent study by Yang and others ( 2012 ) elucidated the possible mechanism of this technology on protein changes after treatment. The putative mechanism of this novel technology was due to the photothermal, photochemical, and photophys ical


18 effects ( Shriver and others 2011 ; Yang and others 2012 ) It is suggested that proteins are modified during the illumination of PL, leading to protein cross linkage, protein solubility reduction, and protein hydrolysis at the high instantaneous temperature environment ( Shri ver and others 2011 ; Li and others 2013 ) As a result, PL has gained more attention bec ause of the effects of protein denaturation and epitope masking. The overall goal of this study is to explore the viability of PL technology for reducing immunogenic potential of whole peanut products that ha ve a similar quality to traditional ly roasted pe anuts. If successfully conducted, this study is of significant importan ce because the data can provide a base for further clinical trail s and the quality evaluation of the final products that will have a substantially low immunogenic reactivity This stud y also provides a possib le alternative for approaching the peanut allergy problem for peanut allergy patients without using immuno intervention or other chemical interruption, such as epinephrine ( Turk and others 1983 ; Achaorbea and others 1988 ) So far, PL reduc tion of major peanut allergen s has not been full y explored This study will reve al more about this technology on the allergenic protein changes and even on the changes of those treated peanut proteins aft er gastric and intestinal digestions T he results could suggest for changes in food processing techniques, especially in the peanut industry and pave the way leading towards producing hypoallergenic peanut and peanut derived products. Justification Severa l studies have shown that the prevalence of peanut allergy both in the United States and Canada has increased from 1997 to 2007 ( Finkelman 2010 ) One study showed that 1.4% of subjects in the United States reported they have peanut or tree nut allergy in a random telephone surv ey ( Sicherer and others 2010b ) This


19 percentage has not increased for the adults, but increased for children less than 18 years old from 0.6% in 1997 to 2.1% in 2007. Also, around 1% of individuals reported pean ut allergy in a similar survey in Canada ( Ben Shoshan and others 2010 ; Sicherer and others 2010a ) To reduce the IgE reactivity to peanut allergens several methods have been effectively demonstrated on different peanut allergens such as enzymatic hydrolysis, heat processing, chemical method, and high intensity ultrasound ( Chung and others 2005 ; Chung and Champagne 2006 ; Chung and Champagne 2007 ; Chung and Champagne 2008 ; Sales and Resurreccion 2009 ) However, Maleki and others ( 2000 ) reported that roasted peanuts have bee n demonstrated to increase the IgE binding reactivity compared to the same variety of peanut when processed by other thermal processing methods This means that roasted peanut s which are the most common peanut product in the United States because of the ir flavor and taste, are more likely to trigger a severe ly anaphylactic reaction than fried or boiled peanut s ( Beyer and others 2001 ) PL is regarded as a non thermal, high peak power technology that consists of intense flashes of broad spectrum ( Rowan and others 1999 ) Although no treatment is currently available for allergies except total avoidance emerging technologies like PL have shown positive outcomes for allergen ic foods such as peanut and shrimp ( Shriver and others 2011 ) With successful development, this technology potentially could be used in commercial processing to create roasted hypoallergenic peanut s Objective Objective 1: To investigate whether PL technology is effective in reducing major peanut allergens Ara h 1, Ara h 2 and Ara h 3 in whole kernel peanuts through in vitro tests including Western Blot and indirect ELISA.


20 Objective 2: Examination of IgE immunoreact ivity of insoluble fractions of PL treated pea nuts by Novex NUPAGE electrophoresis system. Objective 3: Examination of the effect s of PL treatment time and distance from the light source and their interactions on the allergenic properties and color development Objective 4: To verify the reduced allerg en reactivity of the PL roasted whole peanut by in vitro simulated gastric and intestinal digestions. Objective 5: To examine the quality changes ( texture, color, antioxidant capacity, and lipid oxidation ) of the whole peanuts before and after PL treatment s.


21 CHAPTER 2 LITERATURE REVIEW Current S ituation of F ood A llergy Food allergy is defined as an adverse immune response to food proteins Symptoms range from acute, possibly life threatening, allergic reactions such as atopic dermatitis, to chronic debilitatin g diseases such as eosinophilic gastroenteropathies. Epidemiology studies indicate the continuing rise of food allergies. In industrialized countries, 2% of the adult population and 5 8% of children are affected ( Ortolani and others 2001 ; Kagan 2003 ) In the United States, approximatel y 3.7% of adults and 6% of children are known to have one or more food allergies ( Sicherer and Sampson 2010 ) It is believed that one in 25 Americans is susceptible to food allergies (Westphal and others 2004; Sicherer and other 2004). Among adults, the major food allergen in the United States is shellfish (2%) while the major food allergen in European countries is fruits (0.5%) ( Kanny and others 2001 ; Sicherer and Samps on 2006 ; Meyer 2008 ) The major allergen for children in the United States and the Europ ean countries are milk and egg with 2.5 3% ( Kanny and others 200 1 ; Sicherer and Sampson 2006 ; Meyer 2008 ) Despite being considered as the fourth most important public health problem by the World Health Organization, the only effective treatment for food allergens is total avoidance of the allergen containing food. More than 160 foods can cause allergic r eactions. Eight of them are considered major food allergens and include milk, egg, walnuts, fish, shellfish, soybean, wheat, and peanuts ( Krska and others 2004 ) Food Allergy and Anaphylaxis The major food alle rgens are identified as class 1 type food allergies because they are represented by peanut, egg white, and cow's milk; their allergens are heat and


22 acid stable glycoproteins with those allergic sensitization via gastrointestinal tract and cause systemic reactions ( Nowak Wegrzyn 2007 ) They are known to be water soluble glycoproteins with molecular sizes ranging from 10 to 70 kiloDaltons (kD ) and are stable to heat, acid and proteases. Examples in clude proteins in peanut, milk, and egg ( Bargman and others 1992 ; Breiteneder and Radauer 2004 ) Breiteneder and Radauer 2004 further suggested that most of plant food allergens are either storage or defense related proteins. Food allergy is a relatively rare and violent reaction of the immune system towards food proteins. It has been defined as an immunologically based adverse reaction in response to dietary antigens ( Beyer and Teuber 2004 ) The allergen evokes an initial IgE antibody response followed by a secondary IgE antibody response, which is the signal to reflect an allergic reaction ( Babu and others 2001 ) Allergy symptoms range from a spectrum of severity. The most fatal reaction is anaphylaxis. Anaphylaxis is a life threatening reaction caused by the rapid release of mediators (histamine) from mast cells and basophills due to the interaction of the allergen with the IgE that is attached to the cell ( Morritt and Aszkenasy 2000 ) It impacts many areas of the body, including the skin, respiratory tract, gastrointestinal tract, and cardiovascular system. As a result of food allergies, it is estimated that 30 people per every 100,000 sensitive indivi duals are affected and about 30,000 cases of emergency room treatments in the United Sta tes are due to food allergies (The Food Allergy and Anaphylaxis Network 2006 ) Sathe and others ( 2005 ) listed several ways in which allergens can be removed from the fo od s These methods included physical removal of the specific allergen


23 (protein filtration or separation ) chemical modification of the allergens, genetical suppression or elimination of genetical expression, and genetic modification or mutagenesis of the targeted allergen s Chemical modifications involve the alteration of the amino acids with the goal of potentially changing the epitopes conformation on the targeted allergen ( Sathe and others 2005 ) However, one disadvantage of this method is that it is not specific. On the other hand, mutagenesis allows for selective substitution of specific amino acid from the targeted epitopes and thus is more advantageous than the chemical modification. Also, gene suppression has been reported to be successful in elimination of allergens in soybeans while gene silencing has been successfully employed in rice, soybean, and ap ple all ergens (Sathe and others 2005). Another study on trying to inactive food allergens involved the use of proteolytic bacteria from ferment ed foods. Phromraksa and others ( 2008 ) used crude extracted bacterial enzymes from B. subtilis (SR and BD strains ) and B. subtilis (BD strain) from fermented products to reduce wheat flour and milk immunoreactivity respectively. They found that enzymes derived from these two bacteria could hydrolyze the major allergenic fragments of wheat flour (gliadin) and major a Lactoglobulin) without influencing the nutritive value. The M echanism of I nteraction between Antigens and A ntibodies Allergic disorders are typically characterized as an abnormal or hyperactive immune response to environmen tal agents or food allergens. High levels of IgE in human plasma are responsible for the immune response ( Galli and others 2008 ) Ig E is a conserved member of the immunoglobulin fami ly, and the titer of IgE is quite low ( ng to mg / ml) in plasma of normal healthy individuals. IgE is widely distributed in epithelia


24 and mucosa, where it is bound to specific receptors o n eosinophilic granulocytes and mast cells. When bound to these cells, IgE has a long half life that can maintain from weeks to months, but unbound, free IgE in the plasma has a shor t half life of about 6 h This suggests that IgE plays a role in local imm une defense mechanisms ( Gould and others 2003 ) Food proteins bind to the allergen specific IgE molecules residing in the mast cells and basophils, causing them to release inflammatory mediators, inclu ding h istamine (Beyerand and Teuber 2004). Small regions of allergenic proteins known as epitopes, composed of 5 7 amino acids or 3 4 sugar residues, cause the IgE mediated allergy induced by antigen s ( Hendry and others 2001 ) Specifically, the cry stallizable fragment of IgE binds strongly to high affinity receptors on mast cells and basophils, and together with the antigen, mediates the release of inflammatory ag ents from these cells Afterwards, the allergen provokes an initial IgE antibody response followed by a secondary IgE antibody response, which signals an allergic reaction in the organism ( Babu and Arshad 2001 ) The level of IgE is found in ext raordinarily low concentrations in the serum of humans, varying from 20 to 500 ng / ml. Therefore, IgE is important since its biological activities are greatly amplified by binding to receptors on mast cells and basophils ( Schiffer and others 1995 ) An IgE molecule contains two identical light chains (23 kD ) and tw o identical heavy chains (72 kD ). Between the two antigen binding fragments surface has a depression that forms the antigen binding site Individual protein allergens can have several recognition sites (epitopes) per allergenic protein. According to Ayuso and others ( 2002 ) the epitopes are not only fully characterized by their primary


25 structure, but also by their tertiary structure conformations. Food allergens and their epitopes are able to resist the effects of digestion and enzymatic reactions ( Dollery and others 1987 ) and individual allergen systems are affected differently by the processing methods. This is because foo d allergens are complex mixtures of potentially immunoreactive proteins. A short sequence of amino acids that lies at the base of each of the heavy chain regions of an immunoglobin is called a hinge region ( Chintalacharuvu and others 2003 ) The hinge region is replaced by a constant domain so that each heavy chain contains four constant domains A s a result, IgE h as a molecular weight of 190 kD The structure of e pitopes is genera lly categorized as either linear or conformational. Linear epitopes have a contiguous stretch of amino acids whereas conformational epitope involves noncontiguous amino acids forming a three dimensional or structural motif. Individual patients may differ s ignificantly in their sensitivity toward an allergen; however, the basis of such differential sensitivities remains to be elucidated. More than one epitope or IgE binding site is required per fragment of an allergen to cause IgE cross linking. Therefore a molecule with a single IgE binding site must be bound or cross linked to another molecule with an IgE binding site in order to cause histamine release. Understanding mole cular properties of the epitopes is therefore important in learning the nature of IgE allergen interaction. In case of linear epitopes, amino acid residues that determine whether allergen would bind with IgE or not are known as critical amino acid residues. Any modification, deletion, or substitution of such critical amino acid residues may result in loss of IgE binding and may potentially result in reduction or elimination of IgE immunoreactivity to peanut


26 allergens If the epitope is conformational in nature, change in epitope conformation may permit modulation of allergic activity. Food p rocessing, under appropriate conditions, offers opportunities to alter the nature of epitopes. For example, epitope conformation may be modified as a result of protein denaturation treatments (e.g., various thermal processing treatments) leading to reducti on or elimination, or in some cases, an increase, in IgE binding. Acid or enzyme hydrolysis of an allergenic protein may help delete critical amino acids of an epitope. Whether caused by protein denaturation or hydrolysis, loss of epitope and thus loss of IgE binding may help reduce or eliminate the bioactivity of an allergen. It should be emphasized here that processing, depending on the allergen and the processing method, may not affect the allergenic properties of all allergens. Physicochemical changes w ill alter the way in which allergens are broken down during digestion and may modify the form in which they are taken up across the gut mucosal barrier and presented to the immune system. Certainly, the structure of the food matrix can have a great impact on the elicitation of allergic reactions and fat rich matrices may affect the kinetics of allergen release, potentiating the severity of allergic reactions ( Grimshaw and others 2003 ) Understanding the impact of food processing and food structure on allergenic reaction is central to managing allergen risks in the food chain. However, our current knowledge of the impact of food processing on allergen structure indicates that there are no clear rules regarding how different allergens respond to food processing. History of P eanuts Peanuts are also named as groundnuts, earthnuts, and monkey nuts. The peanut plant ( Arachis Hypogea ) is belonged to Leguminosae family other than nuts family. Peanuts are self pollinated plants and their pods are harvested by pulling the pods from


27 the ground. Normally, each peanut pod contains 2 3 oval shaped seeds with 2 lobes. Peanuts are originally from South America around 4000 to 5000 years ago and then exported to Africa and then moved to North America during the beginning of slave trade ( Saavedradelg ado 1989 ) By the nineteenth century, peanut industry had considerably expanded, which was then followed by a rapid demand for peanut oil, roasted and salted peanut, peanut butter and confections in the new twe ntieth century (The Peanut Institute 2007) George Carver Washington, a researcher in Tuskegee University, Alabama, is regarded as the father of the peanut in dustry in United States He started peanut research in 1903 and suggested farmers rotate their cotton farms with peanut and developed more th an 300 different uses of peanut from recipes to industrial products (The Peanut Institute 2007) Virginia type has the largest kernel and is mostly sold as roasted or processed in shell. It accounts for 15% of total United States peanuts p roducts and the r oasted product is widely used as a snack ( Conkerton and Ory 1976 ) Spanish t ype peanuts have comparatively smaller kernels and are characterized by a reddish brown skin and account for only 4% of the total production in United States. But they have the highest oil content compared to other varieties and are mostly used for candies and significant amount used for snacks, also for peanut butter. Valencia is characterized by having three or more kernels to a pod and is covered by a bright red skin. It is the least produced in the United States accounting for only 1% of total productio n (The Peanut Institute 2007) Runner type peanuts have good flavor and better roasting


28 characteristics, which are widely grown in southeastern United States region ( Pattee and others 1990 ) Seven states are responsible for almost 99% of all peanuts grown in the United States. Georgia is the major producer (41%) followed by Texas (24%), Alabama (10%), North Carolina (9%), Florida (6%), Virginia (5%) and Oklahoma (5%) ( Koppelman an d others 2001 ) About 75% of the American peanut crop is used domestically, a nd the remainder is exported mainly to Canada, Japan and Western Europe. On average, the average consumption of peanut butter in USA is 2 kg per year ( FDA/CFSAN 2000 ) Except its main use as food, peanuts can also be used for several other purposes, and there are about 300 derivatives of peanuts ( Morritt and Aszkenasy 2000 ) Hulls or pods can be used as fuel or in kitty litter; kernels can be crushed to obtain peanut oil, which can be used to soap manufacture Peanut skins were used to make beverages at National Peanut Research Laboratory ( FDA/CFSAN 2000 ) Peanuts can also be found in other products like cheese, milk, coffee, medicinal oil, flour, soap, cosmetics, dyes, plastics and linoleum ( Hourihane 1997 ; Hill 2004 ; Husain and Schwartz 2012 ) The Nutrients and Health B enefits of Peanut Peanuts have more protein s than other legumes or nuts. It contains around 21 36% protein s in its seed ( FDA/CFSAN 2000 ) One study indicated that the consumption of a peanut oil diet can reduce 17% of the risk of heart disease (The Peanut Institute 2007) It was further found that a peanut oil diet did not increase the tryglycerol concentrations or lower the good high density lipoprotein (HDL) cholesterol levels compared to the other low fat diets. Peanuts contain highly beneficial monounsaturated and polyunsaturated fats that have the potential to lower blood


29 cholesterol levels and in turn reduce the risk of coronary heart disease. High levels of phytochemicals in peanut could also lower the risk of coronary heart disease ( Awad and others 2000 ) One of the phytochemicals found in peanuts was resveratrol. Phytosterols not only lower the risk of heart disease by interfering with the abso rption of cholesterol, but also help lower the risk of cancer by inhibiting the tumor cell growth ( Awad and others 2000 ) Other studies also found that phytosterols inhibited the cancer cell growth by 70% and reduced the prostate tumor growth by 40% (The Peanut Institute 2007) One of the most common forms of phytosterols is beta sitosterol (SIT). Peanut oil contains more protective SIT than refined olive oil ( Awad and others 2000 ) Vitamin E content is rich in peanuts and has been associated with cancer reduction, immune system improvement and cardiovascular diseases prevention ( Karunanandaa and others 2005 ) Important essential minerals like zinc, magnesium, phosphorus and potassium are also abundant in peanuts. Moreover, Harvard or peanuts could redu ce the risk of developing type two diabetes (The Peanut Institute 2007) Phenolic phytochemicals in foods obtained increased interest from consumers and researchers for their antioxidant activity and health benefits. Their ideal chemic al structure for free radical scavenging activities showed increased antioxidant activity compared to vitamin E and C on a molar level ( RiceEvans and others 1997 ) Peanuts not only have healthy fatty acid profiles and serve good sources of fibers and protein, but also contain a number of components that are capable of directly scavenging free radicals. In peanuts, various polyphenolics (coumaric acid, derulic acid resveratrol),


30 tocopherols, flavonoids (procyanicdins, catechin) and folate are found ( Blomhoff and others 2006 ; Isanga and Zhang 2007 ) Peanut flours contain high protein but low fat ingredients prepared from roasted peanut seed. Peanut skins are established to be of rich source of phenolic comp ounds, including various procyanidins among others, which suggests that there is potential to produce neutraceutical ingredients from peanut skin ( Nepote and others 2005 ; O'Keefe and Wang 2006 ) Peanut seeds contain 51.9% oil in total mass. The composition of peanut fatty acid includes higher proportions of oleic acid (43.2%) and behenic acid (3.2%), which are higher than soybean seeds (28.8% and 0.3%). Peanuts also contain much lower proportions of linoleic acid (35%) and linolenic acid (0.1%) than soybean seeds. Overall, peanut oil is less susceptible to oxidation than soybean oil because of fewer double bond s present in peanut oil ( Wang and others 2012 ) Peanut A llergy Among all the food allergies, peanut allergy is considered as one of the most serious allerg ies Peanut allergy is the most severe fatal allergic reactions for patients who are sensiti ve, accounting for the majority of severe anaphylactic reactions for foods ( Clarke and others 2006 ; de Jonge and others 2007 ) Bock and others ( 2001 ) also reported that peanut was the most dangerous food allergen source with responsi bility of over 63% of the fatal reactions. In the United States alone, peanut induced anaphylaxis affect s around for 1.5 4.4 million people and causes about 50 125 deaths every year ( Leung and Bock 2003 ; Sicherer and Leung 2006 ) Moreover, unlike other food allergies which tend to be outgrown in childhood, peanut allergy tends to be a lifelong problem for around 80% of the peanut allergic population ( Viquez and others 2001 ; Clarke and others 2006 ; de Jonge and others 2007 ) However, avoidance of peanut s consumption


31 in foods is difficult and those food products without proper l abel may have some contamination with peanuts Allergic individual s always have several symptoms including oral allergy syndrome, mild hives, facial swelling, abdominal cramps and hypertension with anaphylactic shock. P eanut A llergens Peanut allergens are storage proteins belonging to two major globulin families, arachin (legumin) and conarachin (vicilin) ( Viquez and others 2003 ) They are classified as either a member of the cereal prolamin superfamily or of the cupin superfamily (Dunwell and others 2001). Previous research has shown that there is no naturally occurring allergen free peanut among a wide variety of commercially grown peanuts (Viquez and others 2001). Among the eleven known peanut allergens Ara h 1 Ara h 11 Ara h 1, Ara h 2 and Ara h 3 are responsibl e for allergic reactions in more than 60% of peanut allergic patients ( Pele 2010 ) Moreover, out of these 3 major allergens, Ara h 1 and Ara h 2 are responsible for over 90% of the reactions in peanut hypersensitive individuals (Viquez and others 2001). Ara h 1 with a molecular weight of 63.5 kD i s resistant to heat, stable to digestion, and highly structured at the secondary level (Koppelma n and others 2003). This protein is affecting over 90% of the peanut sensitive population. Ara h 1 is known to have 30% to 45% similarity with other vicilin sto rage legumes such as soybeans, bea ns and garden pea (Pomes 2003). Ara h 1 has 2 3 allergenic epitopes that are immunodominant being recognized by more than 80% of patie nts. This protein is very abundant in peanut representing around of 12 16% of the tot al protein. Ara h 1 has thermal stability and its allergenic properties are unaffected by thermal denaturation. It


32 is also resistant to proteolytic hydrolysis ( Maleki and others 2000b ; Wichers and others 2004 ) Ara h 2 i s a glycoprotein of about 17 kD with at least two major bands and an isoelectric point of 5.2 (Viquez and others 2001). Its amino acid sequence is composed of high percentage of glutamic acid, aspartic acid glycine, and arginine which shows similarity to seed storage proteins of the conglutin family and the protein has at least 10 IgE epi topes (Viquez and others 2001). It also has 8 cystein residues that could form up to four disulphide bonds and is predomi helical protein (Lehmann and others 2003; Sen and others 2002), which is said to play an important role in its structure. Sen and others (2002) also found that the native non reduced Ara h 2 protein migrated in a 12% SDS PAGE as a doublet pro tein with an a verage molecular weight of 12kD while the reduced Ara h 2 protein migrated as a slightly larger doublet with an av erage molecular weight of 17 kD It is the most potent allergen and is recognized by the serum IgE of more than 95% of peanut se nsitive individuals (Kleber Janke and others 2001). Ara h 2 has been reported to be active even at very low concentrations (< 100 pg/ml), while Ara h 1 and Ara h 3 are only active at 10 to 100 fold higher concentrations compared to Ara h 2 (Palmer and othe rs 2002; Koppelman and others 2003; Knol and others 2003). Ara h 3 belongs to the glycinin storage protein family. It consists of basic and acidic subunits with the molecular weight ranging from 14 to 45 kD. Koppelman and others ( 2003 ) reported that the ba sic subunits and to a lesser extent, the acidic subunits, bind to the IgE and may therefore act as allergenic peptides. They also found that the major IgE binding epitope is found on the N ter minal fragments of 42 and 45


33 kD Ara h 3 has been reported to ha ve homologous epitopes as soybean allergen Glycinin (Pomes 2003). Peanut allergens have been reported to be difficult to avoid by sensitive individuals. This has been authenticated by reports showing that accidental ingestion of peanut by sensitive individ uals has been increasing in recent years. Extensive use of peanuts in the food industry has made dietary avoidance very difficult. A study showed that about 55% of American children allergic to peanuts had accidental ingestion of peanut in 2005, indicating an annual increase rate of 33% ( Yu and others 2006 ) This problem has been accelerated by the inability of ma ny parents to carefully read the food labels. Kemp and Deshazo ( 2004 ) reported that around 54% of parents of allergic children fail to correctly read the labels, thus ending up with accidental ingestion of peanuts by children. Dodo and others ( 2002 ) found that there was no significant difference in allergen content on four types of peanuts in their study on allergen content in various market type peanuts. Also Maleki and others ( 2000 ) found no difference in the level of IgE reactivity between high ol eic peanuts (80% oleic acid) and normal peanut varieties (50% oleic acid). This implies that the risk of fatal reactions is the same for sensitive individuals, regardless of the peanut varieties consumed. The severity and existence of peanut allergy in the Unites States has been explained in many different ways. It is thought that the development of peanut allergy has to do with the processing method they are handled. In the United States, roasting is the main processing technique while boiling is most com mon proces sing method in China and Indian. Cases of allergy in China and India are considerably lower as


34 compared to the United States ( Sampson 2002 ) Therefore, roastin g has been associated with the increasing cases of peanut allergy in the United States. This theory is supported by a study conducted by Al Mussawir and others ( 2002 ) who investigated the differences in the allergenic potency between boiled and dry roast ed peanuts. Their results showed that boiling reduced the allergenic potency of peanuts while dry roasting increased it. Anot her study by Maleki and others ( 2000 ) found that extracts from roasted pe anuts bound to serum IgE was 90 folds higher in peanut pat ients than that from raw peanuts. They concluded that the Maillard reaction was responsible for the observed effect. Further studies of Maleki and others ( 2003 ) pointed out that roasting peanuts caused a major peanut allergen, Ara h 2, to become a stronger inhibitor (at least 3.5 folds increase ) to a digestive enzyme trypsin, thus making it more resistant to digestion. This study also found that Ara h 2 protected Ara h 1 from digestion, an action which is enhanced by roasting. In general, this shows that th ermal processing such as roasting, curing and various types of cooking can cause non enzymatic, biochemical reactions in foods, with the predominant reaction responsible for allergenic behavior being the Maillar d reaction. Maleki and others ( 2003 ) further established that heating causes alterations to the protein structure mainly due to the interactions of sugar components with amino acids, forming carboxymethyl lysine, malanoidin, and other compounds. These and other modifications to the protein are belie ved to have detrimental nutritional, physiological, and toxicological cons equences. Chung and Champaigne ( 2002 ) reported that the protein sugar complex formed during Maillard reaction may cross link with proteins to form advanced glycation end products (AG E,) which increase IgE binding.


35 They added that the formation of carboxymethyl lysine modification on the surface of the protein resulted in higher binding levels of allergens Ara h 1 and Ara h 2 with IgE. To develop an effective processing method for inactivation of peanut allergen, several studies focused on investigating alternative treatments and prevention of peanut allergy. Nelson and others ( 1997 ) found that injection of peanut extracts can significan tly improve the tolerance of anaphylactically sensitive subjects to the ingestion of peanut proteins. However, this tolerance cannot be maintained for individuals who are highly sensitive because of strong systemic reactions to injections. However, there i s no evidence so far of its clinical application because of the risk of systematic reactions. Activated charcoal has been investigated for treatment of peanut allergy. Routon and Kopper ( 2004 ) reported activated charcoal could absorb peanut allergens withi n seconds and prevent further serious reactions to IgE binding Other methods which have been investigated to reduce the allergenic potency in peanuts include enzyme treatment. Chung and Champagne 2003 studied two enzymes, transglutaminase and peroxidase. They found peroxidase could reduce the allergenic potency of Ara h 1 and Ara h 2 in roasted peanuts while transglutaminase was not effective. However, both enzymes were not effective on raw peanuts. Chung and others ( 2005 ) treated peanut extracts with two metals, copper and iron, in order to assess their efficacy in allergen reduction. Treatment of roasted peanut extracts with copper in the presence of hydrogen peroxide resulted in the reduction of Ara h 1 and Ara h 2 levels. However, no effect was observed with iron treatment. Furthermore, in order to study the role of other peanut components on IgE immunoreactivity to peanut allergens Chung and


36 Champagne ( 2006 ) studied the effect of phytic acid on the IgE binding to peanuts. In this study, they compared ph ytic acid containing peanut allergens to native allergens. Their findings showed that the IgE binding for phytic bound allergens was similar to that of native allergens, indicating that other peanut components such as phytic acid might not have any effect on the IgE binding of the peanut allergens. A follow up study on the effect of phytic acid in masking peanut allergens was done by Chung and Champagne ( 2007 ) They attempted to explore the effect of phytic acid on reducing the allergenic potency of peanut butter. New findings were shown that phytic acid could form insoluble complexes with the major peanut allergens to interrupt digestion and subsequently preve nt absorption. Phenolic compounds which have effects on reducing the allergenic potency of peanuts has been explored by Chung and Champagne ( 2008 ) In this study, they used three different phenolic compounds: caffeic acid, chl orogenic acid, and feluric ac id They assessed their effectiveness on the reduction of peanut allergens in peanut extracts and peanut butter slurry. Results showed that out of the three compounds, only caffeic acid formed insoluble complexes with the major allergens Ara h 1 and Ara h 2 in treated samples, thus lowering their allergenic potency. However, chlorogenic acid and feluric acid had no effect. All these studies demonstrated the potential treatments in reducing peanut allergy in different ways. However, so far there has never be en any practical application of these findings in the food industry, prompting the investigation of other technologies, which might be readily and easily applied in the food industry. Moreover, the reviewed studies did not list nutritive value changes afte r these treatments.


37 P rocessing M ethods for P eanut A llergen Reduction A number of thermal and non thermal techniques have been investigated for allergens reduction in peanut and other foods. Thermal processing may alter the IgE binding sites of allergenic proteins in foods with increased or decreased allergenic effects ( Wal 2003 ) The outcome depends on the structure and chemical properties of the allergen, the type of thermal processing applied (dry or wet), the temperature, and the duration of heating ( Davis and Williams 1998 ) Thermal treatments by wet thermal methods include boiling, frying, extrusi on, autoclaving, and retorting while dry heat methods include baking, roasting, and microwaving. As mentioned earlier, roasting treatments enhance IgE binding capacity while boili ng peanut decreases IgE reactivity to peanut allergens by western blot ( Maleki and others 2000a ) In Chapter 2 only roasting, frying, boiling, and autoclaving methods will be reviewed on peanut allergen reduction with these thermal processing methods. Non thermal processing methods exist and are labeled as emerging technologies because they have been used in recent years to reduce allergen reactivity of different foods. Non thermal trea tments provide several benefits, such as preserving the natural characteristics, nutritional values, and flavors as compared to the conventional thermal processing. Effect of T hermal T reatment on P eanut A llergens T hermal processing Thermal processing was developed for enhancing food safety (pasteurization or sterilization), change of physical attributes or texture (drying, gelation), or to induce flavor profile modification (baking) ( Davis and Williams 1998 ) However, thermal processing also affects protein structure. The rationale can be simply explained from the standpoin t of altering protein structure Significant alterations in protein structure occur


38 during heat treatments and these changes depend on two major factors: temperature and intrinsic characteristics of the protein and physic chemical conditions of its environment (such as pH). Typically, loss of tertiary structure is starting from unfolding of proteins. In general, loss of secondary structu re (55 70C), cleavage of disulphide bonds (70 80C), formation of new intra /inter molecular interactions, rearrangements of disulphide bonds (80 90C) and the formation of aggregates (90 100C) as shown in Figure 2 1 ( Davis and Williams 1998 ) At the molecular level, linear epitopes of proteins are more difficult to eliminate than conformational epitopes by thermal processing or any other technique. Linear epitopes belong to the primary structure that is hard to be destroyed even under temperatures as high as 125C. On the other hand, conformational epitopes are relatively easy to be cleaned out by altering the tert iary or secondary protein structure, which can occur within reasonable temperature ranges of 50 125C (Lee 1992). Moreover, at higher temperatures, covalent bond formations between protein lysine residues and other constituents of the food matrix may take place and lead to the formation of other adducts ( Chung and others 2003 ) Protein digestibility and ga strointestinal tract absorption a re normally increase d before heat treatment. In some cases, thermal processing may reduce the digestibility of a particular allergen, such as Ara h 1 and Ara h 2 in peanuts ( Schmitt and others 2010 ) Thermal treatment may also lead to neo antigen formations that are not originally exist ed The generation of neo antigens may enhance the allergenic issue in sensitized peanut allergy patients. One of the factors responsible for this effect is the Maillard reaction. Due to the interaction of sugar and protein components upon heating, which generates sugar conjugated protein deriv atives, will increase the IgE immunoreactivity of the


39 protein effect ( Dav is and others 2001 ) Generally, roasting peanuts has shown to increase the IgE reactivity while boiling and frying have shown to decrease allergenic effects of peanuts. Roasting Peanuts are consumed either fried or boiled in China whereas peanuts are t ypically dr y roasted in the United States ( Beyer and others 2001 ) Runner type peanuts and the Florunner are the most common peanut varieties in the United States which a ccount for 73% of the total United States production (The Peanut Institute 2007) Runners are always consumed in a roasted form and used heavily in peanut butter production. Valencia, another common var iety, is usually consu med in a roasted form in the United States ( Beyer and others 2001 ) It was shown that roasting may strengthen the allergenic properties of peanuts as compared to unprocessed, raw peanuts ( Maleki and others 2000a ) In the industry, peanuts are normally processed using dry roasting methods that include both batch and contin uous processes. Batch roasters are usually natural gas fired revolving ovens and the rotation of this oven produces evenly roasted peanuts products. The oven temperature is around 430C (800F) and internal temperatures of the peanut kernels reach to aroun d 160C (320F) after 40min to 60min treatment Different roasting temperatures and heating durations are used depending on the desired characteristics of the peanut batch. Several types of continuous dry roasters are available such as cross flow roast er, vertical continuous roaster ( Davidson and others 1999 ) Generally, peanuts move through an oven on a conveyor of by gravity feed. One design involves peanuts being f ed on a conveyor into a stream of countercurrent hot air that roasts the peanuts.


40 The peanuts are agitated, allowing air to pass through the individual kernel and ensure an even roast. The major advantages of continuous systems are include higher capacity, greater control over the roasting conditions, and low labor costs due to an automated system ( Fellows 2009 ) For laboratory purpos es, the roasting temperature ranges from 160C to 176C with different treatment times depending on the desired characteristic s ( Schmitt and others 2010 ) used a temperatur e of 160C (320 F), for 5 50 min to investigate the thermal eff ects on peanut proteins. Protein aggregation and solubility reduction were observed during roasting process. Since the IgE binding signal of roasted peanuts is higher than the boiled or fried peanuts, Maleki and others ( 2000 ) indicated that Maillard reaction is responsible for the higher IgE immunoreactivity The Maillard reaction is important in the development of flavor and color of roasted peanuts. The amino groups of proteins are modified with reducing sugars, ketose, or al dehydes to form Amadori products. Subsequently, Amadori products are degraded into dicarbonyl intermediates. These compounds are more reactive than the parent sugars and react with other amino groups to fo rm stable end products, called that named as Mailla rd reaction products (MRPs) or advanced glycation end products (AGEs). In addition, Maillard reaction may lead to the loss or modification of amino acids, such as lysine and cysteine, and can cause malanoidin formation and other non cross linking modificat ions to proteins ( Baynes 1991 ; Kristal and Yu 1992 ) Maleki and others ( 2000 ) demonstrated that such collective modifica tions accounted for the increased IgE binding in roasted peanuts. Other byproducts that may contribute to the higher levels of IgE binding, such as lipid oxidation products, were not


41 investigated. Therefore, it is suggested that the increased allergenic pr operties of Ara h 2 in roasted peanuts result from the Maillard reaction. The modified protein products can further evoke an IgG response, which is known to be related with IgE production ( Ikeda and others 1996 ; Duchateau a nd others 1998 ) Boiling Boiled peanuts are mainly consumed in China and other Asian and Middle Eastern countries, and have been popular also i n the southern region of the United States In recent years, boiled peanuts are found in grocery stores as a type of snack ( Chukwumah and others 2007 ) Numerous studies point out that the sensitization and reactivity of boiled peanuts is much low er as compared to roasted peanuts as we discussed above Typically, the peanuts are boiled at 100C for approximately 20 min ( Schmitt and others 2010 ) The IgE binding of monomeric Ara h 1 and Ara h 1 trimer of boiled peanuts show a significant reduction compared to roasted peanut on Western blots. Heated Ara h 1 may form stable dimmers, trimers and larger complexes. The Ara h 1 trimer has a high stability and resists digestion processes that protect IgE binding sites. The IgE binding to Ara h 2 and Ara h 3 was, in parallel with Ara h 1, significantly lower in boiled peanuts ( Beyer and others 2001 ) Furthermore, Maleki and others ( 2010 ) detected both soluble and insoluble fraction activities with IgE, which revealed that boiled peanuts have a sign ificantly less IgE reactivity in insoluble fraction than roasted peanuts. The overall decrease in IgE binding reactivity in boiled peanuts is not associated with structural modifications of peanut proteins, but mainly because of loss of the conversion of s oluble low molecular weight proteins or peptide fragment into insoluble complexes. This would


42 explain the low prevalence of peanut allergy in some countries where peanuts are boiled ( Mondoulet and others 2005 ) Frying The low prevalence of peanut allergy in China is also related to another popular f orm of peanut product, which is fried peanut ( Beyer and others 2001 ) The cooking temperature is approximately 120C. It is shown that the monomeric form of Ara h 1 is reduced in fried peanuts as compared with roasted pean ut. The IgE binding intensities to Ara h 1 monomer and trimers were also significantly reduced in fried peanut relative to roasted peanuts ( Beyer and others 2001 ) A further study detected both soluble and insoluble proteins in fried peanuts on SDS PAGE and the IgE binding intensi ties on Western blot Fried peanuts had higher or the same level of IgE binding as roasted peanut, but the soluble bands had a notable reduction of IgE reactivity ( Schmitt and others 2010 ) A recent study provided strong e vidence for the disintegration of boiled, roasted, and fried peanuts in simulated gastric environment to investigate the influence of different thermal processing methods on peanut proteins, esp ecially for allergens ( Kong and others 2013 ) The absorption of moisture and the swelling of peanuts during gastric digestion were measured using magnetic resonance imaging (MRI). The disintegration rate of peanut proteins was rated as: fried > roasted > boiled > raw peanuts. Fried peanut s had the fastest disintegration rate with a half time of 3.6 h, but the raw sample extended to 10.7 h. Furthermore, the textures of boiled and fried are more softener than the roasted one i n simulated gastric environment ( Kong and others 2013 )


43 Autoclaving Previous studies have shown that autoclaving at 2.56 atmosphere (atm) for 30min could produce a relevant decrease in the IgE binding capacity of lentil and chickpea allergens. However, several immunoreactive proteins were still present ed in these legumes upon harsh autoclaving ( Cuadrado and others 2009 ) Instantaneous controlled pressure drop treatment, which combines heat and steam pressure, has shown a remarkable decrease in the all ergenic proteins of peanut at 5. 92 atm for 3min ( Beyer and others 2001 ) In peanuts, one study sought to investigate the e ffects of autoclaving on the allergenic protein reactivity. Extreme conditions were applied at 2.56 atm for 30min at 138C in this study. The results showed a significant reduction of IgE reactivity co mpared to conventional roasted peanut based on Western blotting and ELISA. These helical structure. Many of the IgE binding epitopes in the major peanut allergens (Ara h 1, Ara h 2, Ara h helical regions ( Shin and others 1998 ; Barre and others 2007 ; Mueller and others 2011 ) About 22% of the sera from skin prick test results could recognize autoclaved peanut samples ( Cabanillas and others 2012 ) The study demonstrated that the structure of peanut allergens has been changed and some of epitopes were destroyed after autoclave treatment. In addition, the texture of autoclave d peanut is softer than that of roasted peanut. The digestibility of autoclaved peanut is more extensive by trypsin after 10 min treatment than that of roasted peanut. This study has demonstrated that heat treatments can increase the susceptibility to enzy matic digestion of peanut allergens ( Morisawa and others 2009 )


44 N on T hermal T reatment on P ea nut A llergens Non thermal processing is as an alternative method to conventional thermal processing. The goals of non thermal processing are to strike a balance between food safety and minimal processing and to combine novel approaches with current processes installations to optimize resources. Non thermal technologies can be used for decontamination, pasteurization and sterilization, but another emerging application of some non thermal technologies is to inactivate the allergenic proteins in foods. Several non thermal process ed methods, such as PL power ultrasound HHP ionized radiation, PEF. Pulsed light PL consists of a broad spectrum of white light with wavelengths from 200nm in the ultraviolet region to 1000nm in the near infrared region. The spectra are emitted within several nanoseconds from ionized inert gas, such as Xenon. The PL light is thousands of times more intense than conve ntional, continuous mercury UV light and sunshine at the sea level ( Dunn and others 1995 ; Krishnamurthy and others 2007 ) There are two types of PL systems used for food processing : a batch unit and a continuous unit. In the batch system, foods are manually loaded to the sample rack that can be adjusted by the operator to reach a proper distance from the light source. The pulse frequency is typically around 1 20 pulses / s. In a continuous system, a conveyor belt carries foo d samples through one or more lamps. The distance to the lamp conveyor speed, pulse frequency, treatment duration, and lamp tilt angle of the belt can be adjusted to modify the process. A continuous unit is also capable of large r sample treatment and it c an be used as a batch unit when the conveyor speed is set to zero.


45 PL is considered more effective in food processing due to its instantaneous high energy pulses and greater capability to penetrate. PL is regarded as nonthermal technology with a short dur ation (e.g., seconds). However, some studies showed that extended PL treatment c ould incur a significant temperature increase and moisture loss of a food sample, which is due to the infrared spectrum ( Yang and others 2012 ) In a PL system, electrical energy is captured and stored in a capacitor and is ultimately released in short pulses as UV light, infrared light, and visible light (around 54%, 20%, and 26% respectively ) ( Shriver and Yang 2011 ) The resultant bursts can be several thousand times more intense t han continuous UV light ( Krishnamurthy and others 2007 ) As Shriver and Yang 2011 indicated, PL processes might increase the temperature for treated shrimp up to 68.5C. Another previous study reported that pulsed UV light can reduce the human IgE reactivity on almond, soybean extract, peanut butter ( Yang and others 2010a ; Li and oth ers 2011 ; Yang and others 2011 ) could alter allergen con formation (Krishnamurt hy and others 2007) or cause p rotein aggregation (Chung and others 2008; Yang and others 2010), destroying conformational and linear epitopes. Furthermore, radicals may be produced during PL treatment due to molecular ionization under its high energy burst of lights. The visible and infrared waves in the PL spectra are responsible for vibration and rotation of molecules, res pectively (Krishnamurthy and others 2009) UV light can also lead to protein aggregation and fragmentation. Infrared light can cause tem perature increases in food sample and produce thermal effect on treated samples. Due to the protein aggregation, the digestibility of those aggregate has been reduced and the possibility to


46 cause allergy may reduce due to this reason. S ince most epitopes o f p eanut allergens are linear form. PL can fragment the protein mol ecules to form smaller peptides. T he linear epitopes of peanut allergens may be destroyed after PL illumination To obtain more ev idence to support PL on the changes of allergenic proteins h igh performance liquid chromatography (HPLC), n uclear magnetic resonance (NMR), skin prick test, oral administration studies are still needed. Friedman and Brandon ( 2001 ) found that extended PL treatment caused formation of insoluble complex in food, depolymerization of starch, peroxidation of unsaturated fatty acid, carbohydrate crosslinking, protein crosslinking, and protein fragmentation. The PL treatment could not easi ly break the peptide bonds in protein. Photons absorbed by cystine had a higher chance of inactivating a protein than photons absorbed in the aromatic amino acids. The absorbed photons ionized the protein ( Setlow 2002 ) Aromatic amino acids (e.g., tyrosine and phenylalanine) can absorb UV radiation and recombine to form covalent cross links in proteins ( Gennadios and others 1998 ) Chung and others ( 2008 ) conducted a study that was to investigate the effects of PL treatment on peanut extracts. Ara h 1 (63 kD ) showed a significant disappearance in SDS PAGE that indicated they form insoluble aggregates or pre cipitates. Ara h 2 (10 20 kD ) allergens did not form aggregates because they still remained soluble form. Aggregates that formed in the treated peanut samples accounted fo r the peanut allergens disap p earance High pressure microfluidisation Dynamic high pressure microfluidisation is a new technology that combines mixing, ultrarefining, homogenization, emulsification and other units with the basic function of a microjet homogenizer and ultrahigh pressure homogenizer. This


47 equipment has a stron g shear force, high speed bump, instantaneous pressure release, and cavitation effect. The pressure of this unit can achieve 200 MPa. A recent study explored the effects of this unit on Ara h 2 of peanut extracts. The results showed that the primary struct ure of Ara h 2 is not affected by high pressure. However, the teriary and quaternary structure of Ara h 2 have shown unfolded and more hydrophobic regions exposure. Under a pressure of 180 MPa, exposed hydrophobic groups form indicating the second structure of Ara h 2 has been destroyed. The disulfide bonds of Ara h 2 were converted into sulfhydryl groups, resulting in a reduction in the overall IgE binding signal ( Hu and others 2011 ) irradiation Irradiation is an ionic, no heat process that is considered to be a preservation and functional modification method in polymer r esearch ( Abu and others 2005 ) In comparison with other processing methods, such as microwave, UV light, ultra high hydrostatic pressure and hydrothermal treatment s irradiation treatment is rapid, convenient, but more expensive ( Bao and others 2005 ) Among non thermal processing irradiation has been used to monitor food borne pathogens, reduce the population of microbial load and insect infestation, inhibit the germination of root crops and prolong the shelf life of perishable products. Furthermore, irradiation has been reported to have effects on reducing the IgE binding sites of ovalbumin, bovine serum, albu min and milk proteins, and shrimp tropomyosin ( Kume and Matsuda 1995 ; Lee and others 2001 ; Byun and others 2002 ) Previous studies showed that a treatment on a shrimp allergen with a molecular weight 35 kD did not affect its activity ( Hoffman and others 1981 ; Hefle 1996 ; Besler and others 2001 ) irradiation was combined


48 with heat treatment to have a reduced IgE immunoreactivity effect on shrimp ( Li and others 2007 ) On the contrary, a combinat irradiation (1 25 kGy) alone or followed by various thermal treatments would not change their allergenic potential ( Su and others 2004 ) Another study reported that boiling followed by irradiation reduced IgE binding of treated peanuts, up to 26 folds ( Kasera and others 2012 ) Later t o investigate the effects of gamma irradiation on the structure changes of Ara h 6, isolated Ara h 6 and irradiated at 1, 3, 5, or 10 kGy. A whole peanut protein extract was also treated by irradiation. The IgE immunoreactivity was measured by immunoblotti ng and indirect ELISA with anti Ara h 6 polyclonal anti bodies Results s howed that irradiation induced significant changes in the secondary and tertiary structures of Ara h 6 and the IgE reactivity of Ara h 6 gradually decreased with the increased irradiation dose ( Kasera and others 2012 ) Pulsed electric field P ulsed electric field ( EF ) is a novel processing technique used for food pasteu rization PEF involves the application of high voltage pulses (electric field strength of 0.1 40 kV/cm and total energy input of 0.5 1000 kJ/kg) to electroconductive foods that are located between the electrodes. The electric field could induce the movements of ions and permeablilization of cell membranes called electroporation. This biological effect is related to several applications, such as microbial inactivation ( Garcia and others 2007 ) enhanced extraction ( Schilling and others 2007 ) and improved mass transfer processes ( Guderjan and others 2005 ) Results in this study showed that PEF treatment did not significantly affect the secondary structure of peanut allergens. It is more probable that highly localized thermal effects were res ponsible for the minor observed structural changes. The scarcity of studies on the effects of PEF on protein


49 structure is difficult to predict the principle of this method on allergenic proteins ( Johnson and others 2010 ) Chemical and biological methods Peroxidase Peroxidase (POD) is a heme containing enzyme catalyzing the oxidation of a variety of phenolic compounds, such as converting ferulic acis into o quinones. Th e o quinones react with other phenolics, amino, or sulfhydryl compounds to form new cross linked compounds. For instance, peroxidase helps to improve the functional properties by catalyzing the cross linking of polysaccharides and/or proteins ( Labat and others 2001 ; Dunnewind and others 2002 ) Proteins can become cross linked with other protei ns or polysaccharides in the presence of peroxidase. Proteins contain typrosine resides that carry a phenol group. It can react with the ferulic acid moieties of polysaccharides or the tyrosine residues of another protein to form protein polysaccharide or protein protein cross links ( Faergemand and others 1998 ; Labat and others 2 001 ; Oudgenoeg and others 2001 ; Dunnewind and others 2002 ) One previous study reported that transglutaminase has an e ffect on the reduction of immunogenic and allergenic properties of soy proteins ( Babiker and others 1998 ) This reduction in reducing IgE reactivity of soy proteins is due to the cross linking effects that mask the binding sites on the allergenic proteins. As mentioned earlier, roasted peanut have higher allergenic reactions than raw peanuts. Another study investigated the effects of POD on the IgE reactivity to the allergens of roasted peanuts. Protein extracts of roasted and raw peanuts were treated with POD for 1 16 h. SDS PAGE results exhi bited a significant decrease in density or levels of Ara h 1 and Ara h 2. Western blots and competitive inhibition ELISA results showed a significant reduction of IgE reactivity on Ara h 1 and Ara h 2 of roasted


50 peanuts. This reduction was due to the cross linkage of these allergenic proteins. This may result in masking the IgE epitopes and, consequently reducing IgE binding. This study provided evidence that POD helped to reduce the IgE reactivity to the allergens in roasted peanuts, but not raw peanuts ( Chung and others 2004 ) Phytic Acid Phytic acid is the major storage form of phosphate and inositol in mature oilseeds (Reddy and others 2002). On a dry basis, around 1% phytic acid is stored in the whole oilseeds ( Porres and others 2004 ) Phytic acid binds to and forms soluble and insoluble complexes with proteins ( Okubo and others 1976 ; Lombardi Boccia and others 1998 ) Insoluble complexes reduce t he bioavailability of proteins. Chung and others ( 2007 ) investigated the effects of phytic acid on the allergencity of peanut allergens. Results in this study demonstrated that phytic acid assisted the complexes with the major peanut allergens (Ara h 1 and Ara h 2) and these complexes were insoluble at neutral or acidic pH. The levels of soluble allergens and IgE binding of a peanut extract were reduced 6 fold in allergenic potency. The similar effect was also observed in peanut butter, indicating that phyt ic acid may be a method for development of hypoallergenic peanut based products ( Chung and Champagne 2007 ) Polyphenol oxidase and caffeic acid Polyphenol ox idase (PPO) or tyrosinase is a copper containing enzyme that is widely distribution in fruits and vegetables ( Seo and others 2003 ; Matuschek and Svanberg 2005 ) It catalyzes the oxidation of phenols and converts them into o quinones, which then polymerize with other phenolic compounds (caffecic acid) or amines to form browning products ( Iyidogan and Bayindirli 2004 ) In proteins, PPO catalyzes the oxidation of tyrosine residue, which contains a phenol group, converting to an o quinone d erivative ( Hurrell and others 1982 ; Matheis


51 and Whitaker 1984 ) The derived quinine on the protein can react with the amino group, sulfhydryl group, or the tryptophan residue of another protein to form cross link complexes (Chung and others 2005). However, protein complexes would not form a cross link if phenolic compounds such as caffeic acid or chlorogenic acid are present ( Hurrell and others 1982 ; Matheis and Whitaker 1984 ) Researchers explored the effects of PPO and/or caffeic acid and discovered that it can improve the functional properties of soy proteins ( Rawel and others 2002 ) and also increase the heat stability of milk proteins ( O'Connell and Fox 1999 ) Another study examined the role that PPO/caffeic acid played on peanut allergens Results showed cross link formation could mask the epitopes of peanut allergens after PPO/caffeic acid treatment. The IgE binding was markedly reduced based Western blots and ELISA results ( Chung and others 2005 ) Tannic acid Tannic acid is another chemical that can cause protein cross links. Tannic acid is a water soluble polyphenol compound and an antioxidant that is normally stored in barks, legumes, coffee, tea, and other food products ( Schofield and others 2001 ; Barbehenn and Constab el 2011 ) .Tannic acid is used in the leather industry for manufacturing tanning leather, in burn treatments as a topical agent ( Halkes and others 2001 ) as anti bacterial agent in medical studies ( Akiyama and others 2001 ) and as anti ulcer agents ( Souza and others 2007 ) Due to its ability to form protein cross link complex, tannic acid has shown effectiveness in eradicating mite allergens and cat allergens in house dust ( Woodfolk and others 1995 ) and deproteinizing bacterial genomic and plasmid DNAs ( van Huynh 2008 ) Tannic acid has a higher affinity for proline rich proteins ( Luck and others 1994 ) This study demonstrated that complexes


52 formed from tannic acid and peanut allergen interactions would be very stable under acidic and alkaline conditions. Results showed that tannic acid removed peanut allergens from peanut butter extracts at a pH of 7.2 by forming insoluble complexes with the allergens. The major peanut allerg ens (Ara h 1 and Ara h 2) also formed complexes with a tannic acid concentration over 0.5 mg /ml at pH 2 and 8 The data indicated that the insoluble proteins may be stable under gastric and intestinal conditions and ultimately pass through without any all ergenic issue ( Chung and Reed 2012 ) To verify the actual effect s of all these enzymes and acids on peanut allergy, clinical trials and animal tests are still needed. No report exists as of now Figure 2 1 Thermal effect s on the protein structure changes.


53 CHAPTER 3 INVESTIGATION OF THE EFFECTIVENESS OF PUL SED LIGHT ON PEANUT ALLERGENS Introduction Recently, novel processing technologies such as PL have been shown to effectively reduce the allergen level of different foods. F or example, Chung and oth ers ( 2008 ) have shown that PL can effectively reduce the major allergens Ara h 1 and Ara h 2 in both peanut extracts and peanut butter slurry. Yang and others ( 2011 ) reported that PL significantly reduced the immunoreactivity of the major allergens Ara h 1 Ara h 2 and Ara h 3 in raw and roasted peanut extracts and peanut butter slurry. Yang and others ( 2010 ) showed a significant reduction of allergen levels by PL treatment of soybean extra cts. A study by Shriver and others ( 2011 ) indicated that PL caused a notable reduction of allergen in shri mp extracts. Li and others ( 2011 ) have also demonstrated that PL can significantly reduce the IgE immunoreactivity of almond extracts. The PL technology uses pulses of intense broad spectra containing approximately 54% UV, 20% infrared and 26% visible light ( Shriver and Yang 2011 ) During PL emission, inert gas, such as Xenon, contained inside the PL lamp is excited by high voltage from a ground state to an excited state. When the Xenon molecules come back to a ground state, energy is released in photons and absorbed by the food molecules, which can lead to photo thermal, photo physical, and photo chemical effects on foods ( Shriver and others 2011 ) The foregoing triple photo effects are res ponsible for the putative mechanism for the interactions between PL and the food molecules ( Krishnamurthy and others 2007 ) Previously, PL was considered as a non thermal technology used for inactivation of


54 microorganisms in foods a fter a short exposure (e.g., seconds). However, recent research has pointed out that PL may also have photo thermal effect besides its non thermal nature, since extended exposures (e.g. minutes) can cause a pronounced temperature rise and moisture evaporat ion on shrimp and almond ( Yang and others 2012 ) Furthermore, the photo thermal effect can cause modification of protein allergen reactivity via protein cross linkage, protein solubility reduction, and protein fragmentation under instanta neous high temperature (Li and others 2011; Yang and others 2012 ). In Chapter 3, the pr eliminary study on the variation of allergens in pea nut kernels after PL treatment has been investigated. Material and M ethods Materials The materials used in preparing the peanut samples were aluminum weigh ing dish obtained from Fisher Scientific.Inc. ( Allentown PA USA), Whatman filter paper #4 from Wha tman ( DeKalb County IL USA ), acet one Fisher Scientific Inc. (Allentown, PA USA ), ethylene glycol tetraacet ic acid (EGTA) from Sigma ( St. Louis ., MO USA ) and hexane from Fisher Scientific Inc. (Allerntown, PA USA ). The reagents used in the immunologic al tests included Coomassie Plus Protein assay from Bio rad ( Hercules CA USA), bovine serum albumin (BSA) from Invitrogen ( Carlsbad, CA US A ), Ge lCode Blue gel staining reagent from Invitrogen ( Carlsbad, CA US A ), Tris buffered saline (TBS) from Fisher Scientific Inc. (Allentown, PA USA ) phosphate buffered saline (PBS) from Fisher Scientific Inc. (Allentown, PA USA ) o phenyl endiamine dihydrochloride (OPD) and Starting Block TBS/T 20 blocking buffer and Gelcode Blue stain reagent and bicin ochoninic (BCA) protein assay kit from Invitrogen ( Carlsbad, CA US A ) betam ercaptoethanol and Tween 20 from Pierce Chemical ( Rockford, IL USA ). Fresh


55 human plasma (IgE), which is specific to peanut allergy, was measured by ImmunoCAP testing and was shown as 86 kilounits per liter (kU/L). Precast trisglycine minigels (4 20%) and reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) were obtained from BioRad Laboratories (Hercules, CA USA ). Costar 96 well plate produced by Corni ng Incorporated (Corning, NY USA ) was used for the enzyme linked immunosorbant assay (ELISA) and the immobilon P blotting membrane was obtained from Millipore Corporation (Bedford, MA USA ). Horseradish peroxidase conjugated mouse anti human antibody was purchased from Invitrogen (Carlsbad, CA USA ). Super Signal West Pico Chemiluminescent Substrate was p urchased from Thermo Scientific ( Rockford, IL USA ). P ulsed L ight Treatment of Peanut K ernels A PL applicator of Model# LHS40 LMP HSG from Xenon Corp ( Wilmington, MA USA ) was used in this study for the PL treatments. The PL system consists of two lamps filled with Xenon gas, two cooling blowers, one treatment chamber with a conveyor belt, and a control module. Raw runner type peanuts w ere purchased from the local market and shelled by hand to yield the peanut kernels for use in this study. The PL system produces a broadband spectrum between 100 to 1100 nm and operates at 3 % energy in the ultraviolet, visible, and infrared regions, respectively. As per the centimeter squared (J/cm 2 ) per lamp at 7.6 cm below the central axis of the PL lamp. Th e PL parameters for treating the whole peanuts included the PL duration and the distance of the sample to the quartz window of the PL lamp. Shriver and others ( 2011 ) used the same system for allergen reduction of shrimp protein extracts, which


56 were placed in an aluminum dish of 7.2 cm diameter and illuminated at a distance of 10 cm from lamp. T wo distances from lamp were chosen, 10 cm and 7 cm, along with PL durations ranging from 5 min to 9.5 min. The treatment times were selected based on the degree of ro asting that occurred among the peanut samples, ranging from lightly roasted to burnt, during the preliminary tests. In order for both sides of a peanut kernel to expose to PL illumination, there are two possible options for placing a kernel on the aluminum dish. One is to treat the whole kernel for half the preset duration and then flick the kernel upside down to continue with the 2 nd half treatment. The other is to naturally split the whole kernel into two halves and then treat both half kernels all at onc e for half the preset time as for a whole kernel. In this study, the second option was adopted. Three peanut kernels (cotyledon) were separated into two halves and placed linearly in the center of an aluminum dish. The dishes were loaded below the PL quart z window along its central line. The surface temperature of the samples was recorded using an Omega OS423 LS no n contact infrared thermometer from Omega Engineering Inc. ( Stamford CT USA ) before and after PL treatments. The samples were cooled by placing the aluminum dish o n ice immediately after the surface temperature was recorded Variations in moisture w ere also recorded by measuring the peanut mass on a balance before and after PL treatments. Triplicate measurements were conducted for PL treatment, t emperature recording and moisture variations. Preparation of Peanut E xtract after PL T reatment To perform the immunoassays, it was required that crude protein extracts be prepared in a manner that did not change the native properties of protein in the pean ut kernels. The extraction followed the method by ( Chung and Champagne 2001 ) For


57 each time treatment the samples were ground with a Wiley mill in cold acetone until they become finely milled. After that, the meals were placed on the filter with Whatman filter paper #4 using hexane as an eluted solvent until they become white. These defatted peanut meals were air dried at RT for 1 2 h. To extract the soluble peanut proteins, the dry peanut powders were n ormalized by stirring 0.1 g of meals in 0.7 ml of 0.02 M sodium p hosphate containing 10 mM EGTA obtained from Sigma ( St. Louis ., MO USA ) with the final pH adjusted to 7.4. These samples were placed at 4C for 1 h r to fully collect soluble proteins and the n centrifuged at 8500 x g for 10 min. The resultant supernatants were stored at 20C for further immunoassays. Bradford protein assay was used to determine the concentration of soluble proteins in the PL treated samples. SDS PGAE of Peanut E xtracts To ex amine the soluble proteins in different samples, SDS PAGE was performed based on the Laemmli method ( Laemmli 1970 ) Basically, peanut extracts were loaded on 4% 20% Tris glycine gels (5 g / well) combined with a sample buffer, which included 62.5mM Tris HCL, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue mercaptoethanol. This mixture was boiled for 5 min. The gel was subject to electrophoresis at 150 V for 1.5 h. Subsequently the gel was stained with GelCode Blue reagent for 2 h and de stained afterwards with distilled water for 2 h until there was a sharp contrast between the stained protein bands and the clear background. Western Blot of Peanut E xtracts Western blot assay was performed according to the method described by ( Towbin and others 1979 ) After separation of the proteins on SDS PAGE gel the protein bands were transferred to a PVDF blotting membrane for 30 min at 15V. On the


58 completion of transferring proteins, the membrane was washed by 20mM Tris saline buffer (TBS) with pH 7.4 and then blocked with StartingBlock blocking buffer. The me mbrane was incubated overnight at 4C with pooled human plasma containing anti peanut IgE antibodies (1:500) diluted in blocking buffer. After that, three separate washes with TBST (TBS 1X with 0.05% Tween 20) were done, using 5 min time periods. The blot was then incubated with a secondary antibody (1:1000) at room temperature (RT). Another series of washes was done with TBST, but this time, the time points we re 5 min, 10 min and 10 min at room temperature On the completion of the washing step, the membra ne was inc ubated by SuperSiganl West Pico Chemiluminescent substrate for 5 min at RT. The protein bands signal was developed on X ray film. Dot Blot of Peanut E xtracts For dot blot analysis, a nitrocellulose membrane (0.45m) was blotted with 2.5 g of raw and PL treated peanut samples and then dried at 4C. This was followed by blocking the whole membrane with StartingBlock blocking buffer. After that, the blot was incu bated for 1 h at 37C. After incubation with StartingBlock blocking buffer and subsequent washing, the blot was incubated with pooled human plasma IgE (1:500) and then with goat anti human IgE HRP (1:1000) for 1 h at RT for each step. Washes were done betw een each incubation step and after the final incubation. Protein dots were then developed on X ray films following the exact procedure described above for Western blotting. The diluent used for the IgE and goat anti human IgE HRP was StartingBlock blocking buffer.


59 Indirect ELISA of Peanut E xtracts Diluted peanut extract prepared to a final concentration of 2 g / 100l with PBS buffer (pH 7.4) was coated on to a polystyrene 96 well plate for 2 h at 37C. Each protein sample was added in triplicates with 10 0 l per well. The plates were then washed with TBST three times and blocked with StartingBlock blocking buffer (200 l per well) on a rocker at RT for 2 h. After another round of washing, 100 l of pooled human plasma containing IgE antibodies (1:500 dilu tion in 1X PBS) was added to each well. The plate was incubated at 37C for 1 h r After the TBST wash, mouse anti human IgE conjugated HRP antibody (1:3000) was added next in the same manner, with 100 l per well. Incubation followed for 1 h. After another washing step, the protein antibody bonded plate was developed with OPD substrate (0.5mg/ml) that was prepared by dissolving 0.1M citrate buffer (pH 5.5) and 0.03% hydrogen peroxide (100 l per well). The developed time was between 15 30 min and was stop ped with 2.5 N sulfuric acid (100l per well). The absorbance was measured at 490nm using Spect ramax 340384 spectrophotometer that was from Molecular Devices Inc. ( Sunnyvale, CA USA ). Results and D iscussion SDS PAGE of PL Treated Peanut K ernels Shown in Figure 3 2 is the soluble protein profile of raw and PL treated peanuts. Protein bands in the raw sample were differentiated as Ara h 1 (63 kD), Ara h 2 (17 20 kD), Ara h 3 (14 45 kD), Ara h 4 (37kD) and Ara h 5 Ara h 11 (9.8 17kD) (Finkelman 2010; Pele 2010), with the major four marked in the figure Since Ara h 3 and Ara h 4 share a 91% identical sequence, the band at 37 kD could be Ara h 4 or a mixture of both ( Kleber J anke and others 1999 ) The two bands between 20 and 37 kD belonged to Ara h 3, which may consi st of a series of polypeptides ranging from 14 45 kD.


60 For the 10 cm distance to lamp (left), compared to raw, 5 min and 6 min PL illumination did not cause a considerable reduction in the band density of Ara h 1, Ara h 2, Ara h 4 and the lower band of Ar a h 3, but for the upper band of Ara h 3, pronounced reduction was observed at 5 min and total disappearance occurred from 6 min and up. From 7 min to 9.5 min, all the bands related to Ara h 1 to h 4 were either substantially reduced or totally disappeared For the 7 cm distance to lamp ( the right group ), 5 min PL illumination did not cause a considerable reduction in the band density of Ara h 1, Ara h 2, Ara h 4 and the lower band of Ara h 3, but for the upper band of Ara h 3, PL treatment for 5 min and up caused a total disappearance of this band. From 6 min to 7 min, all the bands related to Ara h 1 to Ara h 4 were either substantially reduced or totally disappeared. No experiments were carried out beyond 7 min, because the sample was already nearly burnt at 7 .5 min. The reduced or disappeared protein bands of Ara h 1 to Ara h 4 were considered to have formed insoluble aggregates or been cleaved into smaller polypeptides, because PL treatment can lead to insoluble complex formation between molecules, de po lymerization of starch, peroxidation of unsaturated fatty acid, carbohydrate cross linking, protein cross linking and protein fragmentation ( Fiedorowicz and others 2001 ) The PL treatment could also cause aromatic amino acids to form cross links in proteins due to the absorption of UV radiation, such as tyrosine and phenylalanine ( Gennadios and others 1998 ) In a study reported by Chung and others ( 2008 ) peanut protein extracts were PL treated for 4 min and peanut butter slurry for 3 min, both at a distance of 14.6 cm to lamp. Their results indicated that the soluble p eanut proteins could form


61 cross links and consequently aggregates or precipitates, rendering them insoluble. However, those small soluble peanut proteins (17 20kD) still remained in a soluble state after PL treatment. The insoluble aggregates usually sho w up on high molecular weight area of the gel if the entire homogenized peanut extract (without centrifuge) is loaded (Li and others 2011; Yang and others 2011), but they did not show up in Figure 3 2 because only the soluble proteins after centrifuge were used in this study and the insoluble aggregates were removed during centrifuging. However, in Figure 3 2 many new low molecular bands (below 17 kD) were generated after 7 min illumination (for 10 cm distance to lamp) and after 6 min illumination (for 7 cm distance to lamp), which is a clear indicat ion of the fragmentation effect of PL on the peanut allergen proteins. The PL illumination may also have an instantaneous photothermal effec t during the treatment. Wekhof ( 2000 ) proposed that temporary overheating was produced during the PL illumination du e to the absorption of all the UV light from the flash lamp when the overall energy exceeded 0.5 J cm 2 Another study indicated that the overheating c ould be attributed to a difference in UV light absorption by bacteria and the surrounding medium ( Takeshita and others 2003 ) Maleki and othe rs ( 2010 ) demonstrated that thermal processing of peanuts alters the solubility of proteins and can lead to structural and chemical modifications, giving rise to a change in immunoreactivity of IgE. Thermal conditions can accelerate the Maillard reaction between reducing sugars and free am ino groups of proteins, causing specific chemical modifications to protein molecules or inter and intra molecular covalent cross linking or degradation of bonds ( Maleki and others 2000a ; Maleki and Champagne 2001 ; Maleki


62 and others 2003 ) However, the exact mechanism of PL treatment on protein structural changes is still not clear. Western Blot of PL Treated Peanut K ernels The foregoing SDS PAGE resu lts indicate that PL illumination was capable of reducing the immunoreactivity with Ig E for major allergens in the PL treated peanut kernels to a low level. To characterize the immunoreactivity of IgE with the antigenic proteins of the PL treated peanuts, Western blotting was performed in vitro using pooled human plasma that contained the IgE antibody specific to peanut allergens. The raw peanut sample was used as control. Figure 3 3 shows the immunoreactivity signal of IgE on the Western blot membrane after X ray film exposure. The raw peanut protein bands were detected at 63 kD (Ara h 1), 17 20 kD (Ara h 2), Ara h 3 (14 45 kD), Ara h 4 (37 kD) and Ara h 5 h 11 (10 20 kD). For the 10 cm distance to lamp (left), except for 5 min and 6 min PL illumination times where the allergens Ara h 1 h 4 showed a marginal reduction, 7 min to 9.5 min PL treatments caused the major allergen bands to substantially diminis h or even disappear. For 7 cm distance to lamp (right), Ara h 1 Ara h 4 was significantly reduced or disappeared beginning from 5 min PL treatment. For Ara h 5 Ara h 11, 5 min and 6 min PL illumination resulted in a considerable reduction, as can be se en from the much reduced band density in lane 5 and 6 in the 10 cm section (left). However, as the PL duration increased to 7 and beyond, the band intensity at 10 20 kD increased, because the protein fragments as discussed previously in Figure 3 2 were formed due to the degradation of Ara h 1 h 4 caused by PL illumination. Some of the fragmental polypeptides might still carry effective epitopes that reacted with the I gE. However, from


63 Figure 3 3 (left) it can also be seen that when the PL duration further increased, the band intensity in the 10 20 kD range tended to diminish. This is especially evident for 7 cm distance from lamp ( right). Results in both Figure 3 2 and Figure 3 3 signify that with a shorter distance to lamp and longer durations of exposure, which could result in a higher energy release to the peanuts, PL illumination could significantly minimize the levels of allergen Ara h 1 11 in whole peanuts, even including minimizing the IgE reactivit y of the fragments decomposed from Ara h 1 Ara h 4 due to PL treatment. Maleki and others ( 2010 ) indicated that the decreased levels of allergens may be due to oligomerization or degradation resulting from the over processing effects. Although in this st udy the PL treated peanuts might not have been over processed, it is believed that oligomerization or degradation of peanut proteins might have contributed to the significant reduction of IgE immunoreactivity of PL treated peanuts. Dot B lot of PL T reated P eanut K ernels Dot blot is a technique for detecting, analyzing and also identifying proteins. It is similar to the Western blot in general, but differs in regard to the loading of protein samples as a dot on the membrane rather than separating them into di fferent protein bands. Compared to the Western blot, Dot blot has an advantage in that it can detect more proteins in peanut extract samples, including the smaller proteins under 10 kD that would have otherwise missed in the Western blot or the bigger prot eins that were unable to enter the SDS PAGE gel and would not get transferred to the Western blot membrane. In this experiment, the p eanut protein extracts of the PL treated and the raw peanuts (control) were loaded on a nitrocellulose membrane. The distan ce for the PL treatment was set at 10 cm with the durations being 5, 6, 7 and 7.5 min.


64 From Figure 3 4 it is evident that the IgE reactivity to total peanut allergens was significantly reduced at 7.0 min PL illumination and basically undetectable at 7.5 min. As discussed previously, the phot ophysical, photochemical and also photothermal effects of PL could alter the protein structure to reduce the immunoreactivity with the human IgE antibody. To support dot blot immunoassay, indirect ELISA was performed to provide further evidence. Indirect E LISA Figure 3 5 shows the results of immunoreactivity for the PL treated peanuts at different durations for both 10 cm (A) and 7 cm (B) distances from lamp. The raw peanut was used as positive control and normal human plasma was used as negative control. starting at 6 min PL exposure. When the PL duration reached 7.5 min, peanut allergens al ready diminished to a level similar to the negative control. starting at 5 min PL exposure. When the PL duration reached 6.5 min, peanut allergens already diminished to a level si milar to the negative control and no significant increase in allergen reduction was observed with a further increased PL duration beyond 6.5 min. This in vitro ELISA data shown in Figure 3 5 were consistent with the results of SDS PAGE, Western and dot blot analyses stated previously, which all demonstrated that PL treatment could remarkably reduce the allergens of whole peanut kernels to a level near negative control.


65 Influence of PL D uration and D istance on IgE Binding Reactivity to Whole peanut allergens For PL treatment of foods, the sample distance from the light source (which also relates to the PL intensity), number of pulses, thickness of the sample, and duration of illumination are considered as critical parameters for the optimization of a PL system ( Soliva Fortuny and others 2010 ) For the PL applicator used in this study, the number of pulses was set at 3 pulses/s, the thickness of treated sample was also constant, but the PL duration was varied between 5 min and a maximum of 9.5 min, depending on the distance to lamp, and the intensity of the light was varied by two distances to lamp: 7 cm and 10 cm. Shriver and others ( 2011 ) used a distance of 10 cm to achieve shrimp allergen reduction at different PL durations and a significant reduction was detecte d at 4 6 min. Chobert and others ( 2008 ) found that PL treatment at 4 cm distance to lamp led to protein aggregation and lipid oxidation. Based on the literature, distances of 10 cm and 7 cm w ere used in this study for PL roasting of whole peanuts for allergen reduction and quality retention as assessed by color and texture analyses. From the SDS PAGE ( Figure 3 2 ) and Western blot analyses ( Figure 3 3 ), the total protein profile as well as the specific protein bands related to Ara h 1 Ara h 4 shows a significant redu ction at 7 min PL treatment with 10 cm distance to lamp and at 6 min PL treatment with 7 cm distance to lamp. As the illumination energy increased, i.e., closer distance to lamp and longer PL duration, all the protein bands related to the 11 peanut allerge ns were minimized to an undetectable level. Li and others ( 2011 ) also showed that distance clearly influenced the reduction efficacy of PL on almond allergens due to the changes of energy incidence. It has been shown that the longer the distance between the sample and the lamp, the lower the lethality is for the process


66 ( Gomez Lopez and others 2005 ) and the longer the treatment time and the shorter the distance between the sample and lamp, the higher the inactivation ( Gomez Lopez and others 2007 ) Summary and C onclusion s P ulsed light t echnique was investigated in this study for its efficacy on reducing the immunoreactivity of whole peanut kernels at two sample distances to light source (7 cm and 10 cm), a PL frequency of 3 pulses/s and PL durations of 5 min to 9.5 min. Results of SDS PA GE, Western Blot, Dot Blot and ELISA with pooled plasma of allergenic patients show that PL was capable of reducing all major allergens of whole peanut kernels, including the polypeptide fragments degraded from Ara h 1 Ara h 4 during PL treatments, to an undetectable level, when a proper combination of illumination durations and sample to lamp distances was chosen. A closer distance (7 cm) between the PL lamp and the sample resulted in stronger reduction of IgE immunoreactivit y than 10 cm. To optimize this equipment, more trails of processed peanuts are required. Statistical analysis is needed to determine the interaction between treatment time and distance these two major factors of this PL applicator. The i nsoluble fractions in treated peanut seeds could not be detected by normal SDS PAGE gels. The IgE reactivity of those insoluble fractions needed to be explore d for the PL validation


67 Figure 3 2 SDS PAGE profiles of raw and PL treated pea nut extracts at different durations of illumination. The l eft shows the peanut samples PL treated at 10 cm distance to lamp, while the right 7 cm distance to lamp. The major peanut allergens Ara h 1 (63 kD), Ara h 2 (17 20 kD), Ara h 3 (14 45 kD) and Ara h 4 (37 kD) are labeled on the gel.


68 Figure 3 3 Antigenic protein banding patterns in raw and PL treated peanuts via western blot in vitro analysis with pooled human plasma. Four major allergens Ara h 1 to Ara h 4 a re labeled in figure and R is raw peanut sample. The left arrow is for 10 cm distance to the quartz window and the right arrow is for 7 cm distance to the quartz window.


69 Figure 3 4 Dot blot analysis of raw (control) a nd PL treated peanuts. The total proteins loaded on the nitrocellulose membranes were 2.5 g and 1.25 g, respectively. The PL treatment was conducted at 5, 6min, 7 and 7.5 min at 10 cm distance from lamp.


70 Figure 3 5 Indirect ELISA of PL treated peanuts at 10 cm ( A) and 7 cm ( B) distance to lamp with pooled human plasma containing IgE at different illumination durations. Absorbance readings were obtained at 450 nm. Raw sample was used as positive control. Normal human plasma was applied for negative control as shown in the last column. A = Absorbance of treated samples; A 0 = student t


71 CHAPTER 4 EXAMINATION OF IGE I MMUNOREACTIVITY OF I NSOLUBLE FRACTIONS O F PULSED LIGHT TREATED PEANUTS BY NOVEX NUPAGE ELEC TROPHORESIS SYSTEM Introduction The objective of this experiment was to investigate the IgE immunoreactivity of insoluble fractions formed during PL treated peanuts. A previous study indicated that PL was a suitable non thermal technology for inactivation of bacteria, virus and molds consequently, this technology has been applied to a lot of food industries, such as the following: beverage pasteur ization and surface sterilization of vegetables ( Koutchma 2008 ) However, studies about allergen reduction in different food samples, such as shrimp and almond, has indicated that PL can no longer be considered as a non thermal technology since temperatures significantly increase during processing ( Yang and others 2012 ) The energy simultaneously released within a short time and infrared light portion contributes the in creased temperature of the food sample. Pulsed light has a very high energy as evident by the fact that the intensity of pulsed light is 20,000 times more than the sunlight ( Mimouni 2001 ) .The energy output of a PL applicator was ranging from 1.8 to 5.7 J/ cm 2 per pulse at 1.8 cm below the lamp surface when the voltage was varied from 2000 to 3800 V ( Krishnamurthy and others 2009 ) The applicator has been used for this study was 2.7 J/ cm 2 per pulse at 10 cm ( Shriver and others 2011 ) In the case of heat denaturation of proteins, the process is generally considered irreversible. In brief, denaturation is defined as the process in which a protein is transformed from an ordered to a less ordered state that is due to the rearrangement of hydrogen bonding without any change to covalent bonds. Denaturation is used to refer


72 to aggregation, coagulation, and gelation. Aggregation is a term to describe protein protein intera ctions with formation of complexes of higher molecular weights that are normally occur in thermal processing ( Tanford 1968 ; Maleki and others 2000a ) PL processed peanuts were analyzed using SDS PAGE an d western blot as described in C hapter 1. The proteins extracted from PL treated peanu ts are soluble proteins that were quantified by the Bradford protein assa y The cross linked proteins were hardly detected as de scribed in Chapter 1 that is also because of the limitation of SDS PAGE gel we purchased from Bio Rad (the maximum protein molecules was around 250 kD) and the centrifuge step in protein extraction had filtered the insoluble fractions in protein samples. The denatured proteins and those amino acid residues trapped in the big complex via Maillard reaction during PL illumination cannot be detected with the gel for a general protein profile. To solve this p roblem, NuPAGE t ris acetate gels were selected. This type of polyacrylamide gel was designed to give optimal separation of large molecular weight proteins during gel electrophoresis. To detect the native state of PL treated proteins, the novex tris glycine native running buffer was applied for native proteins detection and NuPAGE Tris acetate SDS running buffer was used for denatured gel system in which the methods are described below. Materials and Methods Sample P reparation Raw runner type p eanuts were pu rchase from a local fresh market (Gainesville, Florida USA) The same applicator as shown in Chapter 3 was used in this study for PL treatment s A motor driven device was designed in this experiment as shown in Chapter 5 Eight kernels were placed in a transparent glass test tube with a hole (used for releasing the vapor pressure) at the bottom. This glass test tube with a stopper was


73 positioned at the top of the motor. The motor was placed on the conveyor belt of PL machi ne and adjusted to ensure that the rod of the motor was located in the center of the quartz window of the PL equipment. The treatment times were performed at 4 min, 5 min, 5.5 min, 6 min and 6.5 min. respectively. Protein E xtraction The extraction follow ed the method performed by Chung and Champagne ( 2001 ). For each treatment time, the samples were ground to a fine powder using a Wiley mill (Model number and manufacturer address needed here) in cold acetone around 0 C Subsequently, the meals were placed on the filter with Whatman filter paper #4 using hexane as the eluting solvent until the meal was white in color. The defatted peanut meals were air dried at room temperature for 1 2 h. To extract the soluble peanut proteins, the dried peanut powders we re normalized by stirring 0.1 g of meals in 0.7 m l of 0.02 M sodium phosphate containing 10 mM EGTA that obtained from Sigma Aldrich ( St. Louis, MO, USA) with the final pH adjusted to 7.4. These samples were stored at 4C for 1 h to fully generate. The hom ogenizer from ESGE Ltd. ( Mettlen Switzerland) was applied for the total protein homogenization. The Coomassie protein assay was used to determine the concentration of proteins in the PL treated samples for further SDS PAGE and western blot assays. Denaturing E lectrophoresis NuPAGE Novex Tris Acetate gels produced by Invitrogen ( Carlsbad, CA USA) were used for electrophoresis. The types of the comb of the gels were 1.0 mm and 1.5 mm with each well of 25 l and 37 l. The gels were in pre cast gel c assette with the size of 10 cm x 10 cm and then placed in XCell surlock TM Mini cell unit purchased


74 from Invitrogen (Carlsbad, CA, USA) for gel electrophoresis. The prepared protein samples were mixed with LDS sample buff er (4 x ) obtained from Invitrogen ( Carlsbad, CA, USA) to get final concentration at 2.5 g/ml and then the mixer of each sample was cooked fo r 10min before loading the gel. Each sample of different treatment samples was loaded as 25 l and triplicate for each of them. The concentration of t he gels we applied in this study is 3% 8%. 1 x NuPAGE SDS Running buffer obtained from Invitrogen (Carlsbad, CA, USA) was prepared by using 20 x NuPAGE SDS Running bu ffer, which was using 50 ml 20 x NuPAGE SDS Running buffer and mixing with 950 ml dei onized water. After assembled the XC ell unit, the voltage was set at 150 V constant. The expected current for the whole gel was around 40 55 mA / gel. When the protein bands (blue color) achieved to the bottom of the gel, the voltage should be cut off b y then. The gel will be stained by coomassie R 250 that was from Invitrogen (Carlsbad, CA, USA) for 2 h. Deionized water was used for background clearance after staining. Western B lot after D enaturing E lectrophoresis After separation of the proteins on SDS PAGE gel, the protein bands were transferr ed to a PVDF blotting membrane purchased from Millipore Corporation ( Billerica MA, USA) for 30 min at 15V. On the completion of transferring proteins, the membrane was washed by 20mM TBS at a pH 7.4 and then b locked wit h StartingBlock blocking buffer. The membrane was incubated overnight at 4C with pooled human plasma containing anti peanut IgE antibodies (1:500) diluted in blocking buffer. After that, three s eparate washes with TBST (TBS 1x with 0.05% Tween 2 0) were performed in 5 min time periods. The blot was then incubated with a secondary antibody (1:1000) at room temperature (RT). Another series of washes we re performed with TBST.


75 However in thi s experiment, the washing time s were 5 min, 10 min and 10 m in at RT. On the completion of the washing step, the membrane was incubated by SuperSiganl West Pico Chemiluminescent substrate for 5 min at RT. The protein bands signal was developed on X ray film. Non Denaturing E lectrophoresis The Novex Tris Acetate ge ls were also applied in this experiment. The mini cell unit was the same as the denaturing electrophoresis system. Novex tris gl ycine native running buffer (2 x ) was used for the sample preparation. The ratio of native sample buffer for sample preparation was 1:1 v/v. Each well was loaded with 25 L of sample and sample buffer mixer. After assembled the XCell unit, the voltage was set up at 150 V constant. The expected current for the whole gel was around 40 55 mA / gel. When the protein bands reached th e bottom of the gel, the power supply was turned off. The gel was stained by coomassie R 250 for 2 h. Deionized water was used for backg round clearance after staining. Western B lot after Non Denaturing Electrophoresis After separation of the proteins on SDS PAGE gel, the protein bands were transferred to a PVDF blotting membrane for 30 min at 15V. On the completion of transferring proteins, the membrane was washed by 20mM TBS with pH 7.4 and then blocked wit h StartingBl ock blocking buffer. The membrane was incubated overnight at 4C with pooled human plasma containing anti peanut IgE antibodies (1:500) diluted in blocking buffer. After that, three 5min separate d washes with TBST wer e done The blot was then incubated wit h a secondary antibody (1:1000) at RT Another series of washes was done with TBST, but this time, the time points were 5 min, 10 min and 10 min at RT. On the completion of the washing step, the membrane was incubated by


76 SuperSiganl West Pico Chemilumines cent substrate for 5 min at RT. The protein bands signal was developed on X ray film. Indirect ELISA Diluted total peanut extracts prepared to a final concentration of 2 g / 100l with PBS buffer (pH 7.4) was coated on to a polystyrene 96 well plate for 2 h at 37C. Each protein sample was added in triplicates with 100 l per well. The plates were then w ashed by TBST for three times and blocked with StartingBlock blocking buffer (200 l per well) on a rocker at RT for 2 h. After another round of washing, 100 l of pooled human plasma containing IgE antibodies (1:500 dilution in 1X PBS) was added to each w ell. The plate was incubated at 37C for 1 h. After the TBST wash, mouse anti human IgE conjugated HRP antibody (1:3000) was added next in the same manner, with 100 l per well. Incubation followed for 1 h. After another washing step, the protein antibody bonded plate was developed with OPD substrate (0.5mg/ml) that was prepared by dissolving 0.1M citrate buffer (pH 5.5) and 0.03% hydrogen peroxide (100 l per well). The developed time was between 15 30 min and was stopped with 2.5N sulfuric acid (100l p er well). The absorbance was measured at 490nm using Spect ramax 340384 spectrophotometer. Results and D iscussion Denaturing E lectrophoresis D enaturing SDS PAGE results were shown in Figure 4 1 Roasted and raw peanut samples were considered as controls that were used to compare with the PL treated samples. The protein profile ranged from 37 kD to 460 kD. Raw peanut sample had different molecular proteins in the seeds, which was shown on SDS PAGE. The smaller proteins (< 37 kD) were eluting from the gel. Roasted peanut s were prepared at


77 165C for 15 min. The protein protein cross linkages were formed during the roasting. The trimer of Ara h 1 was formed and could be detected on the SDS PAGE, which had the molecular weight around 190 kD. The PL treatment times were selected at 4 min, 5 min, 5. 5 min, 6 min, 6.5 min respectively The smear was formed during the PL processing, which was observed on the SDS PAGE. The blue protein smear was dyed by coomassie blue and shown an increased amount with a longer treatment on SDS PAGE. The treatment at 6. 5 min did not have many sharp protein bands as compared to the raw samples. The smears were distributed from the top to the bottom of the gel. The smear s were considered to result from the photo thermal effect of PL that caused the aggregates or protein cr oss linkage formation. The aggregations of proteins were macromolecules and some of them could not enter into gel system even the Novex system was utilized that was designed for large molecules electrophoresis. From the Figure 4 1 it was illustrated that some proteins stayed on the top of the gel without entering into the electrophoresis system. Western B lot The Figure 4 2 showed the results of western blot that generated from Novex gel electrophoresis. The IgE immunoreactivity of the samples treated for 4 min was shown to exhibit a strong signal from 460 kD to 37 kD. The extended treatments after 4 min showed a distinct reduction of amount on overall protein bands However, for the 6.5 min treatment, it was difficult to detect the IgE reactivity. The roasted peanut was considered a control. The dark area of the roa sted sample of the western blot result illustrated that a strong IgE immunoreactivity of those protein protein cross linkages that were detected during the thermal processing. A study conducted by Maleki and others (2000) explained that the reas on for this phenomenon is that thermal processing such


78 as roasting, curing, and various types of cooking can cause multiple, non enzymatic, b iochemical reactions in foods. A major reaction that is related to these reactions is Maillard reaction browning, which i s considered as an important method in the development of flavor and color in peanuts. The modifications of the amino groups of proteins by Schiff bases formation with reducing sugars undergo rearrangement to form Amadori products. Amadori products would f inally degrade into dicarbonyl intermediates. These compounds are more reactive than the parent sugars and they could assist the amino groups of proteins to form cross links, stable end products called advanced Maillard reaction products (MRPs) or advanced glycation end products (AGEs). Proteins including allergenic proteins in peanut seeds could also be trapped in these macromolecules. This study also demonstrated that the smears that appeared on the SDS PAGE were formed during the thermal processing were due to the cross linkage and non cross linkage adducts produced by Maillard reaction. Western blot results showed that Ara h 3 or Ara h 4 could be detected in the 4 min, 5 min, 5.5 min, 6 min treated samples but not in 6.5 min. The reasons for this phenome non attributed to the amou nt proteins of loaded sample were comparatively large or some 37 kD protein cross linkage may formed during the PL process or PL could not inactive Ara h 3 or Ara h 4 bef ore 6 min treatment. Chapter 3 illustrated that soluble pean ut allergenic proteins were inactivated after PL treatment, which demonstrated that PL treatment could notably mask all the allergen proteins in treated peanuts due to the fragmentation and cross linkage o f treated proteins. The temperature profile we also measured during the processing will be discussed in Chapter 7 T he significant increase of temperature after PL treatment could be observed on each peanut kernel Infrared thermometer was used


79 to record the temperature changes before and after treatment. Normally, the room temperature was around 23 25C. The burnt peanut s amples could achieve 130C. These data will be presented and discussed in Chapter 7 The putative mechanism of PL can be explained as photothermal, photochemical and photophysical effect s on the food samples, such as shrimp, peanut, soybean ( Krishnamurthy and others 2007 ; Chung and Champagne 2008 ; Yang and others 2010b ) Protein fragmentation and cross linkage were two major reasons for destroying and masking epitopes of peanut allergen proteins. Infrared light was one composition of PL, with which the temperature of the food samples could be increased after processing. Previous studies have been shown that roasted peanut proteins such as Ara h 1 and Ara h 2 bind higher levels of IgE than raw peanuts ( Koppelman and others 1999 ; Maleki and others 2000a ) Another study revealed that thermal processing of peanuts could alter the solubility of the prote ins and leads structural and chemical modifications that contribute to the IgE binding increase. Overall, the thermal effect caused the peanut proteins to be less soluble in addition to large molecular weight smears being formed with increased time of heat ing. Aggregation of proteins was due to the protein covalent modifications other than disu lfide linkages. They have been covalently lin k ed since proteins held together with non covalent interactions such as hydrophobic, electrostatic, or disulfide bonds wo uld separate into monomeric components after boiling with SDS sample dye containing dithiothreitol ( Schmitt and others 2010 ) Structural and chemical alternations that occur during heat treatment cause d the peanut proteins to become less soluble contribute to enhanced IgE binding. Enhanced IgE binding was detected in t he


80 insoluble fractions which were believed to result from the Maillard reaction ( Gruber and others 2005 ) In addition, h eat transfer occurs through one of three methods, conduction, convection, and radiation. Foods and biological materials are heated primarily to extend their shelf life or to enhance taste. In conventional heating, heat is produced outside of the object to be heated and is conveyed to the material by convection of hot air or by thermal conduction. Under the infrared (IR) radiat ion, the heat energy generated can be absorbed by food mat rixes This radiation method transfers thermal energy in the form of electromagnetic (EM) waves and encompasses that portion of the EM spectrum ( Krishnamurthy and others 2008 ) Recently, more focus on thermal processing because of its higher thermal efficiency and fast heating rate/response time in comparison to conventional heating. IR radiation can be classified into 3 regions, near infrared (NIR), mid infra red (MIR), and far infrared (FIR), corresponding to the spectral ranges of 0.75 to 1.4, 1.4 to 3, and 3 to 1000 m ( Sakai and Hanzawa 1994 ) Amino acids, polypeptides, and proteins reveal ed two strong absorption bands localized at 3 to 4 and 6 to 9 m. Water and organic compounds such as proteins and starches, which were the main components of food, can absorb FIR energy at wavelengths greater than 2.5 m ( Sakai and Hanzawa 1994 ) Thermal inac tivation could damage DNA, RNA, ribosome, cell envelope, and proteins in microbial c ell Sawai and others ( 1995 ) demonstrated that the inactivation of E.coli was due to sub lethally injured cells production after irradiation. IR heating showed that the cell wall was more vulnerable than conventional heating. The order of magnitude of infr ared damages was shown as follow ; protein > RNA > Cell wall > DNA. However, FIR radiation q uantum


81 energy was reported it was insufficient for hydrolysis of hydrogen links in proteins and other biological macromo lecules. FIR irradiation caused destruction o f either polypeptide chain of protein or appearance of o ligopeptide. The s econdary structure of three proteins including alcohol dehydrogenase, peroxidase and trypsin has demonstrated that there was no significant change after FIR radiation. However it wa s detected that changes occurred in albumin helice s ( Govorun and others 1991 ) Ultraviolet radiation (UV) is a part of the non ionizing region of the electromagnetic spectrum that encompasses about 8 9 % of the total solar radiation ( Coohill 1989 ) UV is traditionally divided into three wavelength ranges: UV A (320 400nm) represent ing approximately 6.3 % of the incoming solar radiation and is the less hazardous part of UV light; UV B (280 around 1.5 % of the total spectrum, but can induce a variet y of damaging effects in plants; UV C (200 280nm) is extremely harmfu l to organisms, but not relevant under natural conditions of solar irradiation. UV irradiation can cause not only the modification or destruction of amino acid residues, but also leads to inactivation of whole proteins and enzymes. Inactivation of proteins and enzymes by UV light is due to photolysis of aromatic amino acids or disulfide groups if affected residues are included in active site ( Grossweiner 1984 ) UV C irradiation is known for its lethal activity against most microorganisms including bact eria and viruses. As one important study indicat ed UV C could change the structure of whey proteins and increased concentration of free thiol groups with UV exposure. This study also demonstrated that UV C irradiation can significantly change the tertiary and quaternary structure of whey proteins, accessibility of thiol groups, and


82 oxidation of tryptophan and tyrosine ( Kristo and others 2012 ) Moreover, another report indicated that UV C irradiation cause off flavors production on the whole goat milk ( Matak and others 2007 ) The UV light portion of pulsed light is consisted of most UV C. This UV C has a harmful effect on the enzymes and proteins in the food samples. Non Denaturing S ystem To obtain the actual effect on the native allergenic proteins rather than the denatured forms after dithio threitol treatment, a non denaturing system was utilized ( Figure 4 3 and Figure 4 4 ) In this study, the native SDS PAGE sample buffer was applied and there was no step for boiling sample/ sample buffer mixture to uncurl the protei n molecules. The samples loaded on the gels were maintained in their native states as the proteins were stored in treated peanut seeds. This study provided a further evidence for IgE reactivity examination of PL treated peanut allergens. SDS PAGE Figure 4 3 showed a protein profile of PL treated peanut protein samples at 4 min, 5 min, 5.5 min, 6 min, 6.5 min on Novex gel Roasted and raw peanuts were designated the co ntrols. The raw peanut sample illustrated sharp protein bands than roasted peanut that was primarily due to the Maillard reaction in roasted peanut sample. The smears were clearly detected in PL treated samples and roasted sample. The 37 kD was detected in each peanut sample, which may be Ara h 3 or h 4. The alle r gen Ara h 1 (63 kD) was also detected in each sample except the 6.5 min treatment. However, Ara h 1 cannot be detected after 5.5 min treatment. This may be due to the fact that some protein cross linkages and complex were formed during the processing and those macromolecules have sim ilar molecular weight around 63 kD. Moreover, the fragmentation of Ara h 1 occurred but those native proteins did not separate when


83 mixed with the sample buffer. The whole Ara h 1 was still appeared o n the SDS PAGE gel. Ara h 2 can not be detected on this g el because the smaller molecular weight that was held in this gel was 37 kD. Western B lot The results of w estern blot were illustrated in Figure 4 4 The IgE binding of each sample was detected on the transferred membrane. The signals were detected before 6.5 min through the top area to the bottom area of the PL treated samples. However, roasted peanut samples were darkened in color on their surfaces. The specific band of different allergenic protein could not be differentiated on the designated Western blot membrane. These observations could have been related to the different molecular size of protein cross linkage formation. Most of the proteins were trapped in the Ma illard reaction and finally had different molecular weight and distributed through the entire gel. All these proteins were curled and maintained as native state. The linear epitopes were masked inside without exposure. The conformational epitopes were the most important factors that produce the IgE reactivity. Indirect ELISA As previously discussed, the SDS PAGE and western blot were applied to detect the insoluble fractions above 37 kD. The smaller insoluble fractions were easily eluted from the gel. To include all the proteins and investigate the IgE reactivity to allergenic proteins the total peanut proteins were extracted by using a homogenizer. The roasted and raw peanut proteins were designated as the controls. The roasted peanut sample was normaliz operated with pooled human IgE. The results were presented in Figure 4 5 It was observed that the IgE reactivity of the roasted sample was significantly higher than the other samples with student t test


84 examination. The samples treated for 6.5 min showed the lowest signal value than the others. Overall, the results of this study clearly demonstrated that PL is quite effective on both solu ble and insoluble protein fractions inactivation. Summary and conclusion T he foregoing evidence s pointed to the inactivation effect of PL on insoluble allergenic proteins. In conjunction w ith the previous results on soluble proteins, it could be concluded that the inactivation effects of PL on both soluble and insoluble fractions were eminent In order t o further verify th e PL effect in vitro digestion study should be conducted to investigate the changes of IgE binding reactivity and immunogenic potential of the allergenic proteins when they are in digestive environment, such as different pH values and different enzymatic hydrolys e s.


85 Figure 4 1 Protein profiles of insoluble fractions of PL processed peanuts were analyzed by Novex denaturing electrophoresis. R was referred to a raw peanut sample. Ro was referred to roasted peanut sample. Different durations of PL treatment were expressed as 4 min, 5 min 5.5 min, 6 min, and 6.5 min. The range of protein standard was from 30 kD to 460 kD. The extended treatment s were at 5 min, 5.5 min, 6 min, 6.5min


86 Figure 4 2 Western blot of insoluble fractions of PL processed peanuts samples were compared to roasted peanut. Antigenic protein bands wer e labeled from 30 kD to 460 kD. Notable reduction of IgE binding can be detected in extended time pointed, such as 5.5min, 6min, and 6.5min.


87 Figure 4 3 Protein profiles of insoluble fractions of PL processed peanuts were analyzed by Novex non denatur ing electrophoresis R was referred to a raw peanut sample. Ro was referred to roasted peanut sample. Different durations of PL treatment were expressed as 4min, 5 min, 5.5min, 6min, and 6.5min. The range of protein standard was from 30 kD to 460 kD.


88 Figure 4 4 Insoluble antigenic protein fractions were analyz ed by western blot on non denaturing gel The IgE binding of large molecules that formed during PL treatment and roasting were examined up to 460 kD. Notable reduction was detected at 6.5min PL treatment. Ro was referred to roasted peanut sample. PL treatment was at 4min, 5min, 5.5min, 6min and 6.5min.


89 Figure 4 5 Effects of PL treatment o n total protein of peanut kernels with different duration at 10cm were determined by Indirect ELISA with pooled human plasma containing IgE antibodies. Absorbance readings were at 450nm. Roasted sample was considered as a positive control. Normal human pla sma was applied for negative control which is the last column in the graph. A= Absorbance of treated samples; A 0 = Absorbance of control. Results are significant signal reduction ( = 0.05) 0 0.2 0.4 0.6 0.8 1 1.2 Roasted Raw 4min 5min 5.5min 6min 6.5min Control Absorbance (A/A 0 ) *


90 CHAPTER 5 THE OPTIMIZATION OF PL EQUIPMENT AND THE INTERACTION EFFECT O F MAJOR PARAMETERS: TI ME AND DISTANCE Introduction The limitation of PL applicator in this study was observed when we conducted in itial experiments that were in C hapter 3 The non uniform irradiation of this equipment on the treated peanut kernel was observed, which meant some part of peanut had deeper roasting than other parts of one peanut kernel. The reason for this phenomenon was because the singl e lamp in quartz window, which indicated the light resources were scattered from the lamp. The unit area of peanut surface had received different amount PL and then induced different roasting effects of peanut surface. In Chapter 5 a new designed motor wa s applied for uniformly roasting. This motor would be fixed in a certain location on the conveyor belt to ensure the constant of PL treatment. The time treatment and distance were two major factors that could affect the effects of PL roasting. To optimize these two factors and obtain proper quality of processed peanuts were important in this study because our goal for this technology is to produce hypoallergenic peanuts that could replace the conventional roasted ones. Statistical analysis for color develo pment and IgE reactivity readings could be applied for this optimization. Proper roasted color with low IgE reactivity was an ideal state for PL treated peanut samples. From this analysis, the best treatment time and proper distance could be confirmed in t his study. The peanut products with these two fixed parameters were used for next digestion study. Another benefit for this optimization study is to choose a good peanut product for further hypoallergenic peanut butter producing


91 In this part of the experi ment the optimal treatment time and distance would be determined The treated peanuts would be further used for the digestion study. Material and M ethods Raw runner type p eanuts were purchase d from a Publix store in Gainesville, FL, USA. The same PL applicator was used in this study for PL treatment as indication in Chapter 3 A motor was assembled in the Egg Processing Lab of University of Florida with a steel rod, control pad, wires, motor, and a steel plate. It would allow for sample ro tation during the treatment. Normal glass tubes from Fisher Scientific ( Rockford, IL USA ) used to hold the sample during the treatment. Color A nalysis To quantify the color changes, a machine vision system consisting of a Nikon D 200 digital camera housed inside a light box [42.5cm (W) x 61.0cm (L) x 78.1 cm (H)] (Wallat and others 2002) was used to measure the color of peanut kernels with a D65 (daylight) lamp and 10 observer angle. Each image includes traditional roasted peanuts (control) and PL peanuts as measured samples. Each image was calibrated against a yellow color reference tile (L=87.09, a=7.71, b=70.75) that was obtained from the Lab Sphere X Rite Company (North Sutton, NH USA ). T he software LensEyeSK v10.0.0 from E ngineering and Cyber Soluti ons Inc. (Gainesville, FL USA ) was applied to collect and analyze the images. To obtain precise calibration, the initial image was obtained using the yellow round reference tile and then converted to a rectangular (~150 x 95 pixels) processed image that w as without interference from the black border of the yellow tile. Next, the processed images were subjected to background corrections to produce the corrected images. The final corrected images were calibrated with a standard yellow color (L ref a ref b ref ). The L, a, and b values from the Hunter color


92 system were recorded individually for each peanut sample. The Hunter L, a, and b color space is organized in a cube form. The maximum L is 100 (white) and the minimum L is 0 (black). A p ositive a value denote s red, a negative a value for green ; a positive b value for yellow, and a negative b value for blue This color analysis was triplicate d for each sample. Indirect ELISA All the reagents and the methods are the same as those mentioned in Chapter 3 Experimental D esign The two major treatment conditions investigated were treatment time and the distance between the sample and the central axis of source of PL lamp. A factorial analysis was applied in this research. Factorial analysis is used for analysi s of the effect of multiple factors at different levels and helps find out whether the two main factors are significant. In addition, the interactions between these two factors were also analyzed. The factors used in setting this design are discussed below Two distance 10 cm and 7 cm from the light source were applied. Treatment time points were selected at 5 min, 6 min, and 7 min. This time point selection was based on the maximum treatment time at 7 cm, which is 7 min. At this point, the peanut samples w ere considered burnt. Medium roasted peanut kernels were normally judged by the Hunter color Lightness (L) of 50 1.0 ( Greene and others 2006 ) The color analysis of the treated peanuts was used for kernel was measured for peanut color evaluation. Indirect ELISA was another test to evaluate the allergenicty of treated peanuts. The reading value 0.4 was considered as a threshold, below this value was considered as low allergenic reaction. Both color analysis and indirect ELISA were two types of indicators for PL treatment evaluation.


93 Treatment time was evaluated first since previous studies showed that the effect of PL on the food samples is highly dependent on the treatment time. Distance from the central axis of the PL lamp was the second factor considered. The intensity of PL is proportional to the distance from the PL source. The intensity of light energy is highest at the closest position of the sample to the center axis of the lamp and decreases as the distance between the two increases. Since the durations and distance have been selected, they were expected to provide significant differences on peanut allergenic po tency and provide an insight on the most effective energy levels on the reduction of peanut allergenic potency with proper color generation. After factorial analysis, the distance would be fixed at 10 cm for the further study. To explore the proper treatm ent time that could produce a low IgE reactivity with a good color after roasting, a one factor complet e ly randomized statistical design was applied for this purpose. Multivariate ANOVA analysis was also conducted using JMP Pro 10.0 ( Cary NC USA ). Result s and D iscussion Motor Mechanism A significant challenge was to achieve uniform treatment over the roasted peanut samples. A motor driven device was designed to handle the peanut samples. As mentioned previously, some parts of the peanut samples had darker spots than other parts of the peanut if the kernel were not rotated during PL treatment The components of this motor device are shown in Figure 5 1 The motor shaft was connected to a steel rod. On top of the steel rod, a glass tube with a stopper made of aluminium foil was placed. Peanut seeds were placed inside the glass tube prior to


94 treatment. Once the motor is powered on, the steel rod rotates. Se ven or eight full peanut seeds are rotated in glass tube with full exposure to the PL After the preliminary optimization of the distance and durations of this rotator, another problem that caught our attention was the inner pressure gener ated from the tem perature increase of the peanut seeds. The glass tube was always detached from the steel rod during the treatment. Then another improvement of the glass tube was to release the inner pressure during the PL treatment. Each glass tube was burning on the alco hol lamp to make a hole at the bottom. This remodeled glass tube was prepared before the PL treatment. Factorial Analysis Statistical analysis was performed using JMP Pro 10.0 software. Statistical analysis is based on two factor factorial complet e ly randomized statistical design at p < 0.05. In this analysis, distance was considered as first factor and time was considered as the second factor. The color analysis and indirect ELISA were considered as two responses. The combinations of this factorial a nalysis were 2x3=6 treatments. The interaction of distance and time was also determined at p <0.05. Color analysis The results of the F test in the color analysis show that distance and time effects are significantly different (p < 0.05). In addition, ther e is a significant interaction between the distance and time. All these results are shown in Table 5 1 Table 5 2 shows the least significant diff erence (LSD) that is ap plied for means separation. From the separated means, they present different levels A, B, and C. Time as another factor can cause signif icant effect on the color generation. The means of L value in each different time group show a significant difference at p < 0.05.


95 Table 5 3 gives the Tukey honestly significant difference (HSD), which examined the interactions between time and distance factors. There are six combinations: 7 cm with 5min, 6min, and 7min treatments and 10 cm with 5min, 6min, and 7min treatments. The least significant means describe the effects caused by these two factors, which are significantly interacted ( Figure 5 2 ) There is some interaction observed between the two factors. The T ukey HSD results indicate that 5min treatment at 10 cm and 5min treatment at 7 cm do not have significant differences; however, the rest of the combinations show a notable significant difference between the groups. Indirect ELISA The output of factorial analysis of indirect ELISA is shown in Table 5 4 T he alternative hypothesis is true and the null hypot hesis is rejected at p < 0.05. The significant difference betwe en two factors is shown in Table 5 5 The significant interaction between these two factors is also significant, which indicates that these two factors do not have parallel effects on the reduction of IgE reactivity to the allergenic proteins in the treated peanuts. The means of indirect ELISA readings are categorized as two groups by distance, 7 cm and 10 cm. The means of these two groups are displayed in Table 5 5 The first one is the mean of each group. The IgE react ivity is significantly low in the 7 cm group. The plot gives a clear comparison with these two means. This result shows that lower distance has a positive relationship with lower IgE reactivity In other words, the potency of PL has an increased effect on allergen reduction. Time was also examined as a factor by the tukey HSD test. These three groups are separated as A, B, C, which means they are significantly different. The mean of each time group is presented in Table 5 6 The IgE reactivity readings of the 7 min


96 group at two different distance parameters is significantly low than the other 5min and 6min groups. The interaction of time and distance is shown in Figur e 5 3. The two lines in the graph do not run parallel. Thus, the interaction between time and distance is significant in ELISA test. The results of the tukey HSD analysis are shown in Table 5 7 The 7 min group at 10 cm and the 6 min group at 7 cm treatment group do not have a significant difference, but the rest of combinations are significantly different. It is worth mentioning that the 5 min treatment groups at two d ifferent distances are significant different The same results are observed at 6 min and 7 min treatment groups. Overall, these results show that the effect of time is not the same as the effect of distance. In summary, the color analysis of the 7 min treatment peanuts is similar to the medium roasted peanut with a L mean value around 58, as compared to standard value of 50. The ELISA analysis shows treatment at 6 min and 7 min have more than 60 % IgE reactiv ity reduction as compared to the raw samples. This factorial analysis demonstrates that the 7 min treatment at two distance parameters (7 cm and 10 cm) is more similar to medium roasted peanuts us ed in the industry. Since the 7.5 min treatment at 7 cm dist ance from light source produced burnt peanut samples, it is impossible to extend the time treatment beyond 7min for a given distance of 7 cm. However, it is possible to extend the time treatment at a 10 cm distance because these produce peanuts with better texture and color and low IgE reactivity Previous studies about PL inactivating allergens in different food samples applied 10 cm as a fixed distance since it has a reasonable duration from food treatment. The maximum energy level of the emitted radiati on was 0.27 J/cm 2 per pulse, as per the


97 fact ory calibration. In Chapter 5 another one factor completely randomized design was applied to explore the optimization of distance and time at 10 cm to find out the time treatment with low IgE reactivity readings and proper texture. Multiv ariate ANOVA A nalysis After optimizing the motor driven rotator, 10 cm was selected as a fixed distance for this analysis and further study. The time periods for this treatment were at 10 min, 11 min, 12 min, and 15 min. The out put of statistical analysis is shown below. Color a nalysis Table 5 8 shows that the sum of squares of model is 2438.290 and t he error is 311.994. The degree of freedom is 15. The mean squares of model and error are 812.763 and 26.00, respectively. The p value is less than 0.0001, which is significant at p < 0.05. This means that it rejects the null hypothesis and accepts the alternative hypothesis. A Tukey H SD test ( Table 5 9 ) demonstrated that the separation of the means of each group between 12 min treatment and 15 min treatment groups. However, the 10 min and 11 min t reatment groups are similar in the color analysis. For the means of L value in the different groups, the 12 min treatment group has an average L value of 52.33. This is the closest value that is achieved with commercial medium roasted peanuts. The 15 min t reatment group was over roasted (burnt) and is indicated in Table 5 9 Indirect ELISA a nalysis The output is shown as Table 5 10 The sum of squares of model and error are 0.0023 and 0.002. The total degree of freedom is 23. The mean squares are 0.008 and 0.0001. The F ratio is shown as 77.19. This number is over 1, which means that it


98 rejects the null hypothesis but accepts the alternative hypothesis. Furthermore, different groups are significantly differen t in the F test calculations. The least squares means is listed in Table 5 11 The overall means of each group are quite low. The 12 min and 15 min groups could not be detected at observable values on the ELISA plate. The plot of the means of each group demonstrated that a longer treatment is positively related to the lower IgE reactivity of peanut samples. The tukey HSD test results are shown in Table 5 11 and show that the separated means in each time tre atment are significantly different. The 10 min, 11 min, 12 min, 15 min treated groups are significant difference of IgE binding. Based on this, a longer treatment is more p otent on allergen inactivation. Summary and C onclusion s In summary, the 12 min treatment group showed the best color with an average L value of 52.33 as compared to other groups. In addition, the 12 min treatment group presented a significant ly low IgE reactivity compared to the 10 min treatment group and the r aw peanut samples. The reduction at 12 min was 90% with respect to the raw peanuts. Thus, the 12 min group was selected for the in vitro digestion study because of its low IgE reactivity readings and the possibility of use in commercial settings.


99 Table 5 1 F test of distance and time interaction based on the L value Source Nparm DF Sum of squares F ratio Prob > F distance (cm) 1 1 831.1689 86.7176 <.0001* time (min) 2 2 62.2860 62.2860 <.0001* distance* time 2 2 12.6550 12.6550 0.0001* Table 5 2 Tukey HSD results describing 5 min, 6 min, and 7min treatment groups of L value Level Least sq Mean 5 min A 72.066667 6 min B 63.7858 7 min C 58.0358 Table 5 3 HSD of interactions between combination groups of L value Level Least sq mean 10, 5 A 74.001667 7, 5 A B 70.131667 10, 6 B 68.043333 10, 7 B 66.258333 7, 6 C 59.5283333 7, 7 D 49.813333 Table 5 4 F test output of indirect ELISA readings Source DF Sum of squares Mean square F Ratio Model 5 0.39431196 0.078862 927.0173 Error 12 0.00102085 0.000085 Prob > F C. Total 17 0.39533282 < .0001* Table 5 5 F test of distance and time factors based on Indirect ELISA readings Source Nparm DF Sum of squares F ratio Prob > F distance (cm) 1 1 0.13304201 1563.892 <.0001* time (min) 2 2 0.25922388 1523.572 <.0001* distance* time 2 2 0.00204607 12.0256 0.0014

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100 Table 5 6 Means separated by treatment time: 5min, 6min, and 7min of Indirect ELISA readings Level Least sq mean 5 min A 0.59106667 6 min B 0.47553333 7 min C 0.29921667 Table 5 7 Tukey HSD of interactions between combination groups (p <0.05). Level Least sq mean 10, 5 A 0.66946667 10, 6 B 0.55400000 7, 5 C 0.51266667 10, 7 D 0.40026667 7, 6 D 0.39706667 7, 7 E 0.19816667 Table 5 8 F test of distance and time interaction based on the color readings (p< 0.05). Source DF Sum of squares Mean square F Ratio Model 3 2438.2904 812.763 31.2607 Error 12 311.9941 26.000 Prob > F C.Total 15 2750.2845 <.0001* Table 5 9 Means separated by treatment ti me of color analysis L value (p< 0.05) Level Least sq Mean 10 min A 73.76500 11 min A 65.582500 12 min B 52.337500 15 min C 41.512500 Table 5 10 F test of time and distance interac tion based on Indirect ELISA (p< 0.05). Source DF Sum of squares Mean square F Ratio Model 3 0.02387682 0.007959 77.1922 Error 20 0.00206211 0.000103 Prob > F C.Total 23 0.02593893 <.0001*

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101 Table 5 11 Means separated by T ukey HSD of indirect ELISA readings. Level Least sq Mean 10 min A 0.13766667 11 min B 0.10455000 12 min C 0.08373333 15 min D 0.05091667

PAGE 102

102 Figure 5 1 A model and a picture of a motor designed for uniformly roasting for PL treatment. A) A motor designed for uniformly roasting is placed in pilot plan of food science department, University of Florida; B) A model describes the structure of this motor. Black box is motor. Yellow part is control pad. The rod is the drive for peanut rotating. The peanut samples are placed at the top of the rod in a glass tube.

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103 Figure 5 2 Factorial analysis: Tukey HSD of interactions between comb ination groups of color analysis values (p <0.05). Figure 5 3 Factorial analysis: Tukey HSD of interactions between combination groups of Indirect ELISA readings (p <0.05)

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104 CHAPTER 6 IN VITRO SIMULATED GASTRIC AN D INTESTINAL DIGESTI ONS OF PL PROCESSED PEANUTS Introduction Although PL ha d a notable effect on the allergen reduction of peanut kernels as discussed in the foregoing chapters, it is still unknown whether P L treated peanuts could sustain the gastrointestinal digestion without changing its immunogenic potential Literature ha s shown that digestion in the gastrointestinal tract by digestive enzymes can lead to changes in the allergenic potency of food allergen s. Therefore it is important to determine the allergenic changes of PL treated whole peanuts following digestion through simulated gastric and intestinal enzymes, which would determine more thorough ly the effect of PL on peanuts allergens when subjected t o gastrointestinal digestion Many of the major allergens in foods are resistant to digestion It is what distinguishes them from minor allergens, which are present in foods, but digested in the GI track. One study demonstrated that the digestion of peanut extract with pepsin did not affect the IgE binding properties, even though substantial proteolytic bre akdown and fragments formed ( Vieths and others 1999 ) The digestion of purified peanut allergens was investigated by ( Koppelman and others 2010 ) However, the digestion of the total protein extract s from the processed peanut kernels, such as roasted peanut and PL treated peanuts ha s not been examined. Some studies have indi cated that the food allergens digested by digestive enzymes in the stomach or intestines could modify the protein conformation ( Untersmayr and Jensen Jarolim 2006 ; Shriver and Yang 2011 ) It is important to

PAGE 105

105 determine the allergenic potency of PL treated peanut extracts to understand the IgE reactivity changes of these peanut samples. As d iscussed in the C hapter 3 Ara h 1 was considered to have molecular weight of 63.5 kD that normally occur s in a trimeric form of around 180 kD ( Burks and others 1991 ; Becke r 1997 ) It is formed via non covalent interactions after thermal treatment s ( Shin and others 1998 ; Koppelman and others 1999 ) The trimeric Ara h 1 often forms aggre gate s forming multimers of up to 600 700 kD ( Van Boxtel and others 2006 ; van Boxtel and others 2007 ) We have demonstrated this in Chapter 3 and Ch apter 4 Ara h 2 migrates as a doublet at around 20 kD ( Burks and others 1992 ) Ara h 3 is a more com plex allergen. It is initially identified as a 14 kD protein ( Eigenmann and others 1996 ) but subsequent p urification of Ara h 3 show s that this protein consists of a triplet around 42 45 kD, and another distinct band at 25 kD, and some bands in the range of 12 18 kD ( Koppelman and others 2003 ; Piersma and others 2005 ) Lastly, Ara h 6 was found to have a molecular weight around 15 kD ( Koppelman and others 2005 ) In this study, the incidence of these allergens in simulated gastric and intestinal fluids w as analyzed by SDS PAGE, W estern blot ting and indirect ELISA. Material and M ethods The runner type p eanuts treated by PL at 12 min were chosen in this study due to the presentable color, texture, and comparatively low allergenic potency Roasted and raw peanuts were considered as controls. The processing methods for peanuts are the same as shown in Chapter 3. Simulated Peptic D igestion This procedure followed Laemmli ( 1970b) The simulated gastric fluid (SGF) was prepared with a pH adjusted to 1.2, and also includ ed 0.063 N HCl, 35 mM NaCl and

PAGE 106

106 4000 U pepsin. The porcine pepsin was purchased from sigma ( St Louis, MO USA). The SGF was pre warmed in the incubator at 37C and 80 L of 5mg/ml extracted peanut proteins were added in 1.52 ml SGF at time point t=0. The p epsin: substrate protein ratio was 10 U of pep sin:1g of substrate protein. The pepsin activity was prepared at 3300 U/ml. A substrate protein concentration of 250 l 1mg/ml peanut protein extracts and 760 g/ml pepsin were applied. 200 l of the reaction samples was collected at different time points : 0.5, 2, 5,10, 20, 30, and 60 min. Digestion reaction was stopped at appropriate times by mixing with 100 l of 200 mM NaHCO 3 (pH=11.0) and also 100 l 2X concentrated electrophoresis buffer that contain ed 40% glycerol, mercaptoethanol, 0.33 M Tris (pH 6.8) and 0.05% bromophenol blue. All the samples were heated for 5 min at 100C. The sample at time point t = 0 was prepared by adding bicarbonate and L aemmli buffer and heating the mixture of SGF prior to the addition of test proteins. Af ter the tested proteins were added, the samples were heated again to ensure full protein denaturation. Potential pepsin digestion was tested by adding 80 l of water to SGF and incubat ed for 60 min. Simulated Intestinal Di gestion This procedure followed Ko ppelman and others (2005b) Simulated intestinal fluid (SIF) was prepared by adding 65 mM Tris buffer at pH 8.3 containing 1 mM EDTA. The trypsin chymotrypsin, having enzyme activities of 325 U/mg of protein and 62 U/mg of protein, respectively, were added into the SIF at a concentration of 3.2 mg/ml. These two enzymes were purchased from sigma ( St Louis, MO USA). They were diluted in the SIF with a ratio of 0.20 U and 0.04 U per g of treated peanut proteins. Digestion was conducted in volumes of 1 ml for each peanut sample. Aliquots of 200 l

PAGE 107

107 SIF mixtures were terminated and then removed at 5min, 10 min, 20 min, 30 min, 40 min, 60 min, and 90 min. The termination was acheived by adding 200 L of 2x SDS PAGE sample buffer (containing 40% glycerol, 20 % SDS, 0.33 M Tris (pH 6.8) and 0.05% bromophenol blue). Three independent trials were performed. Results and D iscussion Simulated Pepsin D igestion Raw peanut The SDS PAGE and W estern blot results of pepsin digested raw peanuts are presented in Figure 6 1 The left lane is the protein standard ranging from 10 kD to 250 kD. From left to right, the first two samples were the protein profile of pepsin. The fra gments were located at 37 kD, 15 20 kD, and 10 kD. The next two lanes are the samples from t = 0. The densities of these two lanes are darker than the first two lanes indicating the incorporation of peanut proteins with pepsin enzymes. No proteins were o bserved at molecular weights above 37 kD due to enzymatic reactions. All the digested samples showed darker and thicker bands at around 37 kD. Compared to the zero time treatment, the smaller bands of proteins disappeared while the 37 kD protein band showe d an increased density The treatment at 2.5 and 5min showed the protein bands of 25 kD, 17 18 kD were not as strong as the 0 min treatment and pure pepsin control, which indicated that pepsin may combine with the peptide substrate and form a bigger comple x. The treatment s at 10 min, 20 min, 30 min did not show a palpable difference of protein profiles as compared to the pure pepsin group However, the smaller area s (under 25 kD) were clearer and sharper. It is believed to arise from the complex formation o r fragments produc ed by enzymatic digestion.

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108 The W estern blot results are shown in Figure 6 1 Western blot provide s image of the IgE binding for the specific proteins. P ooled plasma from five patients was used in this study. The strong signals on the W estern blot memb rane displayed the allergenic potency of the digested protein s The 0 min treated sample had two major bands one was around 37 kD and the band was appearing at 17 kD which was Ara h 2. After two min treatment, the Ara h 2 protein completely disappeared. By 60 min digestion, most of IgE binding signals were not present. Roasted peanut For roasted peanuts SDS PAGE results are presented in the same order as the raw peanut sample ( Figure 6 2 ). The first lane is protein standard ranging from 10 kD to 250 kD. The followin g lanes are the pepsin control group, t=0 treatment group, 2.5 min, 5 min, 10 min, 20 min, 30 min, and 60 min treatment groups. S mears were present on top of each sample and throughout the gel. The treatment group t = 0 ha d an unclear area in the small pr otein range under 25 kD. The 37 kD area was markedly increased due to the combination between enzyme and proteins or protein aggregation as discussed above. The bigger protein bands, such as Ara h 1 (63 kD), could not be detected on the SDS PAGE gel The possibility of this phenomenon can be explained to dis assembled Ara h 1 or protein aggregation Western blotting also provided a further view of IgE binding with allergenic proteins ( Figure 6 2 ) The roasted peanut control had a strong signal of IgE reactivity at 17 kD, which was Ara h 2. The Ara h 1 band was show n at 63 kD. Other various bands were also detected on the membrane. Fo r t=0 and t=20 min treatments, the digested samples showed protein reactivity at 37 kD and 10 25 kD. The treatment at 30 min and 60 min only showed the Ara h 2 signals and no other allergen bands.

PAGE 109

109 Pulsed light sample preparation The 12 min PL treated samp le s were evaluated. The protein profile and allergen reactivity were examined by SDS PAGE and W estern blot (Figure 6 3). The standard protein is located on the left most column, ranging from from 10 250 kD. Next is the pepsin group that shows the protein bands of pepsin. The rest of the samples are the experimental groups from t=0 treatment to t=60min. Compared to the raw and roasted peanuts, two major differences were observed on the SDS PAGE O ne is that the smears were distributed through out the whole gel from the top to the bottom; another is that the smaller protein bands area were blur red which means different size proteins were distributed randomly in this area. Once digestion started, the density at 37 kD was notably increased Extended treatment s at 30 min and 60 min decreased the amount of smaller proteins ( < 25 kD) Ara h 1 disappeared, which may be due to denaturation because of low pH value s or enzymatic digestion. Western blot results ( Figure 6 3 ) demonstrated the IgE immunoreactivity of allergen proteins. PL treated samples was different as compared to raw and roasted peanut samples. The pooled plasma could detect major peanut allerg ens Ara h 1 Ara 3 and also other allergens Ara h 4 Ara h 11. However, Ara h 2 was the only allergenic protein visible on the Western blot. The other bands were not present. The digestion of 60 min had a distinct reduction of Ara h 2 IgE binding. These r esults indicate that PL could inactive most of the peanut allergens. After 60 min pepsin digestion, the IgE binding of Ara h 2 virtually disappeared.

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110 Simulated T C hymotrypsin D igestion Raw and roasted peanut Chymotrypsin is a digestive enzyme found in pancreatic juice It act s in the duodenum as an enzyme for protein proteolysis, the breakdown of proteins and polypeptides ( Chervenka and Wilcox 1956 ) Trypsin is a serine protease that is found in the digestive system of many vertebrates where it hydrolyses protein. Trypsin is produced in the pancreas as the inactive form: proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at carboxyl side of the amino acids lysine or arginine ( Rawlings and Barrett 1994 ) In this study, these two enzymes were used for simulated intestinal digestion. SDS PAGE and western blot results are presented in Figure 6 4 The left lane is a protein sta ndard ranging from 10 kD to 250 kD. The first seven samples were digest ed remains of raw peanut sample s and the second set of seven samples was digested remains of roasted peanut samples. This digestion study did not show a difference between raw peanut s a nd roasted peanut s The large protein molecules (above 50 kD) disappeared on the SDS PAGE gel. Ara h 2 and other peanut proteins were observed under 25 kD. Ara h 1, Ara h 3 and Ara h 4 were not detected on the SDS PAGE gel, which indicated the digestive effect of intesti nal enzymes on these allergens. Western blot IgE binding activity was only detected ( Figure 6 4 ) at 16 18 kD (Ara h 2 band ) of both r aw and roasted samples. The treatment at 60 min and 90 min of roasted peanut had a prominent reduction signal compared to the lower treatment times. The IgE immunoreactivity of raw peanut samples showed a distinct reduction after the 20 min

PAGE 111

111 treatment. Thus the digestion efficiency of raw peanut samples was higher than roasted peanut. Pulsed light treated peanut The PL treated peanut was digested at different time points, starting at 0 min and going to 90 min. The right lane in the SDS PAGE ( Figure 6 5 ) is the protein standard ranging from 10 kD to 250 kD. Next are tr y chymotrypsin control s Different fragments of these two enzymes were detected on the SDS PAGE gel around 70 kD, 37 kD, and 20 kD. The smears of pulsed treated peanut accumulated through out the whole gel, especially for the 0 min treatment. With the extended digestion of intestinal enzymes, the smears were gradually reduced. The sam ple of 90 min treatment did not show smears except for the top area. No specific bands were observed from the PL treated samples. Western blot results ( Figure 6 5 ) matched the results of the SDS PAGE. The starting treatment at 0 min had a strong IgE immunoreactivity of smears through out the whole sample lane on the western blot. Ara h 1 and Ara h 3 co uld not be detected on the western blot but Ara h 2 showed strong signals and band s on the western blot membrane. The 5 min and 10 min treatment groups had distinct IgE immunoreactivity reduction as compared to the 0 min treatment group. After 10 min, the total IgE immunoreactivity of the longer treatment groups almost disappeared Ara h 1 Ara h 11 could not be detected on the W estern blot and e ven Ara h 2 was inactivated completely. No smears were present on the gel after the 0 min treatment. Discussion In the SDS PAGE, Ara h 1 was not shown The trimeric ( Shin and others 1998 ) and the oligomeric ( Van Boxtel and others 2006 ) organization of Ara h 1 on the

PAGE 112

112 quaternary folding level contains disulfide bridges that link the proteins, but there is a dissociation of these complexes. It is not known what is the reason, but multimers are not present on the SDS P AGE of raw and roasted samples. One study pointed out that some peptid es of approximately 5 and 10 kD are stable for up to 2 8 min ( Koppelman and others 2010 ) Our study also has shown smaller peptides were generated during the digestion process. Another study described that the digestion of Ara h 1 wi th low pepsin concentration and found that there were no associated peptides of Ara h 1 by chromatography. They found that peptides of relatively high molecular weight half the mass of the Ara h 1 were detected on the chromatograms ( van Boxtel and others 2007 ) The absence of any band on the W estern blot could be explained as Ara h 1 was degraded rapidly or the loss of immunoreactivity of Ara h 1 after limited digestion. It was shown that the digestion of pepsin leads to Ara h 1 degradation in a very short time. This study also detected the changes of Ara h 3 after pepsin digestion. The SDS PAGE results of raw and roasted peanut samples indicated that the 37 kD area was much thicker than normal A study investigated the changes of purified Ara h 3 after pepsin digestion Their results showed that the acidic and basic subunits were sti ll associated by a disulfide bridge and the molecular weight was 70 kD ( Piersma and others 2005 ) However, our results did not show the b ig molecular bands on the gel Another study conducted by van boxtel and others ( 2008 ) analyzed the size exclu ded proteins chromatography under denaturing conditions. The results demonstrated that the majority of the Ara h 3 was found in the range of 7 14 kD. After 60 min digestion, the majority of molecular weights were centralized at less than 7 kD ( Schmitt and others 2004 ) This explained why the longer digestion samples have more protein fragments in

PAGE 113

113 in the range less 25 kD. The IgE immunoreactivity of these protein fragments were not shown on the W estern blot. However, the 37 kD (Ara h 3) bands were clearly appearing before a 20 min digestion. This may be due to the destroyed epitopes after the digestion process. In c ontrast to Ara h 1 and Ara h 3, Ara h 2 was more stable ( Figure 6 2 Figure 6 3 and Figure 6 4 ) except for th e PL trea ted samples. The pepsin and trypsin/chymotrypsin digestion results demonstrate d that Ara h 2 were not reduce d in roasted peanut samples after both pepsin and trypsin/chymotrpsin digestion. Ara h 2 was markedly reduced after 2 min pepsin digestion and had a complete reduction at 5 min digestion of raw peanut sample. However, the trypsin/chymotrypsin digestion of raw peanut samples only had the Ara h 2 protein bands on the SDS PAGE gel. The W estern blot results showed that IgE immunoreactivity of Ara h 2 existed through out the w hole digestion of the raw samples except at the 90 min mark Sen and others (2002) investigated the digestion of Ara h 2 and their results described a digestion resistant peptide. This peptide is stable with minor differences at the N terminal and/or C ter minal part. This shows that proteolysis is restricted by the Ara h 2 structure rather than the specificity of the applied proteases. Evidence from Thomas and others ( 2004 ) showed that intact Ara h 2 migrated on the SDS PAGE as a single band of approximatel y 14 kD. However, it is known that Ara h 6 has the same molecular weight, but is characterized as having 2S albumin units ( Suhr and others 2004 ; Koppelman and others 2005 ) The Ara h 2 bands of our results did not have migration on the SDS PAGE gel and western blot membrane.

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114 The pattern of Ara h 6 was very similar to Ara h 2. The molecular weight of Ara h 6 was approximately 15 kD based on SDS PAGE and 14.98 kD from mass spectroscopy ( Koppelman and others 2005 ) Ara h 2 and Ara h 6 are both 2S albumins with a high degree of amino acid identity ( Kleber Janke and others 1999 ; Suhr and others 2004 ; Koppelman and other s 2005 ) Our results cannot differentiate Ara h 2 and Ara h 6 because we used crude peanut proteins. One study detected the changes of protein patterns and IgE binding with purified peanut allergens. They reported that Ara h 6 has a very sim ilar pattern to the Ara h 2. The results also revealed that Ara h 6 disappeared with a rate faster than the larger isoform of Ara h 2. Their results described that Ara h 6 was substantially digested after only 1 min at the highest pepsin concentration. Fra gmentation was observed at the lowest pepsin concentration and some Ara h 6 was intact after 30 min. Similar ly to Ara h 2, the intramolecular disulfide brides of Ara h 6 maintain ed the digestion fragments as a single molecule. Although the digestion of Ara h 6 was more rapid than that of Ara h 2, a similar large peptide remained for the experiment even after 1 hour. Combined with our results, the bands of Ara h 6 and Ara h 2 overlapped. No bands migrat ed from our pepsin digestion results. In order to determ ine the protein bands around 15 17 kD, chromatography is needed for future studies ( Koppelman and others 2010 ) chymotrypsin proteolysis effects were evaluated for the intestinal digestion. The r esults in this part revealed that only Ara h 2 and less than 10 kD proteins existed in the raw and roasted peanut samples. For the PL treated samples, there was no specific band but smears were distributed through the whole gel. This phenomenon has been d iscussed in Chapter 3 and 4 which was due to the protein

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115 cross linkage and aggregation. Although Ara h 2 bands were not detected on the SDS PAGE of PL treated peanut samples, the W estern blot showed a strong signal of Ara h 2 and the IgE immunoreactivity of the smear through out the whole gel at 0 min treatment. After the 0 min treatment, the intestinal digestion started and most of the IgE reactivity of protein fragments and bands disappeared. Sen and others ( 2002 ) performed digestion experiments with Ara h 2 using trypsin and found a peptide of Ara h 2 of similar size. Another study conducted in 2010 involved the application of trypsin on purified Ara h 2 digestion and their results matched the study Sen and others ( 2002 ) conducted ( Koppelman and others 2010 ) Koppelman and others ( 2010 ) observed two bands in the molecular region of 9 kD and 4 kD after Ara h 2 and Ara h 6 were digested. On the SDS PAGE of raw and roasted peanut samples, the 9 kD bands were observed. However, the W estern blot did not show any signals on 9 kD bands. Our results matched the previous studies discussed above. Of course, pepsin has a different specificity to that of trypsin/chymotr y psin and all these results cannot be extropolated easily. There are many cleavage sites in the sequence of Ara h 2 and Ara h 6 but only a few of them are cleaved in real practice. This revealed that the 2S albumin structure may be a more important fa ctor than the primary sequence. Summary and c onclusion s By evaluating the potential gastric and intestinal digestibility of allergenic proteins of PL treated peanuts, it is concluded that Ara h 2 and Ara h 6 are more stable allergens than the other allergenic proteins in raw and roasted peanut extracts However, a ll the allergens were reduce d in PL treated pea nut kernels after 60 min peptic digestion and 5 min intestinal digestion Digestion resistant peptides obtained after digestion of Ara h 2 may be responsible for prohibiting the complete digestion of Ara h 2 In the gastric

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116 digestion study, the reduction effect was observed after 30 min digestion. In the intestinal digestion st udy, the reduction effect was observed after 10 min. Overall, PL reduced allergenic reactivity with human IgE of all the peanut allergens based on the in vitro digestion study. So far, all the evidence from C hapter 3 and 4 combined with this chapter have s hown the potential of PL on peanut allergens inactivation.

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117 A) B) Figure 6 1 Protein profiles and Western blot of pepsin digested raw peanut samples. A) SDS PAGE of pepsin digested raw peanut samples. B) Western blot of pepsin digested raw peanut samples. Ara h 1 was hardly detected on SDS PAGE, which was digested by pepsins. Western blot clearly showed that strong signals were detected before 30 min treatment. A ra h 2 react ivity was existed till 5 min treatment.

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118 A ) B) Figure 6 2 Pepsin digested roasted peanuts were determined by SDS PAGE and Western blot. A) SDS PAGE of pepsin digested roasted peanut. B) Western blot of roasted peanut digested by pepsin. This part showed a very strong I gE immunoreactivity signal through 0 min treatment to 60 min treatment. Ara h 2 had an increased IgE reactivity with a longer treatment.

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119 A) B) Figure 6 3 Pepsin digestion of PL treated peanut at 12 min. A) SDS PAGE of pepsin digested PL treated peanut at 12 min The smears appeared on the SDS PAGE indicated that protein aggregates were formed during the PL illumination. B) Western blot of pepsin digested PL treated peanut at 12 min It showed that the IgE immunoreac tivity of Ara h 2 was greatly reduced at 60 min digestion.

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120 A) B) Figure 6 4 SDS PAGE and Western blot of mimic intestinal digestion of raw and roasted peanuts. A) SDS PAGE results. B) Western blot results. Most protein bands were not detected except 17 kD (Ara h 2). The western blot results indicated that the immunoreactivity of Ara h 2 was still strong in both raw and roasted peanut before 90 min treatment. 90 min treatment group has a reduction of IgE binding of Ara h 2.

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121 A) B) Figure 6 5 SDS PAGE and Western blot of mimic intestinal digestion of PL treated peanut at 12 min. A) SDS PAGE results. The smear appearin g on SDS PAGE at 0 min treatment was observed With enzymatic digestions, the smea rs were gra dually disappeared. B) Western B lot results. Weste rn blot showed that the smears had the IgE reactivity through the whole gel After 20 min treatment, the overall signals were hardly detected.

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122 CHAP TER 7 EXAMINATION OF Q UALITY C HANGES ( T EXTURE, C OLOR, A NTIOXIDANT C APACITY, LI PID O XIDATION) OF THE W HOLE P EANUTS B EFORE AND A FTER PL T REATMENT Chapter 7 examines the quality of PL treated peanut kernels. The texture, color changes, moisture, total phenolics, and lipid oxidation of treated kernels were determined The Chapter 7 aims at evaluat ing the edibility and nutrients of PL t reated peanut s, which are important for potential future production of allergen reduced or hypoallergenic peanuts Excessive moisture loss was observed in previous treated protein extracts of peanut and shrimp during PL treatment. Because of the infrared light portion of PL the temperature will increase during PL treatment even for a short time, such as 3 min (Yang and others 2012) As we observed when the kernels were treated in C hapter 1, the surface temperature readings could reach as high as 120 C. The moisture loss and temperature increase are the reasons for texture change after PL treatment. UV light makes 54% of pulsed light ( Kr ishnamurthy and others 2007 ) Both UV and ultrasound have been demonstrated that they can increase the concentrations of antioxidants and polyphenolic compounds ( Sales and Resurreccion 2010 ) The reason for the polyphenols increase is because of increased enzymes that are responsible for the biosynthesis of secondary metabolites, such as flavonoids ( Cantos and others 2000 ) In recent years, phenolic phytochemicals in foods have received increased interests from consumers and researchers because of their antioxidant activity and health benefits. In addition to being important from a nut ritional perspective, the antioxidant capacity is also important for food stability, as oxidation often leads

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123 unacceptable flavors, colors, or loss of nutrients ( StAngelo 1996 ; O'Keefe and Wang 2006 ) Peanuts are rich in energy and are high in oil and protein content but with a low percentage of carbohydrates and ash ( Stangelo and others 1979 ) Peanut contains around 50 55% oil, 30 35% linoleic acid, and 45 50% oleic acid ( Coupland and McClements 1996 ) Lipid oxidation occurs during storage of peanut products and contributes to undesirable flavors in peanuts. The oxidation reactions could produce numerous aliphatic al dehydes, ketones, and alcohols ( Bett and Boylston 1992 ) Finally, off flavors such as oxidized, cardboard, and a painty taste will increase in peanut products ( Gills and Resurreccion 2000 ; Grosso and Resurreccion 2002 ) This process can be accelerated at higher temperatures, such as deep fat frying, with increases in free fatty acid and polar matter contents, foaming, color, and viscosity ( Coupland and McClements 1996 ) As PL technology increases the surface temperature of treated food samples, we speculate that PL may increase the lipid oxidation of treated peanut kernels. Chapter 7 will focus on t he color, texture, total phenolics and lipid oxidation for PL treated peanut kernels. Material and M ethod Sample preparation The PL treatment on the peanut kernels was the same as the Chapter 5. Texture A nalysis The conventionally roasted peanut kernels, which were used as control, were manually split into halves. Both the PL and conventionally roasted half peanut kernels were subjected to a TA.XT Plus Texture Analyzer from Stable Micro System

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124 ( Godalming, UK) with a 2 m m diameter probe of T 52 model Th is instrument was equipped with a 50 kg load cell. The data were recorded and analyzed by the Exponent Stable Micro System TEE 32v software. Each half peanut kernel was oriented with the germ side down. The probe was placed in the center of the pea nut kernel. The probe was set up to travel 2 mm distance into the kernel at the test speed of 2 mm / s. Maximum force during penetration and withdrawal of the needle from the peanut kernel was measured to evaluate the hardness of the peanut kernel. Each pe anut sample had 15 kernels. A deformation curve was obtained from the analysis, which showed a major, sharp peak that produced the maximum force for each peanut kernel. The characteristic of hardness (kg) was compared between the PL roasted peanut and cont rol using the maximum force data. Color A nalysis To quantify the color changes, a machine vision system consisting of a Nikon D 200 digital camera housed inside a light box [42.5cm (W) x 61.0cm (L) x 78.1 cm (H)] ( Wallat and others 2002 ) was used to measure the color of peanut kernels with a D65 (daylight) la mp and 10 observer angle. Each image was calibrated against a yellow color reference tile (L=87.09, a=7.71, b=70.75) that was obtained from the Lab Sphere X Rite Company (North Sutton, NH). The software LensEyeSK v10.0.0 (Engineering and Cyber Solutions, Inc., FL) was applied to collect and analyze the images. To obtain precise calibration, the initial image was obtained using the yellow round reference tile that was then converted to a rectangular (~150 x 95 pixels) processed image that was without inter ference from the black border of the yellow tile. Then, the processed images were subjected to background corrections to produce the corrected images. The final corrected images were calibrated with a standard yellow color (Lref, aref, bref). The

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125 L, a, an d b values from the Hunter color system were recorded individually for each peanut sample Th e color analysis was conducted in triplicate for each sample. The Hunter L, a, and b color space is organized in a cube form. The maximum L is 100 (white), the min imum L is 0 (black). Positive a stands for red N egative a stands for green. Positive b stands for yellow, negative b stands for blue. The coordinates of three color parameters were expressed using C* (chroma), h (hue angle), and total color differenc 7 1, 7 2 and 7 3. ................................ ................................ ............................ ( 7 1) ................................ ................................ ................................ ( 7 2) .................... ( 7 3) Total Antioxidants Capacity E valuation Extraction of total antioxidants The methods for the extraction of hydrophilic and lipophilic antioxidants were followed by ( Prior and others 2003 ) a nd ( Wu and others 2004 ) The ratio of sample to solvent was 1:20 and three extractions were conducted t o facilitate extraction of lipids from peanuts by mixing 0.125 g of grounded peanuts with 2.5 ml hexane/dichloromethane purchased from Fisher Scientific 1:1 v/v ( Fair lawn, NJ USA ) in a 50 ml glass tube (Fisher Scientific, Fair Lawn, NJ). The mixture was vortexed for 1 min and sonicated for 10 min. Samples were centrifuged at 24,000g for 15 min at 4 C to obtain a stable residue. All hexane extracted was combined in another glass tube and then evaporated under nitrogen in a dry block heater at 30 C. The dried hexane extract was used for L ORAC assay. The residue was evaporated under vacuum conditons to

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126 remove hexane and then 5 ml of acetone water acetic acid (AWA) solution containing 70 m l acetone, 29.5 ml water and 0.5 ml acetic acid was added The mixture was vortexed for 2 min. The samples were held at room temperature for 10 min with occasional shaking and then centrifuged as described above. The AWA extract supernatant was transferred to a test tube and used in the H ORAC assay. This assay wa s done under yellow light. Hydrophilic oxygen radical absorbance capacity (H ORAC) assay The H ORAC procedure was followed by Prior and others ( 2003 ) Assays were prepared in Costar polystyrene flatbottom black 96 microwell plates (Corning; Acton, Massachusetts). A sodium salt solution of Fluorescein (Reidel deHa en; Seelze, Germany) was prepare d freshly at a final concentration of 70 nM in 75 mM phosphate buffer. Trolox (Aldrich; Milwaukee, WI) s tandards were prepared from 50 to 3.12 M in phosphate buffer. 2,20 azobis (2 amidino propane) dihydrochloride (AAPH) (Wako; Richmond, VA) was prepared freshly at a final concentration of 153 mM in phosphate buffer before the actual usage. Fluorescence was measured using Spectra Max Gemini XPS microplate reader (Molecular Devices, Sunnyvale, CA). The fluorescence was monitored at 485 nm excitation and 530 nm missions for 40 min at 1 min intervals. The reaction was conducted in 75 mM phosphate buffer at pH 7.4 with a final reaction volume of 250 l. Hydrophilic extracts were diluted using phosphate buffer and fell within the linear region of the Trolox standard curve. Diluted sample extracts and standards, both at 50 l, was added to the w ells followed by 10 0 l of the f luorescein solution Each sample was subjected to triplicate runs The solution was rapidly added with a multi channel pipette. The plate containing only the samples and standard s +

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127 f luorescein was incubated in a plate reader for 10 min at 37 C. 50 l of the AAPH solution was then rapidly added via a multi channel pipette during the incubation. Prior to the first measurement, samples were shaken for 5 s using a medium orbital intensity. The antioxidant capacities of the extracts were expressed as mmol TE/L of peanut samples. Lipophilic oxygen radical absorbance capacity (L ORAC) assay The L ORAC procedure was adapted from ( Prior and others 2003 ) Solutions of 7% pharmaceutical grade randomly methy lated beta cyclodextrin (RMCD) purchased from Trappsol; CTD, Inc. ( High Spri ngs, FL, USA) w as prepared in 1:1 acetone/water (7% RMCD). Trolox standards were prepared from 200 uM to 1.56 M in 7% RMCD. Lipophilic extracts were diluted using 7% RMCD (1:25) to fall within the linear region of the Trolox standard curve. Fluorescein w as prepared at a final concentration of 21.5 nM in 75 mM phosphate buffer. AAPH was prepared freshly with a final concentration of 77 mM in phosphate buffer immediately prior to usage. 25 l of diluted sample extracts and standards were added to the w ells in triplicate followed by 120 l of the f luorescein solution as quickly as possible Then, the mixture was incubated in the plate reader for 15 min at 37 C. 80 l of the AAPH solution was rapidly added by using a multi channel pipette. Prior to the first measurement, samples were shaken for 5 s using a medium orbital intensity. The antioxidant capacities of the extracts were expressed as mmol TE/L of peanut samples. Lipid O xidation For this part, fresh raw runner type peanuts were directly harvested from the field and stored at 4C and 10% moisture. The se fresh raw peanuts were considered to be absent with lipid oxidation occurrence but the conventionally roasted peanut had a high

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128 percentage of lipid oxidation ( Win and others 2011 ) These peanut samples wer e provided by Dr. Barry Tillman in the Agronomy D epartment University of Florida. Peroxide value 2 g of raw peanut sample s or PL treated peanut samples were mixed with 6 ml of chloroform methanol (1:1 v/v) solution. The mixture was homogenized using a homogenizer from the ESGE Ltd. ( Mettlen Switzerland) Each sample was duplicated at this time. Then 7 ml of NaCl (0.5%) was added and vortexed for 30s and followed by centrifugation at 2000 rpm for 10 min in cold room (4C). After centrifug ation 2 ml of the chloroform layer was taken by a syringe (Sigma, St. Louis, MO). Fresh ferro u s solution was prepared by adding 0.2 g barium chloride in 25 ml 0.4 N HCl (ke pt cold and stable). At the same time, 0.1 g ferrous sulphate hepta hydrate was dissolved in 10 m l distilled water. Next 3 ml of each solution was taken using a pipette and centrifuge d for 3min. The supernatant was taken immediately for use. The next step was to add 50 l of ferrous solution and ammonium thiocyanate solution (7.5 g ammonium thiocyana te in 25 ml distilled water). Finally, the mixture was incubated for 10 min at RT The color was developed and recorded by 415 Spectro Master Spectrophotomete produced by Fisher scientific ( Drive Hudson, NH, USA) at 500 nm. Thiobarbituric acid reactive substances (TBARS) assay TCA extracting solution was prepared with 75 g trichloroacetic acid, 1 g propyl gallate and 1g EDTA in 1 liter of distilled water. 2 g of peanut samples were mixed with 5 ml TCA extraction solution. Each peanut sample was triplica te at this step. Then, the mixture was homogenized using a homogenizer f rom the ESGE Ltd. ( Mettlen Switzerland) for 30 s. The mixture was then filtered by Whatman #1 filter paper. At the same time, TBA solution was prepared using 2.89 g of thiobarbitur ic a cid in 1L distilled

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129 water. Another glass tube with 2 ml TBA solution was prepared and 2 ml of filtrate was v o rtexed for 10 s. The tube was incubated in a water bath at 100 C for 40 min. The data w ere recorded at 532 nm using a 415 Spectro Master Spectroph otomete manufactured by Fisher S cientific ( Drive Hudson, NH, USA) Results and D iscussion Texture A nalysis The mean maximum force (hardness) recorded during the compression tests of both PL treated and dry roasted samples are shown in Table 7 1 The roasted peanuts were used as control over raw peanuts, since the PL treated peanuts were also roasted due to the photo thermal effect of PL and may serv e as the starting material to manufacture peanut butter like the dry roasted peanuts. For the dry roasted peanuts, the mean maximum force value was 1.76 0.38 kg. At 10 cm distance from lamp, the peanuts PL treated for 5 min, 6 min, and 7 min exhibited a significant difference (p <0.05) in hardness compared to control, with the maximum force values of the former higher than that of the latter. However, the 8 min PL treated peanut had a similar hardness to the roasted peanut (p > 0.05). The hardness of PL treated peanut sample at 8.5 min was also not statistically different from that of the roasted peanut (p < 0.05), although the former showed a higher compression force value (i.e., 1.83 kg vs. 1.76 kg). The hardness value (1.52 kg) at 9.5 min duration, whi ch was representative of nearly burnt peanut kernels, was statistically different from that of the roasted peanut (1.76 kg), because the burnt peanut kernels tended to soften its texture (Kita et al., 2006). For the data in Table 7 1 it can be seen that for shorter PL durations (e.g., 5, 6 and 7 min vs. 8 and 8.5 min), the peanuts were not roasted enough yet by PL as evident by the significant difference (p <0.05) in t heir hardness compared

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130 to control, because under roasting would generate a harder texture, while well roasted peanuts exhi bit softer texture (Kita and others 2006). At 7 cm distance from lamp, both PL durations of 5 min and 6 min showed a significantly (p < 0.05) higher hardness value (2.2 kg and above) than that of the roasted peanut (1.76 kg). At the 6.5 min mark, the texture was shown to be statistically similar to that of the roasted peanut (p < 0.05). Longer treatment times beyond 6.5 min significantly reduced the hardness of PL treated peanuts (e.g., 1.40 kg), as shown in Table 7 1 Similarly, the significantly lower hardness value at 7 min corresponded to a textu re of nearly burnt peanut kernels that tended to be softened as mentioned earli er. Temperature a nd Moisture C hanges During PL I llumination During roasting, proper moisture reduction helps produce a crispy texture. If the nuts are heavily roasted, the sugar s in the nuts can decrease with an increased temperature. Therefore, higher temperatures and longer periods of roasting can decrease the quality of the nuts ( Ozdemir and Devres 1999 ) A study conducted by Kita and others ( 2006 ) indicated that decrease in moisture can directly affect the mechanical properties (e.g., hardness) of roasted nuts. Roasting at hig her temperatures for the same time could reduce the hardness of walnuts, but extended roasting time did not show any significant effect on their hardness ( Kita and Figiel 2006 ) Demir and others ( 2004 ) found that both tempera ture and time had strong effects on hazelnut texture. Due to the photothermal effect of PL illumination, which can cause a pronounced instantane ous temperature rise (Li and others 2011) and can also be observed in Tab le 7 2 a similar roasting effect on the peanut kernels to that of the conventional dry roasting was noticeable after PL treatments. The surface temperature of PL treated peanuts after 5 m in exposure at both 10 cm and 7 cm distances from lamp was 35.1

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131 1.8C and 50.3 1.1C, respectively ( Table 7 2 ). The surface temperature for the maximum exposure times, 9.5 min for 10 cm distance to lamp and 7 min for 7 cm distance to lamp, were 116.0 2.1C and 108.6 4.6C, respectively ( Table 7 2 ). Comparison of the temperature rises in Table 7 2 between 7 cm and 10 cm di stances to lamp showed that shorter distance generated great photo thermal impact during illumination given the same duration. Previous studies by Krishnamurthy and others ( 2008 ) have also shown th at reduction of microorganisms is proportional to treatment It is worth mentioning that the temperatures in Table 7 2 were detected with a 5 10 s delay due to the time required to remove the sample from the chamber before temperature measurement by the handheld infrared thermometer. So, the instantaneous temperature of the sample during the PL treatment could be higher than the surface temperatures recorded. This inference was supported by the nearly burnt appearance of the peanuts after 9.5 min PL treatment at 10 cm distance to lamp or after 7 min exposure at 7 cm distance from lamp and their surface temperatures of around 110C as shown in Table 7 2 The temperature 110C was below the smoke point of peanut oil (188C to 198C) (Morgan, 1942) and should a lso be lower than the smoke point of the whole peanut kernels, so this temperature was not sufficient to burn the peanut kernels. It is thus reasonable to believe that the instantaneous temperature of the peanuts after 9.5 min PL illumination at 10 cm dist ance to lamp or 7 min exposure at 7 cm distance from lamp must have exceeded 188C. Moisture loss percentages during PL treatments are also shown in Table 7 2 Traditional dry roasted peanuts gave a moisture loss around 3% with uniform heating at

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132 165C for 15 min. The PL treated peanuts at 10 cm distance to lamp resulted in a moisture loss ranging from 1.4% 5.6% for 5 9.5 min PL durations. The samples at 7 cm distance to lamp caused a moisture loss ranging from 2.0% 8.8% for 5 7 min Pl durations. The higher moisture loss encountered in the samples at 7 cm distance to lamp, given the same time, was probably due to higher instantaneous temperatures incurre d in the peanut kernels. Color A nalysis The values for the color dimensions, i.e., treated and dry roasted peanuts are shown in Table 7 3 Since most of the area of the burnt peanuts was charred, the samples related to 9.5 min in the 10 cm group and 7 min in the 7 cm group were not entered for color analysis. The ANOVA was carried out to determine the statistical difference of each co range tests at 95% significance level was used for separating the means. The L* values indicate the lightness of the processed peanut samples. Statistical analysis showed that the dry roasted peanuts had a similar L va lue (p>0.05) to that of 7 min and 8 min PL samples at 10 cm distance to lamp ( Table 7 3 ). Below 7 min, the PL treated peanuts exhibited lighter color than the dry roas ted peanuts, while above 8 min, the PL treated peanuts exhibited darker color than the dry roasted peanuts. However, for the 7 cm distance group, PL treated peanuts at 5 6.8 min all showed a significantly (p<0.05) darker color than the dry roasted peanuts. The a* value parameter, which indicates the rate of red color of processed peanuts, was found to be statistically similar between the PL treated peanuts for 8.5 min at 10 cm distance to lamp and the dry roasted peanuts (p> 0.05). For all other PL durations, the a* value of PL treated peanuts was significantly different (p<0.05)

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1 33 compared to that of the dry roasted peanuts. In general, extended exposure and shorter distance to lamp produced a more red color on the sample. The b* value parameter indicates the rate of yellowness of the sample. The dry roasted peanuts had a similar (p>0.05) yellow color to that of the PL treated peanuts for 8.5 min at 10 cm distance to lamp. Other PL treated samples in the 10 cm group were signifi cantly different (p <0.05) compared to the dry roasted peanuts. Overall, less exposure time and longer distance produced less yellow color and vice versa. For 7 cm distance to lamp, the yellowness of dry roasted peanuts was statistically similar (p>0.05) t o that of PL treated peanuts at 6 and 6.8 min, but statistically different (p<0.05) from that of all other PL treatments. However, for the yellowness, the difference between the roasted and PL treated samples was not large in general. measure the saturation and total color differences from the yellow color reference, respectively. Hue angle ho was used to express the true of the PL treated peanuts for 8 min at 10 cm distance to lamp was similar (p>0.05) to that of the dry roasted peanuts. However, none of the PL samples at 7 cm distance to lighter than the latter. Most hue angle h o values of the PL treated peanuts at both distances to lamp were similar to that of the dry roasted peanuts (p>0.05), except for 5 min at 10 cm distance to lamp. The C* value of the dry roasted peanuts was similar (p>0.05) to that of the 8.5 m in PL treated peanuts at 10 cm distance to lamp and also similar to that of 6 min, 6.5 min and 6.8 min PL treated samples at 7 cm distance to lamp.

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134 Overall, the color results suggest ed that the PL treatment as a potential roasting technique was capable of producing similar color to that of dry roasted peanuts if we set an appropriate combination of PL duration ranging from 5 min to 8.5 min and sample distance to lamp ranging from 7 cm to 10 cm inclusive. The color development of dry roasted peanuts is prima rily a result of the Maillard reaction, which is an important non enzymatic browning reaction in the development of flavor and color in peanuts. This Maillard reaction was also reported to take place during the PL processing of foods ( Maleki and others 2000a ) Antioxidant C apacity H ORAC results were shown in Figure 7 1 Roasted and raw peanuts were considered as controls. The mean separations were analyzed by tukey HSD. The results indicated that roasted peanut and 10 mi n treatment groups were not significant ly different (p > 0.05) R oasted peanuts contained 24888 3513 moles trolox/100g and the 10 min treatment group ha d 24864 339 moles trolox / 100g. Raw peanut s were significantly lower (p < 0.05) in H ORAC than ot her samples. The TE value of raw peanut was 8870 5230 moles trolox / 100g and three times lower than the roasted peanuts The 12 min treated peanut was significantly higher (p < 0.05) than the roasted peanut. The TE value of 12 min treated peanut was 63 830 5841 moles tolox / 100g and it was 2.6 times higher than the roasted one. Although the 15 min treated peanut was also significantly higher (p < 0.05) than both the roasted peanut and 12 min treated peanut samples the 15 min was not edible and the c olor ha d turned black. Overall, hydrophilic antioxidants capacity was significantly increased (p < 0.05) after PL treatment.

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135 L ORAC results described lipophilic antioxidant capacity in raw/roasted peanuts and PL treated peanuts ( Figure 7 2 ) Tukey HSD was applied for statistical analysis. Raw peanut samples had 50393 10 moles trolox / 100g, which was significantly lower than other peanut samples. The 12 min tre atment group contained 80593 14 moles trolox / 100g and it was significantly higher than the raw peanut samples, 10 min, and 11 min treatment groups. Overall, the L ORAC and H ORAC results of raw and roasted peanuts matched the results that were conduc ted by Davis and others ( 2010 ) Another study also demonstrated that UV light could increase total antioxidant capacity in treated peanut about three times more than the raw peanuts ( Sales and Resurreccion 2010 ) The UV light effect on increasing antioxidant capacity was also observed in blueberries with a dose of 2.15 and 4.30 kJ/m 2 ( Wang and others 2009 ) Conventional roasted peanut s also has increased total antioxidant capacity ( Davis and others 2010 ) which agrees with our results. Lipid O xidation The p eroxide value of PL treated peanut samples and the control (raw) is presented in Figure 7 3 The data was plotted from 0 day to 10 days to partially simulate shelf life conditions, at room temperature. Cumene hydroperoxide (CPO) was used as a sta ndard to calculate the treated samples. The results were analyzed by tukey HSD using JMP 10.0. The shelf life of 10 days clearly showed a significant increase d peroxide value (p < 0.05) of all the samples. Raw peanut was the lowest one since we used fresh samples. The 15 min sample was the lowest one among the PL treated peanuts but could not be accounted for accurate evaluation because the

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136 primary oxidation step had passed The 1 2 min sample was significantly more oxidized (p < 0.05) than 9 min tr eatment. TBARS results are presented in Figure 7 4 In this figure, secondary oxidation trends o f each sample were described. Tukey HSD was used for statistical analysis. All the PL treated peanuts showed a linear increase from 0 day to 10 days. The treatment at 15 min had the largest significant oxidation degree (p < 0.05) than other samples after t he samples were collection following PL illumination. Both the p eroxide value and the TBARS results demonstrated that a longer PL treatment resulted in higher oxidation values. The peroxide value is an indicator of the initial stages of oxidative changes. This method could evaluate the total hydroperoxide content and is one of the most common quality indicators of fats and oil during the storage ( Shahidi and Zhong 2010 ) During the lipid oxidation, malonaldehyde (MDA) a minor component of fatty acids was produced. TBARS was used to evaluate the secondary lipid oxidation process ( Ke and others 1984 ) Lipid oxidation produces the oxygen containing compounds such as aliphatic aldehydes, ketones, and alcohols ( Coleman and others 1994 ) In addition, free radicals, hydroperoxides, and secondary products formed during lipid oxidation can interact with nitrogen containing compounds, such as proteins and pyrazines, and modify the precursors for the M aillard reaction ( Williams and others 2006 ) Lipids can be oxid ized by enzymatic (lipoxygenase) and non enzymatic reactions, such as oxygen, light, metal ions ( Kanner 1994 ) In peanut s lipoxygenase acts as a catalyst to oxidize polyunsaturated fatty acids to form hydroperoxides, which then produce secondary products (Fennema 1996). Several non thermal processing methods have been

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137 explored on lipid oxidation of peanuts. Gamma radiation and pulsed electric field can also induce the lipid oxidation of peanut seed s, which shorten s the shelf life of peanut oil and change s the quality of peanut seeds ( Zeng and others 2010 ; de Camargo and others 2012 ) Although the 10 day study on the lipid oxidation was not long enough to warrant a bigger picture on the variation of the lipid oxidation in the PL treated peanut kernels, the current date from this study seemed to suggest that the peanuts roasted by the PL technique were prone to lipid oxidation possibly due in part to more cell rupture occuring under the high instantaneous temperature during PL roasting For future work gas chromatography should be conducted for the profile analysis of oxidized lipids A l onger storage period is required to obtain the whole picture of lipid oxidation in the PL treated peanut kernels by both peroxide value and TB ARS methods

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138 Data are expressed as mean standard deviation (n=46). Means in the same column Table 7 1 Texture comparison between the dry roasted and PL treated peanuts Treatment Hardness (kg) Dry roasted peanut (Control) 1.760.38 a PL treatment (10 cm) 5min 2.040.20 b 6min 2.060.15 b 7min 1.930.24 b 8min 1.740.30 a 8.5min 1.830.40 ab 9.5min 1.520.24 c PL treatment (7 cm) 5min 2.480.31 b 6min 2.200.38 b 6.5min 1.640.24 a 6.8min 1.400.12 c 7min 1.110.31 d Table 7 2 Surface temperature and moisture loss of dry roasted and PL treated peanuts. Duration Surface temperature (C) Moisture loss (%) Dry roasted (Control) 160.02.1C 3.00.5% PL Treatment (10 cm) 5min 35.11.8C 1.40.2% 6min 48.72.3C 2.60.3% 7min 65.32.1C 3.20.3% 8min 98.54.5C 4.40.4% 8.5min 107.0 3.7C 4.60.5% 9.5min 116.02.1C 5.60.5% PL Treatment (7 cm) 5min 50.31.1C 2.00.2% 6min 61.22.4C 2.80.3% 6.5min 88.32.8C 4.00.2% 6.8min 98.53.2C 5.50.3% 7min 108.64.6C 8.80.4% Peanut samples before treatment were at room temperature (23.10.3 C).

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139 Table 7 3 Comparison of color between dry roasted and PL treated peanuts. Duration Color Analysis L* a* b* h C* Control Dry Roasted 64.57 2.57 a 12.43 2.44 a 35.39 0.83 a 42.31 0.96 a 1.23 0.05 a 37.51 1.59 a PL (10 cm) 5min 74.00 1.08 d 0.05 0.23 e 19.78 0.90 d 53.19 0.77 d 0.00 1.71 b 19.78 0.90 e 6min 68.04 1.08 bc 8.20 0.98 d 29.73 1.29 bc 45.26 0.74 bc 1.30 0.03 a 30.84 1.38 cd 7min 66.26 0.83 ab 7.65 0.89 d 28.56 0.67 c 47.07 0.54 c 1.31 0.02 a 29.56 0.83 d 8min 66.94 2.53 ab 8.38 0.67 d 31.69 1.31 b 44.45 0.59 abc 1.31 0.01 a 32.78 1.40 bc 8.5min 59.52 0.87 ef 14.38 1.71 ab 35.24 1.76 a 45.51 0.69 bc 1.18 0.03 a 38.05 2.24 a 9.5min PL (7 cm) 5min 69.91 2.16 c 8.03 0.30 d 28.42 1.58 c 45.65 1.20 bc 1.29 0.01 a 29.53 1.55 d 6min 59.53 3.18 ef 15.12 2.90 b 33.74 2.60 a 46.92 1.12 c 1.15 0.05 a 36.97 3.02 a 6.5min 58.20 2.61 ef 14.65 14.08 b 31.89 1.46 bc 49.04 1.24 bc 1.13 0.08 a 35.09 1.11 a 6.8min 57.41 0.82 f 15.91 1.78 bc 35.12 2.98 a 47.14 2.48 c 1.15 0.01 a 38.56 3.44 a 7min Data are expressed as mean standard deviation (n=46). Means in the same

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140 Figure 7 1 H ORAC of PL treated peanuts. Roasted and raw peanuts were considered as controls. Tukey HSD was applied to analyze the means separation. 0 10000 20000 30000 40000 50000 60000 70000 80000 roasted raw 10 11 12 15 Antioxidant capacity (mol Trolox/100g) Time treatment (min) A B A D E C

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141 Figure 7 2 L ORAC of PL treated peanuts. Roasted and raw peanuts were considered as c ontrols. Tukey HSD was applied to analyze the means separation. 0 20000 40000 60000 80000 100000 120000 Roasted raw 10 11 12 15 trolox / 100g) Time treatment (min) B C D E E A

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142 Figure 7 3 Peroxide value of raw peanut, PL treated peanuts at 9 min, 12 min, 15 min on 0 day, 5 days, and 10 days s helf life at room temperature. Y axis stands for CPO concentration. X axis stands for shelf life. Figure 7 4 TBARS of raw peanut, PL treated peanuts at 9 min, 12 min, 15 min on 0 day, 5 days, and 10 days shelf life at room te mperature. Y axis stands for MDA concentra tion. X axis stands for shelf life. 0 2000 4000 6000 8000 10000 12000 14000 0 day 5 day 10day Peroxide value raw 9 min 12min 15min 0 500 1000 1500 2000 2500 3000 3500 0 day 5 day 10day TBARS raw 9min 12min 15min

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143 CHAPTER 8 OVERAL L CONCLUSION S PL is considered a non thermal technology that is widely used in food preservation. Our study has demonstrated that the extended treatment for allergen reduction could significantly increase the surface temperature of peanut kernel s Due to this phenomenon, this technology is not considered non thermal te chnology, especially in extended treatment s that last several minutes The initial results demonstrated that PL was capable of reducing all major allergens of whole peanut kernels, including the polypeptide fragments degraded from Ara h 1 h 4 during PL tr eatments, to an undetectable level. Due to the thermal effect of PL on the peanut kernels, the initial SDS PAGE results clearly showed that protein ma c romolecules were formed during this proc ess. To validate PL technology, insoluble fractions were examine d using the Novex NuPAGE gel system The Western blot of the denatur ed system showed that treatment below 5.5 min at 10 cm had the strong est IgE binding signals i n the 250 kD to 460 kD or larger range The Western blot results of the non denaturing gel had similar results in that the IgE binding signal of 6.5 min treatment was removed completely, but before 6.5 min treatment, the immunoreactivity of IgE was clearly observed on the top area of the membrane. The overall IgE reactivity of the total protein extr act of PL processed peanuts was shown with indirect ELISA. The signals of all PL treated samples were significantly lower than the raw samples. So far, all the results illustrate that PL treatment may reduce allergens in peanut s via in vitro immunoassay te st s The PL treatment times distance from the light source ( also relates to the PL intensity), number of pulses, thickness of the sample, and duration of illumination are

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144 considered to be critical parameters for t he optimization of a PL system. D uration and distance are two major factors in this system Factorial analysis demonstrated that these two factors interact. To optimize this equipment and obtain constant treatment s a distance of 10 cm was selected M ultivariate ANOVA analysis showed that the 12 min treatment was the best treatment time for generating edible roasted peanut s with low IgE reactivity After the peanuts are swallowed and digested the proteins undergo different enzymatic reactions and enter different pH environments The structures of proteins will be affected The in vitro chymotrypsin revealed that PL treated peanut protein extracts ha ve IgE reactivity through out the whole gel under a pH of 2.0. Gastric digestion removed the immunoreacivity of IgE after a 90 min digestion of PL treated peanuts For intestinal digestion, IgE reactivity through out the whole gel under a pH of 8.3 was also observed. Moreover, the 10 min intestinal digesion showed that the IgE reactivity was completely removed from the PL treated peanuts This study revealed that Ara h 2 and Ara h 6 were more stable allergens than other allergenic proteins in raw and roasted peanut extracts. During the PL illumination, moisture lo ss ha d a positive correlation with temperature and a hard texture was produced during this illumination. Overall antioxidant capacities were significantly higher in 12 min treated peanuts than the raw roasted, 10min, and 11min treatment groups. Also, PL c ould initiate the lipid oxidation occurrence.

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145 In summary PL can notably reduce the IgE reactivity to peanut allergenic proteins based on in vitro testing. To obtain final validation of this technology, a c linical trial is still required. The 12 min treat ment group at 10 cm provided the best quality as compared to conventional roasted peanut s This provides insight for commercial production of hypoallergenic peanut s and peanut butter

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162 BIOGRAPHICAL SKETCH Xingyu Zhao was born in Shijia zhuang, Hebei, China. S he obtained her b achelor degree of biotechnology in Department of B iological Science, N orth China Coal Medical College in 2008. S he start ed doctoral program in Department of Food Science and Human Nutrition at University of Florida in June 2010 and graduat ed in December 2013.