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Reduction of Wheat Allergen Potency by Pulsed Ultraviolet Light, High Hydrostatic Pressure, and Non-Thermal Plasma

Permanent Link: http://ufdc.ufl.edu/UFE0043070/00001

Material Information

Title: Reduction of Wheat Allergen Potency by Pulsed Ultraviolet Light, High Hydrostatic Pressure, and Non-Thermal Plasma
Physical Description: 1 online resource (106 p.)
Language: english
Creator: NOOJI,JYOTSNA K
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ALLERGY -- HIGH -- HYDROSTATIC -- LIGHT -- NON -- PLASMA -- PRESSURE -- PULSED -- THERMAL -- ULTRAVIOLET -- WHEAT
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science REDUCTION OF WHEAT ALLERGEN POTENCY BY PULSED ULTRAVIOLET LIGHT, HIGH HYDROSTACTIC PRESSURE AND NON-THERMAL PLASMA By Jyotsna K Nooji May 2011 Chair: Wade Yang Major: Food Science and Human Nutrition Wheat allergy is known to elicit adverse immune reaction in children and adults. Ingestion and inhalation of wheat proteins can cause Baker?s asthma, urticaria, atopic dermatitis, gastrointestinal symptoms and wheat dependent exercise induced anaphylaxis (WDEIA) in hypersensitive individuals. Allergenic proteins are present in the entire wheat protein fractions which are mainly characterized into; water/salt soluble albumin and globulin fractions and insoluble gluten fraction. Total avoidance has been utilized in an attempt to reduce allergic reactions but, is often impractical or ineffective; thus research focusing on using processing technologies to alter food allergens is gaining more and more attention. Various thermal and non-thermal processing techniques have been utilized to alter the structure of the allergenic proteins. One of the major disadvantage of conventional thermal processing over non-thermal processing is it causes undesirable effects on the nutritive and sensory qualities. Pulsed ultraviolet light (PUV), high hydrostatic pressure (HHP) and non-thermal plasma (NTP) are non-thermal processing methods which can potentially alter wheat protein conformation and hence, reduce the immunereactivity. The main objective of this study was to assess the effect of PUV alone or in combination with heat, HHP and NTP on the IgE binding of wheat protein extract. Wheat protein extract was subjected to PUV at 3pulses for 30 s, 60 s, 90 s, 120 s and 120 s followed by boiling. In addition, HHP (21?C and 70?C for 5 and 15 min) and NTP (1, 3 and 5 min) were also applied to wheat proteins extracts. The control (untreated), PUV, HHP and NTP treated samples were analyzed by SDS-PAGE, western blots, dot blots and indirect ELISA. Allergen potency indicated by IgE binding was determined in blots and ELISA. The PUV, HHP and NTP treatments indicated noticeable difference in the proteins profile demonstrated by SDS-PAGE. A significant reduction in IgE binding was observed in PUV (90 s), HHP (21?C and 70?C for 5 min) and NTP (5 min) treated samples as demonstrated by indirect ELSIA. The maximum reduction in IgE binding was achieved by PUV (46%) and HHP (42%) treatments. These findings indicate that non-thermal processing methods can be implemented to reduce wheat allergen potency.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by JYOTSNA K NOOJI.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Yang, Weihua.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-10-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043070:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043070/00001

Material Information

Title: Reduction of Wheat Allergen Potency by Pulsed Ultraviolet Light, High Hydrostatic Pressure, and Non-Thermal Plasma
Physical Description: 1 online resource (106 p.)
Language: english
Creator: NOOJI,JYOTSNA K
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ALLERGY -- HIGH -- HYDROSTATIC -- LIGHT -- NON -- PLASMA -- PRESSURE -- PULSED -- THERMAL -- ULTRAVIOLET -- WHEAT
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science REDUCTION OF WHEAT ALLERGEN POTENCY BY PULSED ULTRAVIOLET LIGHT, HIGH HYDROSTACTIC PRESSURE AND NON-THERMAL PLASMA By Jyotsna K Nooji May 2011 Chair: Wade Yang Major: Food Science and Human Nutrition Wheat allergy is known to elicit adverse immune reaction in children and adults. Ingestion and inhalation of wheat proteins can cause Baker?s asthma, urticaria, atopic dermatitis, gastrointestinal symptoms and wheat dependent exercise induced anaphylaxis (WDEIA) in hypersensitive individuals. Allergenic proteins are present in the entire wheat protein fractions which are mainly characterized into; water/salt soluble albumin and globulin fractions and insoluble gluten fraction. Total avoidance has been utilized in an attempt to reduce allergic reactions but, is often impractical or ineffective; thus research focusing on using processing technologies to alter food allergens is gaining more and more attention. Various thermal and non-thermal processing techniques have been utilized to alter the structure of the allergenic proteins. One of the major disadvantage of conventional thermal processing over non-thermal processing is it causes undesirable effects on the nutritive and sensory qualities. Pulsed ultraviolet light (PUV), high hydrostatic pressure (HHP) and non-thermal plasma (NTP) are non-thermal processing methods which can potentially alter wheat protein conformation and hence, reduce the immunereactivity. The main objective of this study was to assess the effect of PUV alone or in combination with heat, HHP and NTP on the IgE binding of wheat protein extract. Wheat protein extract was subjected to PUV at 3pulses for 30 s, 60 s, 90 s, 120 s and 120 s followed by boiling. In addition, HHP (21?C and 70?C for 5 and 15 min) and NTP (1, 3 and 5 min) were also applied to wheat proteins extracts. The control (untreated), PUV, HHP and NTP treated samples were analyzed by SDS-PAGE, western blots, dot blots and indirect ELISA. Allergen potency indicated by IgE binding was determined in blots and ELISA. The PUV, HHP and NTP treatments indicated noticeable difference in the proteins profile demonstrated by SDS-PAGE. A significant reduction in IgE binding was observed in PUV (90 s), HHP (21?C and 70?C for 5 min) and NTP (5 min) treated samples as demonstrated by indirect ELSIA. The maximum reduction in IgE binding was achieved by PUV (46%) and HHP (42%) treatments. These findings indicate that non-thermal processing methods can be implemented to reduce wheat allergen potency.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by JYOTSNA K NOOJI.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Yang, Weihua.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-10-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043070:00001


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1 REDUCTION OF WHEAT ALLERGEN POTENCY BY PULSED ULTRAVIOLET LIGHT, HIGH HYDROSTACTIC PRESSURE AND NON THERMAL PLASMA By JYOTSNA KRISHNA NOOJI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE GREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Jyotsna Krishna Nooji

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3 To my parents, Jaikrishna and Vidya Nooji, and my husband, Pradeep Rao

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4 ACKNOWLEDGMENTS I would like to express my gratitude to my major advisor Dr. Wade Yang for h is constant encouragement, and for sharing his knowledge and experience Without his guidance and support this work would not have been possible. I would like to extend my gratitude to my committee members, Dr. Charles Sims, and Dr. Steven Bruner for their continuous support and guidance. I am grateful to Dr. Susan Percival for providing spectrophotometer for my experiments. I would also like to thank my lab mates: Sandra Shriver, Cheryl Rock, Akshay Anugu, and Yiqaio Li for helping me get through the difficult times, and for all the emotional support, entertainment and caring they provided. I have cherished every moment spent with them. Lastly and most importantly I would like to thank my parents, Jaikrish na and Vidya Nooji for their lifelong commitment toward my education. They have always stood beside me during my difficult time, and my mother has always been my best girlfriend They have been my personal cheering squad throughout my life. Most of all, I would like to thank my beloved husband, Pradeep for his unconditional love, encouragement and patience. He reminded me every day how proud he was of my hard work and accomplishments and his love and positive attitude gave me strength to complete this progr am. Finally I want to thank my extended family, and friends for always encouraging me to fulfill this dream.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 2 LITERATURE REVIEW ................................ ................................ .......................... 18 Food Allergy and Allergens ................................ ................................ ..................... 18 History of Wheat ................................ ................................ ................................ ..... 21 Wheat Kernel Structure ................................ ................................ ........................... 22 Wheat Proteins ................................ ................................ ................................ ....... 23 Albumins and Globulins ................................ ................................ .................... 23 Storage Proteins ................................ ................................ ............................... 24 Wheat Allergy ................................ ................................ ................................ ......... 25 ................................ ................................ ................................ 26 Wheat Dependent Exercise Induced Anaphylaxis ................................ ........... 26 Wheat Allergen Proteins ................................ ................................ ......................... 27 Amylase/Trypsin Inhib 5 Gliadin ................................ ......... 28 Lipid Transfer Protein (LTP) ................................ ................................ ............. 29 Research in Developing Reduced Allergenic Products ................................ ........... 29 Ultraviolet and Pulsed Ultraviolet Light Technology ................................ ......... 36 P UV light pasteurization ................................ ................................ ............. 36 Effect of PUV on food allergens ................................ ................................ 37 High Hydrostatic Pressure Technology ................................ ............................ 38 Effect of HHP on food allergens ................................ ................................ 38 Non Thermal Plasma Te chnology ................................ ................................ .... 39 3 MATERIALS AND METHODS ................................ ................................ ................ 48 Chemical Reagents ................................ ................................ ................................ 48 Reagents for SDS PAGE ................................ ................................ ........................ 48 Reagents for Western Blot and Dot Blot ................................ ................................ 48 Reagents for Indirect Enzyme Linked Immunosorbent Assay (ELISA) ................... 48 Wheat Protein Extraction ................................ ................................ ........................ 49 Albumin and Globulin Fraction ................................ ................................ ......... 49

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6 Glutenin Fraction ................................ ................................ .............................. 49 Total Soluble Wheat Protein ................................ ................................ ............. 49 Pooled Human Plasma Samples ................................ ................................ ............ 50 Wheat Allergic Plasma ................................ ................................ ..................... 50 Control Plasma ................................ ................................ ................................ 50 Equipments ................................ ................................ ................................ ............. 50 Pulsed Ultraviolet Light (PUV) Source ................................ .............................. 50 High Hydrostatic Pressure ................................ ................................ ................ 51 Non Thermal Plasma ................................ ................................ ....................... 51 Centrifuge and Spectrophotometer ................................ ................................ ......... 51 PUV Treatment ................................ ................................ ................................ ....... 51 PUV Treatment on Wheat Albumin and Globulin ................................ ............. 52 PUV Treatment on Wheat Gluten ................................ ................................ ..... 52 PUV Treatment on Total Soluble Wheat Proteins ................................ ............ 53 HHP Tre atment ................................ ................................ ................................ ....... 53 NTP Treatment ................................ ................................ ................................ ....... 54 Protein Assay ................................ ................................ ................................ .......... 54 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) .......... 54 Western Blot ................................ ................................ ................................ ........... 55 Dot Blot ................................ ................................ ................................ ................... 57 Indirect ELISA ................................ ................................ ................................ ......... 58 Statistical Analysis ................................ ................................ ................................ .. 59 4 RESULTS AND DISCUSSION ................................ ................................ ............... 64 Eff ect PUV Irradiation on Albumin and Globulin ................................ ..................... 64 Temperature Measurement During PUV treatment and the Effect on Sample Volume ................................ ................................ ............................. 64 SDS PAGE of PUV Treated Albumin and Globulin ................................ .......... 64 Western Blotting of Wheat Albumin and Globulin ................................ ............. 65 Effect PUV Irradiation on Gluten ................................ ................................ ............. 66 SDS PAGE of PUV Treated Gluten ................................ ................................ 66 Western Blot of PUV Treated Gluten ................................ ............................... 67 PUV Treated Total Soluble Wheat Protein ................................ ............................. 68 SDS PAGE of P UV Treated Wheat Proteins ................................ .................... 68 Dot Blot of PUV Treated Wheat Proteins ................................ ......................... 68 Indirect ELISA of PUV Treated Wheat Proteins ................................ ............... 69 HHP T reated Total Soluble Wheat Proteins ................................ ........................... 70 SDS PAGE of HHP Treated Wheat Proteins ................................ ................... 70 Western Blot of HHP Treated Wheat Proteins ................................ ................. 71 Dot Blo t of HHP Treated Wheat Proteins ................................ ......................... 71 Indirect ELISA of HHP Treated Wheat Proteins ................................ ............... 72 NTP Treated Total Soluble Wheat Proteins ................................ ............................ 72 SDS PAGE of NTP Treated Wheat Proteins ................................ .................... 72 Western Blot of NTP Treated Wheat Proteins ................................ .................. 73 Dot Blot Results of NTP Treated Wheat Proteins ................................ ............. 73 Indirect ELISA Results of NTP Treated Wheat Proteins ................................ .. 73

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7 D iscussion ................................ ................................ ................................ .............. 74 Effect of PUV Treatment on Wheat Proteins ................................ .................... 74 Effect of HHP Treatment on Wheat Proteins ................................ .................... 75 Effect of NTP Treatment on Wheat Proteins ................................ .................... 76 5 SUMMARY AND CONCLUSION ................................ ................................ ............ 93 THE ANOVA PROCEDURE ................................ ................................ .......................... 94 REFERENCES ................................ ................................ ................................ .............. 99 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 106

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8 LIST OF TABLES Table page 2 1 Major food allergen isolated and characterized ................................ .................. 41 2 2 Nutritional value of few selected cereals ................................ ............................ 42 2 3 Chemical composition of endo sperm, bran, and germ ................................ ....... 42 2 4 Strength of common bonds in Biomolecule s ................................ ...................... 43 4 1 Temperature and time measured during PUV treatment ................................ .... 77

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9 LIST OF FIGURES Figure page 2 1 Longitud inal view of the wheat kernel ................................ ................................ 44 2 2 Cross sectional view of wheat kerne l ................................ ................................ .. 45 2 3 Cross sectional view of wheat ker nel ................................ ................................ .. 46 2 4 C lassification of wheat gluten ................................ ................................ ............. 47 3 1 PUV Equipments A) Xenon Steripulse XL 3000 batch systen, B) Xenon Steripulse XL 3000 continuous system ................................ .............................. 61 3 2 HHP equipments A) Lab scale HHP unit, B) DASYLab 7.0 software used for HHP treatment ................................ ................................ ............................... 62 3 3 NTP Treatment set up ................................ ................................ ........................ 63 4 1 SDS PAGE analysis of PUV treated wheat albumin an d globulin ...................... 78 4 2 Western blot analysis of PUV treated wheat albumin and globulin ..................... 79 4 3 SDS PAGE analysis of PUV treated wheat gluten ................................ ............. 80 4 4 Western blot analysis of PUV treated wheat glu ten ................................ ............ 81 4 5 SDS PAGE analysis of PUV treated total soluble whe at proteins ...................... 82 4 6 Dot blot results of PUV treated to tal soluble wheat proteins ............................... 83 4 7 Immunoreactivity of PUV treated total soluble wheat proteins determined by indirect ELISA ................................ ................................ ................................ ..... 84 4 8 SDS PAGE analysis of HHP treated t otal soluble wheat protein l ....................... 85 4 9 Western blot analysis of HHP treated to tal soluble wheat protein ...................... 86 4 10 Dot blot results of HHP treated total soluble wheat protei n ................................ 87 4 11 Immunoreactivity of HHP treated total soluble wheat prote ins determi ned by indirect ELISA ................................ ................................ ................................ ..... 88 4 12 SDS PAGE analysis of NTP treated tota l soluble wheat protein ........................ 89 4 13 Western blot analy sis of NTP treated tot al soluble wheat protein ....................... 90 4 14 Dot blot results of total soluble wheat proteins treated with HPP ....................... 91

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10 4 15 Immunoreactivity of NTP treated total soluble wheat proteins determined by indir ect ELISA ................................ ................................ ................................ ..... 92

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11 LIST OF ABBREVIATION S HHP High hydrostatic pressure HMW High molecular weight lgE Immunoglobulin E LMW Low molecular weight NTP N on thermal plasma PUV Pulsed ultraviolet light

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science REDUCTION OF WHEAT A LLERGEN POTENCY BY PULSED UL TRAVIOLET LIGHT, HIGH HYDROSTACTIC PR ESSURE AND NON THERMAL PLASMA By Jyotsna K Nooji May 2011 Chair: Wade Yang Major: Food Science and Human Nutrition Wheat allergy is known to elicit adverse immune reaction in children and adults. dermatitis, gastrointestinal symptoms and wheat dependent exercise induced anaphylaxis (WDEIA) in hypersensitive individuals. Allergenic proteins are present in the ent ire wheat protein fractions which are mainly characterized into; water/salt soluble albumin and globulin fractions and insoluble gluten fraction. Total avoidance has been utilized in an attempt to reduce allergic reactions but, is often impractical or inef fective; thus research focusing on using processing technologies to alter food allergens is gaining more and more attention. Various thermal and non thermal processing techniques have been utilized to alter the structure of the allergenic proteins. One of the major disadvantage of conventional thermal processing over non thermal processing is it causes undesirable effects on the nutritive and sensory qualities. Pulsed ultraviolet light (PUV), high hydrostatic pressure (HHP) and non thermal plasma (NTP) are non thermal processing methods which can potentially alter wheat protein conformat ion and hence, reduce the immune reactivity. The main objective of this study was to assess the effect of PUV alone or in combination with heat, HHP and NTP on the IgE binding of

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13 wheat protein extract. Wheat protein extract was subjected to PUV at 3pulses for 30 s, 60 s, 90 s, 120 s and 120 s followed by boiling. In addition, HHP (21C and 70C for 5 and 15 min) and NTP (1, 3 and 5 min) were also applied to wheat proteins extra cts. The control (untreated), PUV, HHP and NTP treated samples were analyzed by SDS PAGE, western blots, dot blots and indirect ELISA. Allergen potency indicated by IgE binding was determined in blots and ELISA. The PUV, HHP and NTP treatments indicated no ticeable difference in the proteins profile demonstrated by SDS PAGE. A significant reduction in IgE binding was observed in PUV (90 s), HHP (21C and 70C for 5 min) and NTP (5 min) treated samples as demonstrated by indirect ELSIA. The maximum reduction in IgE binding was achieved by PUV (46%) and HHP (42%) treatments. These findings indicate that non thermal processing methods can be implemented to reduce wheat allergen potency.

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14 CHAPTER 1 INTRODUCTION Food allergies are abnormal immunological response s to normally harmless food s or food components which are almost always protein based. R eactions that occur within minutes to an hour are considered immediate hypersensitivity reactions and are mediated by immunoglobulin E (IgE) antibodies. D elayed hypersen sitivity reactions take place between 6 24 hours or more after ingestion and are cell mediated. Various studies suggest that the prevalence of food allergy in United Stat es is approximately 1.0 % 4.0 % (Sampson 1999 ; Sicherer and others 2003) Food allergy is a constant problem in the population which includes c hildren, adolescents and adults who are allergic to certain foods. According to the Food Allergen Labeling and Consumer P rot ection Act (FALPCA ) (FDA 2009), a n estimated 2 % of adults and 5% of yo ung children and infants suffer from allergy in Unites States The prevalence of food allergy amongst children under 18 years has increased by 18% from 1997 to 2007 (Branum, CDC 2008). The FDA (2009) has listed eight ma jor foods that are responsible for approximately 90% of overall food allergic reaction which include s milk, eggs, fish, c rustacean shellfish, tree nuts, peanuts, wheat and soybeans. Wheat is an essential part of the diet in United States and several oth er cou ntries throughout the world, including C hina, Japan, Germany, and India (Pomeranz 1988) According to FAO (2008), China is the leading wheat pro ducing country followed by India, United States, and Russia, with average wheat production at 112, 78, 68, and 63 million metric tons, respectively Wheat is among the major cereals cultivated throughout the world along with rice, maize, barley, rye, sorgh um, oats and millet Wheat is mostly milled into a flour form and, is consumed in the form of bread, biscuits,

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15 pasta products, breakfast cereals, couscous, chapathi (Indian bread), tortilla and many other wheat based products. Wheat is now recognized as a source of allergens and is responsible for various allergenic reactions (Mittag and others 2004; Sicherer 2002; Scibilia and others 2006; Sampson 1999) Allergic reactions to wheat manifest in children as well as i n adults (Battais and others 2005; Matsuo a nd others 2004; Scibilia and others 2006) Although many researchers (Sicherer 2002) (Takizawa and others 2001) have put forward the theory that children outgrow wheat allergy with age, adults were also found to suffer severely from wheat allergens (Scibilia and others 2006; Sampson 1999) Some of the adverse reactions demonstrated by individuals allergic t o wheat are u r ticaria, atopic dermatitis gastrointestinal symptoms and anaphylaxis (Takizawa and others 2001; Pastorello and others 2007; Luis and others 1990; Varjonen and others 1994; Simonato and others 2001) Currentl y there is no medical prevention or cure for food allergies. Therefore the best way in food allergy management is complete elimination of allergenic foods. Food proteins are main food components that are required for both functiona l properties of foods and nutritional importance in the human diet. The functional properties of proteins are numerous, as they are used for emulsifying agents, viscoelastic properties, textural properties, solubility, gelation, thickness swelling, and man y more (Sikorski 2001) In terms of their nutritional importance, food proteins contain amino acids essential for human growth, maintenance, repair and metabolism (Sikorski 2001) However, as previously mentioned they are responsible for adverse

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16 reaction in individuals allergic to them. Therefore, for certain individuals choosing food proteins can be r estrictive. the food label; nonetheless, some of the allergens may be accidently transported in the food product, or may be present in unspecified manner as a derivative of food allergens ( FDA 2005) There are certain cases due to cross contamination where the individual reacts to a minor amount of allergens present in the food. In these instances, most often food products are ma nufactured on equipment which is also used to produce other allergen containing foods; this is called cross contamination, and allergenic proteins are introduced into the food inadvertently Other times, consumers may unknowingly purchase foods which contain allergenic proteins like, children, and adults who have less knowledge regarding allergens (Vierk and others 2007) As described, food proteins, including allergenic proteins, can be used for their functional and organolepti c properties which can be present in the form (hydrolyzed wheat proteins as meaty flavor enhancer, whey and casein powders, soy lecithin and so on) that are not obvious as a food ingredient and or too difficult/technical to assimilate (Vierk and others 2007) There have been numerous attempts to reduce or eliminate food allergens through processing and technology. Some of these met hods have pro ven to be effective and include deamidation of amino acids, enzymatic hydrolysis, step wise polishing, high pressure, irradiation methods, and thermal treatment (boiling, extrusion, cooking) (Sicherer 20 02; Zhenxing and others 2007b; Mondoulet a nd others 2005; Ehn and others 2004; Handoyo and others 2008; Davis and Williams 1998) However the demand for new technologies which can produce less allergenic food products is

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17 becoming more vital. Thermal trea tment, although sometimes effective in reducing food allergens, can make the food products lose their quality by affecting the taste, texture and other organoleptic properties. Non thermal technologies can be advantageous because they often have minor effe (Chung an d others 2008; Yang and others 2010; Messens and others 1997) The main objective of this study was to analyze the effect of pulse d ultraviolet light (PUV), high hydrosta tic pressure ( HPP), and non thermal plasma (NTP) technology on major wheat allergen reactivity The wheat protein extracts were treated with PUV, HHP and NTP and protein profiles were analyzed by gel electrophoresis. The IgE reactivity was investigated by Western blot, dot blot and indirect ELISA (enzyme linked immunosorbent assay) with pooled human plasma from patients with clinical history of wheat allergy.

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18 CHAPTER 2 LITERATURE REVIEW Food Allergy and Allergens Consumption of foods with allergenic c omponents can cause various disorders in hypersensitive individuals. F ood allergy can be caused by inha lation or ingestion of an allergen and, is immune mediated. Non immune mediated reactions are known as food intolerance (Sicherer 2002; Sampson 2004) The immune mediated food allergy can be further categorized into IgE mediated and non IgE mediated allergy (Asero and others 2007) Th e IgE or immunoglobulin E antibody is involved in inducing immediate reacti on s following the intake of food allergen s (Tanabe 2007) During the initial exposure of the food the allergen binds to T cells which subsequ ently activate B cells. The B cells produce and release IgE which is cross linked to mast cells or basophils (Tanabe 2007) This process is ca lled sensitization. Upon re exposure to the allergen IgE cross links the allergen which causes degranulation of the mast cel ls. Chemical mediators such as histamine and prostaglandin are released that ultimately cause allergic reactions in the individual. This process results in the adverse reaction s seen in hypersensitive individual s, like anaphylaxis, urticaria, gastrointestinal symptoms and other various adverse reactions (Tanabe 2007; Sampson 1999, 2004 ) Some o f the most common food allergen and have been mentioned in Table 2 1. Extensive studies have been conducted over the past few decades in order to identify the actual cause of IgE mediated food allergies. According to the se studies and the current insight of food allergy it is now understood that the IgE binds to the specific site on the allergen or the surface of allergen protein known as an epitope (Tanabe

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19 2007) The epitope is categorized into linear and conformational epitopes depending on t he epitope structure (Bannon 2004; Davis and Williams 1998) The cross reactivity between the epitope and the al lergen is determined by the structure of the allergen protein and the sequence of the amino acids (Bannon 2004; Davis and Williams 1998) In order to understand allergen epitopes, a short review on protein conforma tion is required. The structure of the protein is categorized as primary (amino acid sequence), secondary, tertiary and quaternary structure. The primary structure refers to the linear sequence of amino acids connected to each other via peptide bonds. It f orms the back bone for other higher structure of proteins. The secondary structure refers to the three dimensional organizations of segments of polypeptide cha helix is stabilized by hydrogen bonding and is an organized structure where the hydrogen bonding occurs within single protein chain. The sheet st rand i s connected via hydrogen bonds (Damodaran and others 1996) pleated she ets. The random coil arrangement has no ordered pattern along the polypeptide chain. This kind of structure is formed when the amino acid side chains prevent sheet. The tertiary structure is a thr ee dimensional organization where the linear protein chain with secondary structure segments folds into three dimensional arrangement. The quaternary structure contains more than one polypeptide chain. More than one polypeptide chains are linked with each other via non covalent interaction (Damodaran and others 1996) A linear epitope is composed of a short s equence of amino A conformational epitope is composed of various amino acids on the protein that are brought together by

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20 folding or determined by secondary, tertiary or quaternary structure of the protein. Therefore, for the IgE to crosslink the conformational epitope, secondary and tertiary structur e of protein is necessary ; whereas IgE binding to linear epitopes can entail the primary structure of the protein as well (Bannon 2004) During heating, hydrolysis and other physical/ chemical treatment s there can be a great effect on protein structure; some of the proteins secondary and tertiary structu re s are disrupted, expos ing smaller amino acid sequence to the exterior of the protein (Sathe and Sharma 2009) These once buried structures are hydrophobic sites on proteins tha t are hidden from surfacing water or other solvent (Tanabe 2007; Sampson 1999) and can contain linear epitopes that become exposed upon denaturation. Conversely, this conformational change can destroy conformational epitopes According to a study involving patients exposed to milk allergens had significantly higher IgE reactivity to linearized proteins (linear epitopes) compared to proteins in their native state (conformational epitope) (Vila and others 2001) The linear epitopes were revealed by treating the native lactoglobulin) with DTT (dithiothreitol), which denatured the secondary and tertiary structure. Similarly in another study by (Jrvinen and others 2007) IgE binding to linear and conformational epitopes was observed i n 37 patients, in the 16 who had already outgrown the allergy, as well as those with persistent allergy to egg. High IgE reactivity was observed to both linear and sequential epitopes in patients with persistent allergy compared to patients with tolerance. The author also speculated that linear epitope binding must be considered in order to identify the actual allergenic nature of food proteins. Since the

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21 allergen protein undergoes digestion that includes enzymatic hydrolysis the linear epitope is exposed i n the gut due to disruption of secondary and tertiary structure (Pasini and other s 2001; De Zorzi and others 2007) The Application Support Center provides a group of general purpose styles to help you format your document and give your thesis or dissertation a continuity of appearance. Styles cannot do everything but they can be used for general formatting purposes. Each style created by the ASC is listed in Table 2 1. History of Wheat The origin of wheat is believ ed to be from the hybridization of an Eurasion emmer type w heat and wild species of grass. Durum wheat or Triticum turgidum is closely related to Triticum dicoccoides (hexaploid wheat), and is native to wild emmer wheat. Modern cultivated wheat belongs to two species: (1) hexaploid bread wheat, T. aestivum and (2) tetraploid, hard or durum type wheat, T. turgidum Wheat tends to grow well in temperate climate and is grown throughout the year in some part of the World (Pomeranz 1988; Carver 2009) From January to March it is cultivated in Australia, Argentina and India; between April and June it is grown in Mexico, Japan, China, and some parts of United States and from July to September it is grown in France, Germany, England, Canada and Norway; finally from October to December it is cultivated in Finland, South Africa, Burma, and Argentina. Many countries in the world grow wheat as their main source of food supply (Pomeranz 1988) Wheat and related cereals like rice, barley and rye are an important pa rt of the diet in Europe, Asia United States and other parts of the world (Pomeranz 1988) In addition to its nutritional importance, wheat is widely utilized in the baking industry for its excellent viscoelastic properties (Ahmedna and others 1999) Hexaploid wheat; Triticum

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22 aestivum is used for the production of bread flour, and t etraploid; T.turgidum hard or durum type wheat which is mainly used for pasta products Table 2 2 illustrates the nutri tive value of different cereals, which indicates wheat is comparatively high in proteins versus other cereals. Some of the most common wheat based products include bread, biscuits, pasta couscous, pita and chapathi/naan (Indian bread). Wheat is most commo nly divided into winter or spring wheat which refers to the season during which the crop is grown. The other most commonly used commercial classification is hard or soft wheat that refers to the kernel strength and hardness. Durum wheat is the best example of hard wheat and is used in the pasta and noodle products. Soft wheat is preferred for products like bread, biscuits and pastries. Wheat Kernel Structure A wheat kernel is approximately 5 to 8 mm in length and 2.5 to 4.5 in width. In the technological p erspective w heat kernel or seed mainly consists of bran, endosperm and the germ ( Figure 2 1). The wheat bran consists of a pericarp which is the outer most hard layer and closely adheres to the seed coat. Beneath a layer of nuclear tissue is the aleurone layer ( Figure 2 2 and 2 3) The starchy endosperm is beneath the aleurone layer. During milling the germ, bran and the endosperm can be separated and used for varieties of products. The whole wheat flour consists of nutrients from all the layers (Pomeranz 1988) The white flour is produced by separating b ran and germ from the endosperm, and sol ely using the endosperm portion Table 2 3 illustrates, t he chemical composition of endosperm, bran and germ and clearly shows wheat endosperm contains high amount of starch compared to other parts. Therefore, the endosperm is widely util ized for the production of wheat flour for baking The germ is present on t he dorsal side of the wheat kernel and accounts for 2 4% of the kernel

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23 weight. The germ or embryo transports nutrients during sprouti ng and germination of the seed and it will grow into new plant, absorbing nutrients from the endosperm during its developme nt (Eliasson and Larsson 1993) Wheat P roteins Wheat and other related cereals proteins are mainly divided based on their solubility. According to the Osborne c lassification that is most widely adopted, wheat, barley, rye and other cereals are classified into albumin, globulin, prolamin and glutelin protein fraction depending on their solubility in different solvents (Shewry and others 1986; Osborne 190 7) The wheat proteins are also classified into two major groups, the storage proteins which consist of gluten proteins and cytoplasmic or metabolically active proteins i.e. albumin and globulin fraction. Albumins and Globulins Albumin and globulin fracti on are classified as water and salt soluble proteins and together comprises to about 10 to 15% of total wheat proteins. The albumin proteins present in wheat are soluble in water and account for 10% of total wheat proteins whereas salt soluble globulin acc ounts for only 5%. Albumins can be easily extracted by dissolving the whole wheat flour in water and the supernatant derived is considered as albumin fraction. Lockehart and Bean (1995) performed a sequential extraction of albumin in deionized wate r in the ratio 1:5 (w/v). The resulting supernatant was considered as albumin fraction. The globulin fraction is extracted in dilute salt solution (NaCl). The albumin and globulin proteins are also known as cytoplasmic proteins and contain high amount of a mino acids, especially lysine, compared to other wheat proteins. These proteins contain metabolic enzymes, enzymes that hydrolyze proteins,

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24 Storage Proteins The water/salt insoluble g luten protein fraction of wheat is the main storage protein of the wheat kernel and accounts for 50 to 80% of total wheat proteins, also known as prolamins. Gluten can be prepared by washing the whole wheat in dilute salt solution to remove any water/salt soluble albumin and globulin fraction. The resulting sticky mass is known as gluten that can be subdivided into two major protein groups: (1) gliadins and (2) glutenins (Field an d others 1982; Tatham and others 2000; Shewry and others 1986; Osborne 1907) The monomeric gliadins containing single polypeptide chains are soluble in 70 % gliadin s based on their mobility in gel gliadin s have l ess proline, glutamine and phenylalanine but the sulfur containing amino acids cysteine and methionine are involved in intra /inter mole cular disulfide linkage (Tatham and others 2000) The gliadin has little or no sulfur containing amino acids but, has high amount of glutamine, proline and phenylalanine. During the treatment with reducing agent s like DTT (Di thiotriol) mercaptoethanol the disulfide bonds are cleaved resulting in mor e linear structure of amino acid sequence. The remaining proteins can be extracted in dilute acetic acid or dilute alkali and are known as glutenin fraction. Glutenin is a high molecular weight protein consisting of subunits stabilized by disulfide bondi ng Reducing glutenin yields low molecular (LMW) and high molecular weight (HMW) subunits. Both subunits differ in molecular we ight and amino acids. The molecular weight of H MW glutenin subunits can be larg er than 70 kDa whereas, LMW subunits are smaller tha n 50 kDa. The polymeric glutenin fractions are high molecular weight proteins containing polypeptide chains bonded by intermolecular disulfide bonds. During the reduction of the disulfide bonds

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25 the glutenin fraction results in low molecular weight (LMW) an d high molecular weight (HWM) glutenin subunits (Shewry and others 1986; Pomeranz 1988) Another way of distinguishing wheat gluten proteins are sulfur rich (S rich), S p oor and high molecular weight prolamins ( Figure 2 4 ) The S gliadins corresponding to 32 to 42 kDa and LMW glutenin subunits range from 36 to 44 kDa. The S gliadin corresponding to 44 to 72 kDa. The thi rd group is HWM prolamins comprising of HMW glutenin subunits (64 to 136 kDa) (Shewry an d others 1986; Tatham and others 2000) Gluten is usually extracted in acid, bases and a lcohol. Chemical detergents such as sodium dod ecly sulfate (SDS), urea, dithiothr e i t ol (DTT) are often added to assist in extraction; however, these chemicals will disrupt the disulfide bonds resulting in non native proteins. For these reasons extracting native wheat gluten can be problematic if proper sol vent is not used. Furthermore, extraction allow apparition of the wheat gluten into gliadin and glutenin subunits by extracting these fractions separately. Glutenin subunits ( including low and high molecular subunits ) can be extracted in dilute acid (e.g. acetic acid, HCl) and gliadins can be efficiently extracted in 70% alcohol solvent. Wheat Allergy IgE mediated allergy to wheat occurs after the ingestion or inhalation of wheat and related cereals like barley, rye, oats etc. Depending on the route of exp osure, wheat may demonstrate as a classic IgE mediated food allergy affecting skin, respiratory tract, gut, wheat dependent exercise induced anaphylaxis(WDEIA), occupational asthma hildren wheat is the causative factor for several immunological reactions like atopic dermatitis, nausea, abdominal pain and other gastrointestinal symptoms and anaphylaxis (I nomata 2009)

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26 Studies i ndicate that most children outgrow the hypersensitivity to wheat by the age of 3 to 5 years unlike peanut, milk and eggs which have been shown to persist throughout the life time (Sicherer 2002) But the prevalence of wheat allergy in adults can be severe and fatal and therefore when considering wheat as a food allergen both children and adults should be given equal importance in diagnosis and treatment (Mittag and others 2004; Matsuo a nd others 2004; Scibilia and others 2006) inhalational of wheat flour. It is an IgE mediated allergy where the ind ividual has specific IgE to wheat flour and the inhalation of wheat flour results in adverse reaction. The major allergen s s asthma are proteins from water and amylase inhibitor family) (James and others 1997; Luis and others 1990) An i mmunoblotting study by Weiss and others (1993) showed extensive IgE binding to albumin and globulin polypeptides whereas lower IgE binding to gluten protein fractions. Another study by Mittag and others (2004) also showed similar results that i llustrated that the water/salt soluble a lbumin and globulin fraction had high IgE reactivity in indi viduals having the symptoms of b asthma ; however no major allergen was identified. This shows that the individuals suffering from b have adverse reactions to various subf ractions of albumin and globulin proteins. Wheat Dependent Exercise Induced Anaphylaxis Anaphylaxis is a severe form of food allergy, which can be life threatening. Anaphylaxis reactions occurs when intake of foods li ke wheat, shellfish, peanuts, treenuts, egg, and milk (Sampson 1998) WDEIA reaction occurs after the intake of

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27 wheat followed by exercise within 2 to 3 hrs Scientists demonstrated that IgE reactivity of 5 gliadin (65 kDa) was major protein involved in WDEIA using skin prick tests, ELISA and i mmunoblotting (Mittag and others 2004; Palosuo and others 2003; Matsuo and others 2004) Wheat Allergen P roteins According to published literature the allergen responsible for inducing the adverse reaction is glycoprotein range between 10 60 kDa (Watanabe and others 2001; Sanchez and others 1992) Glycoproteins are low molecular weight water soluble protein s and are often stable to heating, acid treatment and proteases. Therefore these allergens are still active in the body even after undergoing extensiv e heat, enzymatic hydrolysis or other chemical treatment. Upon reaching the gut the allergen ic protein can elici t the adverse reaction. Watanabe and others (2001) performed a study on the IgE reactivity of wheat glycopro teins. The IgE reactive protein was found to be a 60 kDa glycoprotein which was present in whe amylase inhibitor ( water /salt soluble protein ) Hypoallergenic wheat flour was produced by the enzymatic treatment of the 60 kDa glycoprotein with actinase and cellula se. However, a 16 amylase inhibitor has shown to have substantial IgE reactivity in patients with B asthma (Sanchez and ot hers 1992; Garcia and others 1996) Several researchers have demonstrated IgE binding to various fractions of wheat. From these different studies it should be noted that entire wheat protein fraction s have been showed to eli cit IgE reactivity in wheat allergic individuals depending on the route and extent of exposure. A study done by Simonato and others (2001) showed IgE binding to soluble and insoluble protein fraction s Th is study examined 20 patients (atopic and non atopic) with irritable bowel syndrome and other symptoms aft er the

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28 ingestion of wheat. The i mmunoblotting results of this study recognized several IgE binding protein bands in water/salt soluble fraction. The a uthor s also mentioned that a 16 kDa protein w as the most frequently observed band in more than 50% of the sera ; whereas in the salt insoluble proteins the IgE reacted to 42 kDa protein band Wheat 5 G liadin amylase/trypsin inhibitor has been frequently reported in i ts involvement in wheat allergi c patients and is confirmed as the major allergen in B Pastorello and others (2007) investigated the IgE reactivity in three wheat protein fraction namely albumin/g lobulin, gliadin and glutenin. The s trongest IgE binding was amylase inhibitor along with IgE binding to lipid transfer protein (LTP) and LMW glutenin subunits. Similarly Simonato and others (200 1) confirmed that amylase/trypsin inhibitor corresponding to 16 kDa w as the major allergen involved in atopic patients with positive results in skin prick test. Individuals with atopic dermat itis showed reactivity to low molecular weight proteins particularly a 26 kDa protein as demonstrated by i mmunoblotting. Other IgE bands which ranged from 7 kDa 84 kDa were classified as minor allergens (Varjonen and others 1994) Five childre n with wheat allergy reacted to 15 kD a wheat protein as analyzed by W estern blotting (James and others 1997) The results confirm that the low molecular weight protein is responsible for eliciting adverse reaction after the ingestion and inhalation of wheat in wheat allergic was observed to 26 38 and 69 kDa in children suffering from atopic dermatitis (AD). The results were confirmed by performing skin prick tests and radioallergsorbent test (RAST) (Varjonen and others 1995) A higher immuno reactivity was also found with gliadins, LMW glutenin subunits and various ban ds in albumin/globulin fraction; yet, the

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29 sera had sign ificantly lower IgE reactivity in HMW glutenin subunits (Battais and others 2003) The immunoblot s of proteins from the albumin/globulin fraction ranged between 15 70 kDa, and amylase/trypsin inhibitor. The allergen reactivity appears to be varied depending on the age of the individual and symptoms. The major allergen associated 5 gliadin, which has reported several times in individuals suffering from anaphylaxis followed by exercise (Tatham and Shewry 2008; Matsuo and others 2004) According to the study by Batta is and others (2005) IgE 5 gliadin was higher in adult patients with WDEIA and more than 50% (adults and children) reacted to the same al lergen with urticaria. I n the case of children there were no specificity in IgE reactivity to single allergen ; however, reac tivity gliadin and extensive reactivity to the albumin/globulin fraction in children with atopic dermatitis was noted Lipid Transfer Protein (LTP) Upon encounter with an allergen,the IgE binds to specific sequence of amino acid on the al lergen which is known as epitope. The major IgE binding epitope has been recognized by Maruyama and others (1998) that consists of Gl n Gln Gln Pro Pro (Gln = glutamine ; pro = proline) which is confirmed as the major IgE binding site in wheat allergic individuals and is present in LMW glutenin subunits. Research in Developing Reduced Allergenic P roducts To date an e xtensive amount o f research has been conducted in the following fields: identifying the IgE mediated allergen associated with wheat allergy, managing the wheat allergy, treatment and va rious processing and methods that could potentially be used to produce non allergenic and/or wheat products with reduced allergen potency. Processing methods used to reduce allergen potency are particularly of

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30 interest because they could potentially help a large population of wheat allergy sufferers. Some of processing method s have shown to be effective ; however; there are no hypoallergenic or reduced allergen wheat products currently in the market. This shows a huge demand for producing safer and healthier wheat based products that can be consumed by adults and children suffe ring from wheat allergy. To date an extensive amount o f research has been conducted in the following fields: identifying the IgE mediated allergen associated with wheat allergy, managing the wheat allergy, treatment and va rious processing and methods that could potentially be used to produce non allergenic and/or wheat products with reduced allergen potency. Processing methods used to reduce allergen potency are particularly of interest because they could potentially help a large population of wheat allerg y sufferers. Some of processing methods have shown to be effective ; however; there are no hypoallergenic or reduced allergen wheat products currently in the market. This shows a huge demand for producing safer and healthier wheat based products that can be consumed by adults and children suffering from wheat allergy. Various efforts have been made to reduce the allergen reactivity of certain foods. During the processi ng conditions the allergen reactivity may be altered due to a change in protein structure. These modifications may ultimately result in the alteration of epitope s to which IgE would normally bind. Thermal treatment on food allergens is extensively researched due to its wide applicability in food processing. Heat treatment alters the native stru cture of proteins that result in denaturation. Denaturation of proteins modifies secondary and tertiary structure and results in formation and breakage of covalent and non covalent bonds. Con formational epitopes are associated with IgE

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31 binding to the secon dary and tertiary structure; t herefore modified structure may reduce an adverse immune response. Heating food proteins result in unfolding which can be reversible or irreversible depending on the type of protein, temperature and extent of heating. The nat ive structure of proteins has hydrophobic bonds buried inside whereas the hydrophilic sites are on the surface. Heating can lead to new inter/intramolecular interaction in proteins that includes, hydrogen bonding, electrostatic interaction, disulfide bondi ng. These interaction s can be ir reversible leading in unfolding and random coil conformation. However this may not apply for all the proteins due to the treatment conditions, and therefore, unf olded proteins may refold resulting in new covalent/non covale nt i nteractions and regaining of allergenic activity. Some food allergens ( proteins ) are stable to heating, and their allergenic properties may not be affected Maillard reaction is a chemical reaction between the free amino group of peptide chain and red ucing sugar during heating at sufficiently high temperature. The stages involved in Maillard reaction are complex are not completely understood. However, some of the distinctive stages are sugar and amine condensation and Amadori re a r rangement. The second stage involves sugar dehydration and fragmentation and the final stage includes the formation of heterocyclic n itrogen compounds. Some of the Maillard reaction intermediate s are Schiff base, hydroxyl methyl furf ural, aldehydes and ketones. The final produ ct s which impart the brown color that is often desired in cooked products are called melanoidins Wheat undergoes the M aillard reaction during baking, boiling, fermentation etc where browning is desired for the flavor compo nents and appeal. Furthermore t he reaction can involve covalent/non covalent modification in

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32 proteins that contribute to allergenicity. De Zorzi and others (2007) studied the effect of pasta drying temperature (60 110C) on the digestibility and allergenicity of wheat allergens. The y found a change in protein solubility due to the formation disulfide and hydrophobic interaction yet the interactions were reversible when they were subjected to digestive conditions. In contrast, heating at ultra high temperature (110C) resulted in decreas ed in proteins solubility that wa s irreversible. The immunological results indicate d a reduction in the IgE r eactivity when pasta was heated up to 80C ; pasta heated above this temperature showed increase in IgE reactivity. Heating proteins at high t emperature in the presence of reducing sugar may result in the formation of new immunologically reactive structures in the protein These new structures that have IgE and/or allergen activity are referred as neo antigens (Davis and others 2001) The temperature at which the proteins are treated can have major effect on its structure and functionality. Bread is one of the examples of heat treated wheat product. During baking the bread crumb and crust reach different temperature. T h e temperature of the crust reaches almost to 200C ; ture reachs approximately 100C. Therefore the effect on proteins can be varied depending on the temperature and treatment time (Wal 2003) It was found that, compared to crumb, the crust contained proteins which showed lowered solubility following baking Since the crust reached very high temperatur e s, there could be new irreversible covalent interaction involving aggregation, cross lin king and Maillard type reaction. I n contrast the crumb was more soluble due to significantly lower temperature. The interaction involved in crumb could be disulfide bo nding and hydrophobic interaction due to

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33 denaturation (Pasini and others 2001) that can be cleaved with a reducing agent (Davis and Williams 1998) eta lacto globulin is one is of the major allergen s present in milk and was shown to have reduced aller genicity when heated at 70C (Ehn and others 2004) The researchers speculated that the IgE reactivity was decreased with increase s in temperature (90C); however the heat tr eated milk still retained allergen reactivity to a minor extent A traditional roasting of peanuts involves heating at 140C for 40 min whereas boiling of peanuts takes place at 100C (Mondoulet and others 2005) for a shorter period of time The roasted peanuts were confirmed to have significantly high er IgE reactivity to Ara h 1 and Ara h 2 ( two major allergen s present in peanuts ) compared to boiled peanuts Thus, it should be noted that heating proteins at certain temperature (80 100C) causes denaturation of proteins resulting in reduced IgE binding. On the other hand, there is considerable difference in the IgE reactivity when proteins are heated at very high (>100C) which may contribute to the formation of peptide fragments and ultimately amino acids that may or may not have adverse immune response (Korhonen and others 1998; Davis and others 2001; Davis and William s 1998) These studies indicate that temperature is a large component involved in changing the allergen reactivity of proteins. Wheat is consumed as whole wheat and white flour. Another processing method which separates the wheat into different components is called step wise polishing. The endosperm of wheat which contains high starch content, i s separated by removing the outer layer and the bran (pericap and aleurone). The resulting high starch wheat flour

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34 is extensively utilized in baked products, pas ta, pastry etc. Whole wheat kernel s that underwent stepwise polishing to remove the outer layer that contained high concentration s of the water/salt soluble albumin and globulin fraction was ultimately separated (Handoyo and others 2008) Immunoblottin g results demonstrat ed a reduction in IgE activity of the inner most fraction due to the lower concentration of albumin/globulin Gamma irradiation is a newer technique that has been researched in recent year for its ability in altering the proteins involved in allergenic res ponse. The effect of lactoglobulin), chicken egg (albumin) and shrimp (tropomyosin) (Byun and others 2002) The irradiation dose ranged between 3 to 10 kGy, and the IgE binding activity was demonstrated by imunoblotting and ELISA. The IgE binding epitope was structurally altered and a reduced IgE binding was observed in a ll of the samples. (Zhenxing and others 2007b) found that irradiated shrimp (7 10 kGy) myosin showed a variati on in the protein structure. A new band at 45 kDa was generated in irradiaded samples at 7 and 10 kGy demonstrated by SDS PAGE. Pen a1, one of the major allergen in shrimp (36 kDa) existed in all the irradiated samples observed in SDS PAGE results. The IgE reacted to the newly generated band (45 kDa) at 7 and 10 kGy, conversely the band at 15 kGy confirmed by Immunoblotting. ELISA and Immunoblotting indicate a reduction in IgE binding to Pen a 1 with increase in the dose level. Gamma irradiated (1 15 kGy) s hrimp in conjunction with heat (100C) has shown to have reduce IgE binding to shrimp allergens(5 30 fold) but, the radiation treatment alone was not effective in reducing the allergenicity (Zhenxing and others 2007a) On the other hand, gamma irradiated (1

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35 25 kGy) followed by thermal treatment (autoclaving, roasting, blanching, micro waving) almond, cashew and walnut showed no difference in the allergenicity (Su and others 2004) This shows the stability of some proteins that is stable to both radiation and therm al treatment. Wheat gliadin fraction 5 gliadin elicit s an immune response in sensitive individuals especially in WDEIA individuals (Maruyama and others 1998; Matsuo and others 2004; Simonato and others 2001) I rradiation treatment with Cobalt 60 (2.2 12.8 kGy) has shown to increase the allergenic response of the wheat gliadin fraction as confirmed by immunoblotting and ELISA. Specifically, IgE reactivity increased with dose level s of 12.2 kGy with higher IgE binding to whole wheat com pared to the gliadin fraction alone, indicating the interaction of proteins in the presence of other proteins. Similar results were obtained by microwave heating (30 150 k J) of wheat gliadin fraction (Leszczynska and others 2003) A high immunologica l activity was confirmed at 40 k J but, the response to allergen pr oteins decreased at 90 and 150 k J detect ed by Immunoblotting a nd ELISA. Deamidation of wheat proteins is performed in order to improve th e functionality of the product namely the emulsion stability, foaming capacity, solubility, and water holding capacity compared to native proteins (Mimouni and others 1994) Deamidation is achieved either enzymatically or chemically. Degree of hydrolysis is the term used to define the number of peptide bonds cleaved during hydrolysis. Increase in the percentage is proportional to increase in the am ount of fragmented proteins and better solubility. Acid deamidation of wheat gluten is found to have reduced IgE binding (M aruyama and others 1990) The gluten was deamidated with 30%, 50% and 90%

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36 demidation. The results indicate d a significant reduction in IgE binding confirmed by dot blot. Ultraviolet and Pulsed Ultrav iolet Light T echnology Ultraviolet (UV) radiation has been widely utilized in the food industries as a pasteurization technology (Krishnamurthy and others 2008 ) Currently there are two types of UV mode: continuous and pulsed. Continuous UV light consists of wavelength region between 200 and 400 nm. The UV spe ctrum is further divided into short wave (UVC) from 200 to 280 nm, medium wave (UVB) from 280 to 320 and long wave (UVA) from 320 to 400 nm. The PUV radiation encompasses a wide range spectrum, from vacuum UV to far infrared radiation with the wavelength r ange between 100 1100 nm Although both methods have been utilized in microbial inactivation, the effectiveness of PUV is two to four times that of continuous UV technology. The inactivation mechanism for PUV includes from UV, infrared and visible range; w hereas, the inactivation mechanism for continuous is comparatively limited (Krishnamurthy and others 2008 ) Another major drawback of continuous UV is the source of UV generation. Typically mercury is used to generate UV radiation and might result in some residual mercury deposition in the food. On the other h and, PUV equipment consists of a discharge lamp filled with xe non or krypton gas. Electrical en ergy is stored in a capacitor and is released into the lamp in short bursts that consists of xenon or krypton The intense energy causes the Ionization of gas wi thin the lamp and results in the release of high energy radiation ranging in w avelength between 100 and 1100 nm. PUV l ight pasteurization PUV has been effectively utilized in inactivation of the microorganisms. Krishnamurthy and others (2007) investigated the effect of PUV on inactivating

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37 S taphylococcus aureus in milk and re ported 0.55 to 7.26 log 10 reduction. The results indicate no significant rise in temperature when the sample was placed at a distance of 5 cms from the quartz window. In another study by McDonald and others (2000) on inactivation of Bacillus subtilis spores P UV was three times more effective compared to continuous UV light. Effect of PUV on food allergens visible region, therefore the synergistic effect of the three regions might be effective in altering many chemical bonds ( Krishanmurthy and others 2008) Table 2 4 shows various chemical bonds that exist i n bio molecules. From this Table (2 4) it is clear that PUV produces sufficiently high energy that can effectively cleave covalent bonds. Therefore, PUV might alter the conformational structure of allergen proteins resulting in reduced immunoreactivity (Chung and others 2008; Yang and others 2010) On the other hand, continuous UV consists of energy f rom UV region only (Krishnamurthy and others 2008 ) Therefore the maximum wavelength in continuous UV irradiation is 400 nm from UV A region. C ho and Koji (2010) r eported that UV irradiation on beta lactoglobulin showed increase in the IgE binding when treated for 4, 8, and 12 hr. However, beta lactoglobulin irradiated for 32 hr indicated decrease in IgE binding. F ew researchers have employed PUV to reduce the allergen potency. Chung and others (2008) have illustrated that the PUV treatment of peanut extracts and liquid peanut butter caused a six to seven fold reduction in IgE binding compared to the control. In a recent study by Yang and others (2010) PUV was applied on soybean extracts and the results indicated a remarkable decrease in IgE binding The researchers noted that the optimal treatment time was 4 min at 13.2 cm from the PUV lamp.

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38 High Hydrostatic P ressure T echnology High hy drostatic pressure (HHP) is a non thermal technology which utilizes water or a dilute aqueous solution (~ 100 to 1000 MPa) as a pressure transmitting medium. The liquid surrounding the food inside the pressure vessel is compressed; and; the resulting high hydrostatic pressure is applied uniformly to the food product. This is a great advantage of this technology, considering that the product is treated evenly. There might be small rise in the temperature due to adiabatic compression; however, the temperature rise is in significant in most cases unless an external heat treatment is applied. Foods subjected to HHP have been shown to have improved fun ctional properties. For example pressure treated ovalbumin gels had better texture and elasticity compared to he at induced gels (Yaldagard and others 2008) Similarly, pressure treated soymilk at 500 MPa for 10 min showed better emulsifying activity and stability compared to heat treated soymilk at 100C for 19 min (Messens and others 1997) In comparison to thermal processing, HHP can preserve the taste, flavor, color and nutritional quality of the treated food. Furthermore, food proteins can be structurally modified upon pressurization causing changes in food allergen reactivity. The proteins under high pressure can undergo reversible and/or irreversible alteration in the structure resulting in denaturation, aggregation or gel formation. Non covalent bonds are highly susceptible to pressure trea tments whereas; covalent bonds may or may not be affected high pressure. Effect of HHP on food allergens High pressure treatment on rice grains resulted in the loss of some of the major allergens and resulted in hypoallergenic rice grains (Kato and others 2000) The results indicated that high pressure in combination with protease treatment was able to

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39 Glb, and Alb 16) and effectively remove t hem from the rice, resulting in less allergenic rice. Soybean sprouts obtained from HHP treated seeds showed reduced antigenicity. Conversely, pressurized soybean seeds and tofu showed increase in the antigenecity compared to untreated ones. Non Thermal P lasma T echnology In the recent years, NTP has been utilized in food processing as an innovative method for microbial inactivation. Plasma is defined as highly energized matter in a gaseous state. The NTP equipment mainly consists of two electrodes with die lectric discharge material, a power outlet and a treatment chamber. A gas, such as air, argon, or nitrogen, is supplied between the electric fields. During plasma discharge, the energized matter reacts with gas or food molecules to generate charged particl es in the form of positive ions, negative ions, free radicals, electrons and quanta of electromagnetic radiation (photons). High energy electrons cause ionization of the energized matter as well as the excitation of the particles present. Therefore, there might physical damage taking place when the biological material which is ultimately ruptured leading to the inactivation of microorganisms. The electrons cause ionization and dissociation of molecules leading in alteration in the orientation of ions and mo lecules. Therefore, it has been speculated that the main mechanism behind the microbial inactivation is destruction of DNA by UV irradiation, volatilization of components by UV photons and erosion of spore by free radicals. Reactive oxygen species (ROS) a re generated by NTP and readily diffuse into the cell surface. It has been speculated that ROS has profound damaging effect on the microbial cell. The oxidative stress caused by NTP can rupture the cell membrane and

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40 hence cell death. NTP was employed in in activating E.coli on almonds and the results reported indicate approximately 5 log reduction after 30 sec treatment at 30 kV and 2000 Hz (Deng and others 2007) The E.coli O 157:H7 is one of common food borne pathogen and was effectively inactivated by pulsed NTP up to 7 log units (Montenegro and others 2002) The power required for NTP is very low compared to other inactivation technology and capital cost is minimal c ompared to other sterilization technology. Therefore, non thermal plasma treatment is considered effective and economical in inactivation microorganisms.

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41 Table 2 1 Major food allergen isolated and characterized (Sampson 1999) s Caseins, Casein, Casein Whey Lactoglobulin, Lactoglobulin Chicken egg white Ovomucoid, ovalbumin, ovatransferrin (Gal d 1) (Gal d 2) (Gal d 3) Peanut Vicilin, conglutin, glycinin (Ara h 1) (Ara h 2) (Ara h 3) Soybean Vicilin, con glycinin (Gly m 1) Fish Parvalbumin (Gad c 1 [cod]; Sal s 1 [Salmon]) Shrimp Tropomyosin (Pen a 1; Pen i 1; Met e 1) Brazil nut 2S albumin (Ber m 1) Walnut 2S albumin (Jug r 1) Rice Amylase inhibitor (Ory s 1) Wheat Amylase inhibitor Barley Amylase inhibitor (Hor v 1) Buckwheat 11 S globulin (Fag e 1) Mustard 2S albumin (Sin a 1 [yellow]; Bra j 1 [oriental]) Celery Pathogenesis related protein Profilin (Api g 1) (Api g 2) Potato Patatin (Sol t 1) Carrot Pathogenesis related protein (Dau c 1) Apple Pathogenesis related protein, Profilin ( Mal d 1) ( Mal d 2)

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42 Table 2 2. Nutritional value of few selected cereals (Pomeranz 1988) Cerea l Energ y (kJ) Protei n (g) Fa t (g) Carbohydrate s +Fiber (g) Calciu m (mg) Iron (mg ) Thiami n (mg) Riboflavi n (mg) Niaci n (mg) Wheat Hard 1,390 13.8 2.0 70 37 4.1 0.45 0.13 5.4 Wheat Soft 1,390 10.5 1.9 74 35 3.9 0.38 0.08 4.3 Rice 1,495 7.5 1.8 77 15 1.4 0 .33 .05 4.6 Maize 1,490 9.5 4.3 73 10 2.3 0.45 0.11 2.0 Barley 1,390 11.0 1.8 73 33 3.6 0.46 0.12 5.5 Rye 1,335 11.0 1.9 73 38 3.7 0.41 0.16 1.3 Oats 1,625 11.2 7.5 70 60 5.0 0.50 0.15 1.0 Millets 1,485 9.7 3.4 73 32 4.5 0.50 0.12 3.5 Table 2 3. Ch emical composition of endosperm, bran, and germ (Eliasson and Larsson 1993) Whole Wheat Endosperm Germ Aleurone Bran (pericarp) 8.2 12.1 5.8 16.2 24.3 31.1 18.4 24.3 2.85 7.60 1.8 0.5 0.8 3.65 9.47 11.1 17.2 1.7 5.1 9.0 1.4 8.6 43.0 17.1 73.3 1.8 1.6 2.2 5.05 18.8 6.0 9.89 0.0 1.03 59.2 63.4 72.6 0.0 0.0 0.0

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43 Table 2 4. Strength of common bonds in Biomolecules (Krishnamurthy and others 2008 ) Chemical Bond Type Wavelength Bond Dissociation Energy (kJ/mole) 129 930 147 816 C=O 168 712 C=N 195 615 C=C 196 611 P=O 238 502 O H 259 461 H H 275 435 P O 286 419 C H 289 414 N H 308 389 C O 340 352 C C 344 348 S H 353 339 C N 408 293 C S 460 260 N O 539 222 S S 559 214

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44 Figure 2 1. Longitudinal vie w of the wheat kernel (Cornell and Hoveling 1998)

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45 Figure 2 2. Cross sectional view of wheat kernel (Cornell and Hoveling 1998)

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46 Figure 2 3. Cross sectional view of wheat kernel (Eliasson. and Larsson. 1993)

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47 Figure 2 4 C lassification of wheat gluten (Tatham Arthur S and others 2000)

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48 CHAPTER 3 MATERIALS AND METHOD S Chemical Reagents The standard reagents phosphate buffered saline (PBS) 10 X solution, sodium acetate tri hydrate, sodium chloride and aceti c acid were purchased from Fisher Scientific (Fair Lawn, NJ). Reagents for SDS PAGE Tris/Glycine/SDS running buffer containing 24 mM Tris, 192 mM glycine and 0.1 % (w/v) SDS, pH 8.3 was obtained from Bio Rad Laboratories (Hercules, CA). Leammli sample buf fer, 2 Mercaptoethanol Electrophoresis Purity Reagent, Precision Plus Protein All Blue Standards were purch ased from Bio Rad Laboratories Gel Code Blue Safe Protein Satin was obtained from ThermoScientific (Rockford, IL). Reagents for Western Blot and Do t Blot Tris/Glycine Buffer containing 25 mM Tris, 792 mM glycine, pH 8.3 was purchased from Bio Rad Laboratories Starting Block TM T20 (TBS) Blocking Buffer, 1 Step TM Chloronapthol, Super Signal West Pico Chemiluminescent Substrate (an enhanced chemilu minescent substrate for detection of HRP) was purchased from ThermoScientific Methanol HPLC Grade, Tris Buffered Saline (TBS) containing Tris 25mM, NaCl 0.13 and KCl 0.0027 M pH 7.4 and Tween 20 was obtained from F isher Scientific Reagents for Indirect Enzyme Linked Immunosorbent Assay (ELISA) Stable Peroxide Substrate Buffer and O phenylenediamine dihydrochloride (OPD) was purchased from Thermo Scientific

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49 Wheat Protein Extraction Albumin and Globulin Fraction The albumin/globulin fraction was extracte d from commercial whole wheat flour (Pillsbury whole w heat flour) purchased from the local grocery store. The extraction procedure was followed according to Simonato and others (2001) with few variatio ns A weight of 5g whole wheat flour was mixed with 0.5 M NaCl buffer. The mixture was stirred with magnetic stirrer for 1 hr at 4C followed by centrifugation at 12000g for 20 min at 4C. The resulting supernatant was saved as water/salt soluble, albumin and globulin fraction at 20C for the experiment. Glutenin Fraction Wheat gluten powder was purchased from Sigma Aldrich (St.Louis, MO) The glutenin fraction was extracted in acetate buffer according to Takeda (2001) with few variations. The gluten powder was disso lved in acetate buffer at pH 4 in a ratio 1:4 (w/v) The mixture was stirred with magnetic stirrer on vortex at room temperature for 5 min. Two variations of samples (supernatant and homogenate) were used to analyze SDS PAGE and Western blot. For the homog enate, the sample was homogeniz ed using a hand held BioHomogenizer (Biospec, Bartlesville, OK) for 4 min at high speed For the supernatant, the samples were centrifuged at 8000 x g for 10 min and only the supernatant was collected for further analysis. T otal Soluble Wheat Protein C ommercial whole wheat flour (Pillsbury Whole Wheat Flour) was purchased from local grocery store to extract the total soluble wheat proteins. The extraction procedure was followed according to (James and others 1997) with few variations. Five gram s of whole wheat flour was dissolved in 100 ml phosphate buffered saline (PBS)

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50 pH 7.4 for 24 hours (4C). The mixture was centrifuged at 1 1,000xg for 20 min at 4C. The resulting supernatant was dialyzed against distilled water for 24 to 36 hours The water was replaced with fresh deionized water every 10 hrs The dialysate was collected in plastic tubes and stored at 20C for subsequent us e in the experiments. Pooled Human Plasma Samples Wheat Allergic Plasma Pooled human plasma from three individuals containing specific IgE antibodies to wheat was obtained from Plasma Lab International, Everett, WA. Wheat IgE specific plasma was used to d etect the IgE binding demonstrated by Western blot, dot blot, and Indirect ELISA. Control Plasma Control plasma of individuals with no known history of any allergy to any food was obtained from Plasma Lab International Control plasma was utilized in indir ect ELISA analysis to ensure there is no non specific binding. The absorbance value of control plasma was negated from the samples to prevent error from non specific binding. Equipments Pulsed Ultraviolet L ight (PUV) S ource Xenon Steripulse XL 3000 (Xen on Corp, Wilmington Ma) was utilized to treat the samples (Figure 3 1 A and 3 1 B ) The PUV unit produced polychromatic light in the wavelength range between 100 and 1100 nm. The energy from the PUV lamp included 54% from UV region 26% from visible regio n and 20% from infrared region. The PUV equipment mainly consisted of a 16 inch linear clear fused quartz PUV lamp, treatment chamber, control module and cooling blower.

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51 High Hydrostatic Pressure A laboratory scale high pressure unit ( Figure 3 2 A ) Avure PT 1 (Avure Technologies, Kent, WA) monitored with DASYLab 7.0 software ( Figure 3 2 B ) (DASYTECH, Bedford, NH) was utilized in the study. Non Thermal Plasma The NTP system included two major components, NTP reactor chamber an d high voltage power supply A dditionally it included electrical parameter measurement and control devices ( Figure 3 3) The NTP was generated by dielectric discharge between the two electrodes. The NTP reactor was the planar configuration of the dielectric barrier discharge reacto r with two dielectric layers. The electrodes in the NTP chamber were covered with dielectric plates (epoxy resin board). The thickness of the dielectric layer was 0.062 inches and the radius of the discharge chamber was 1 inch with height of 0.25 inches. Centrifuge and Spectrophotometer Sorvall RC 5B Refrigerated Superspeed Centrifuge, Du Pont instruments (Wilmington, DE) was utilized to centrifuge samples Spectramax 340 384 spectrophotometer (Molecular Devices, Inc. Sunnyvale, CA) was utilized to read the absorbance of the samples. PUV Treatment The settings for the PUV t reatment were 3 p ulses/sec with the pulse width of 360 s. T he distance between the samples and P UV lamp was adjusted manually to 10cm Joules/cm 2 at 1.9 cm from the quartz window. A volume of 5 ml of sample was placed in round aluminium dish with diameter of 6.5 cm The samples were placed directly under

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52 the PUV lamp To make the sample tray stationary a custom designed holder was used to push the samples such that it gets the maxi mum energy from the PUV source. A cooling blower was switched on during the entire treatment to prevent the overheating of the PUV lamp and to reduce the ozone accumulation in the pilot plant. This method was followed for all the samples to obtain the same energy each ti me. The samples were exposed to PUV with different treatment time. The initial and final temperatures of the samples were recorded using hand held thermometer. PUV T reatment on Wheat Albumin and G lobulin A volume of 5 ml of albumin/globulin proteins were treated with PUV for different time. The initial and the final surface temperatures were measured using a hand held infrared thermometer. The samples were weighed before and after treatment to account for moisture loss caused by heating of the sample. The treatment conditions included control (untreated), boiled (5 min at 100C), PUV 30 s, PUV 60 s, PUV 90 s and PUV (90s) followed by boiling for 5 min at 100C. The samples were subjected to SDS PAGE and Western blotting. PUV Treatment on Wheat Gluten A vol ume of 5 ml of wheat gluten (homogenized and supernatant) was treated under the PUV at a distance of 20.5 cm from the UV lamp. The initial and the final surface temperatures were measured using a hand held infrared thermometer. The samples were weighed bef ore and after treatment to account for moisture loss during treatment. The treatment conditions for homogenized samples included control (untreated), boiled (5 min at 100C), PUV 45 s and PUV 45 s followed by boiling at 100C for 5 min. The treatment condi tions for supernatant gluten samples were control

PAGE 53

53 (untreated), boiled at 100C for 5 min and PUV 45 s. The samples were subjected to SDS PAGE and Western blotting. PUV Treatment on Total Soluble Wheat P roteins Total soluble wheat proteins (5 ml) were trea ted with the continuous PUV unit. A customized ruler designed by the fellow student was used to insert the samples under the UV lamp. The sample s were placed exactly under the PUV lamp every time so that high energy PUV radiation can be absorbed by the sam ples and also for the accuracy of the PUV treatment The distance between the P UV lam p and samples was adjusted to 20.4 cm with 3 pulses/sec. The treatment conditions were control (untreated), boiled at 100C for 5 min, PUV 30 s, PUV 60 s, PUV 90 s, PUV 12 0 s and PUV 120 s followed by boiling at 100C for 5 min. The initial and final temperatures of the samples were measured. The initial and final weight of the sample was measured and the moisture loss was calculated. HHP Treatment The samples were shipped to University of Delaware at controlled temperature (4C) for the HHP treatment. The samples (1.5 ml) were transferred to a sterile polypropylene pouch (Fisher Scientific) heat sealed and placed in another pouch to prevent it f rom leaking. HHP treatments were conducted at 600 MPa and the treatment time included 5 and 15 min at 21C and 70C respectively. Pressure increase rate was approximately 22 MPa/s, and pressure release was immediate. After the treatment the samples were sealed in plastic pouches and shipped to University of Florida at 4 C The samples were transferred into plastic tubes and stored at 20 C until it was used for the experiment

PAGE 54

54 NTP Treatment T he NTP treatment on wheat proteins was conducted in University of Minnesota. The NTP unit was designed by Dr. Ruan and his colleagues. The system included 30 kV power supply with frequency of 60 Hz ( Figure 3 3 ) A volume of 12.87 ml was treated with NTP. The samples were subjected to NTP by placing it between two electrodes and the treatment time i ncluded 1, 3 and 5 minutes. The samples were transferred into glass vial and shipped to Universi ty of Florida. Upon receiving the package at 4 C the samples were transferred into vials and stored at 20 C until further experiments. Protein Assay Protein co ncentration of treated and control samples were measured according to Bradford Assay with the Coomassie Plus Protein Assay Reagent Kit (Pierce Boitechnology, Rockford, IL). The protein concentration of unknown samples was estimated by reference to absorban ces (595 nm) obtained for a series of standard protein Bovine Serum Albumin (BSA) dilu tions (0.2 to 0.8 mg/ml) which were assayed alongside the unknown samples. A standard curve was plotted for ea ch BSA standard vs. its concentration in mg/ml. The standar d curve was used to determine the concentration of treated and control wheat protein samples. Sodium Dodecyl Sulfa te Polyacrylamide Gel Electrophoresis (SDS PAGE) The control and treated wheat samples were separated by a 4 20% Tris HCl precast gels (30 an d 50 l) (Bio Rad Laboratories) according to the method of Laemmli (1970) The protein samples were diluted in loading buffer (2% SDS, 25% glycerol 0.01% Bromophenol Blue, and 62.5 mM Tris HCl, pH 6.8 10 % 2 m ercaptoethano l) in ratio 1:1 and boiled for 5 min at 95C and cooled to room temperature. The precast gels were assembled on to a Mini PROTEAN TetraSystem connected to Power Pac

PAGE 55

55 Basic (Bio Rad ). The electrophoresis tank was filled with SDS PAGE runni ng buffer Tris/Glycine/ SDS Buffer containing 25mM Tris, 192 mM glycine and 0.1 % (w/v) SDS pH 8.3. The standard protein marker (10 l ) was loaded in the first well and the subsequent wells were loaded wi th control and treated samples(~ 20 25 g) using gel loading tips The proteins wer e electrophoresed at ~190 Volts for about 45 min or until the dye front reached the bottom of the well. The bromophenol blue dye pr esent in the sample buffer helped to monitor the electrophoresis process. The gel wa s either s tained with Gel Code Blue Safe Protein Stain (5 10 hrs) for analyzing the protein expression or transferred to polyvinylidene fluoride (PVDF) membrane (Millipore Immobilon P Transfer Membrane, Millipore Corporation, Billerica, MA). Western Blot After perf orming gel electrophoresis the resolved proteins were blotted on to a PVDF membrane. The gel was placed in the deionized water and equilibrated in ice cold transfer buffer (pH 8.3), consisting of 25 mm Tris, 192 mm glycine and 20% aqueous methanol. The me mbrane dimension of 7 x 8 inch was utilized for blotting. The membranes were wetted in methanol to hydrate and rinsed with dei onized water. The membrane wa s incubated in ice cold transfer buffer for 5 10 min. Filter paper of approximately the same dimensio n as membrane wa s used to sandwich the gel and the membrane. The filter paper was submerged in transfer buffer and placed in 4C until ready for transferring. The Tras Blot SD Semi Dry Transfer Cell and Power Pac HC (Bio Rad ) wa s utilized for transferring the proteins. The filter paper wa s placed on the transfer ce ll. The well hydrated membrane wa s placed on the filter place followed by placing the gel on the membrane. To have proper contact between the gel and membrane and for better transfer of proteins the air pockets be tween the gel and the

PAGE 56

56 membrane wa s removed by firmly pressing the membrane gel sandwich with a g lass rod. Another filter paper wa s placed on the gel to complete the sandwiching. The transfer cell wa s closed to begin transferring. The T ran sblot wa s run at 15 volts for 30 min at the room temperature. After transferring the proteins to the PVDF membrane the membrane wa s blocked in the blocking buffer for 1 hr, on the shaker, at room temperature. Blocking the membrane prevents non specific bac kground binding of the primary and/or secondary antibodies to the membrane. The membrane wa s rinsed 2 times for 1 0 min with TBS containing 0.5% Tween 20 on a shaker. Primary antibody with a history of wheat allergy is diluted in blocking buffer in a ratio 1:4 (1 ml of antibody in 3 ml of blocking buffer ) The membrane is incubated in the diluted primary antibody overnight on a shaker at 4C. Care is taken to completely submerge the membrane in the antibody solution to enable adequate homogenous covering of the membrane and shaking is required to prevent uneven binding. The membrane is washed 2 times for 5 10 min with TBS Tween 20 to remove residual primary antibody that could cause high background and poor detection. The horse radish peroxidase conjugated secondary antibody contains enzyme (peroxidase) is used to detect the secondary antibody was diluted (1:1000) in blocking buffer. The membrane was incubated in secondary antibody solution, the amount (3 5 ml) used depending on the size of the membrane to attain complete coverage of the membrane. The incubation time is 1 2 hrs at room temperature, with agitation. The secondary antibody was poured off and the membrane was rinsed 3 times for 10 min with TBS Tween 20. To visualize the protein bound to the memb rane, the membrane was incubated in 1 Step TM Chloronapthol ( ThermoScientific, ) or in Electrochemiluminescence (ECL) reagent (SuperSignal West Pico

PAGE 57

57 Chemiluminescent substrat, ThermoScientific). For visualizing the bound protein with chloronapthol, the mem brane was placed on plain counter. Using the transfer pipets (graduated 0.3 ml Small Bulb, Samco Scientific, Mexico) the membrane was covered with chloronapthol. Once the entire membrane is covered with reagent, it is incubated for 20 30 min at room temper ature. After 30 min of incubation the IgE bound samples can be visualized. The membrane was scanned immediately to analyze the results. Chemiluminescent detection uses an enzyme to catalyze a reaction that results in the production of visible light. The pr ocedure was followed according to Millipore Immunodetection Technique. An equal volume of Supersignal West Pico Luminol/ Enhancer Solution (Thermoscientific) and Supersignal West Pico Stable Peroxide Solution (Thermoscientific) was mixed in a test tube. The membrane was incubated in the ECL reagent mixture for 5 min. After pouring off the excess solution the membrane was placed the cassette and the air bubbles were gently smoothed out and taken to the dark room. In the dark room the autoradiography film ( Classic Autoradiography Film BX, Molecular Technologies, St.Louis, MO) was placed on top of the membrane and the cassette was closed. The exposure time was between 30 sec 4min. Membrane was fed into the developer. The X ray film was scanned immediately fo r analyzing the results. Dot Blot Dot blot is a similar technique as Western blot for detecting, analyzing and identifying the IgE bound proteins. However, in this technique the protein samples are not separated electrophoretically but, are spotted throug h circular templates directly onto the nitrocellulose membranes (0.45 m, 8 cm x 8 cm, Thermoscientific). A grid was drawn with a pencil to indicate the region the blot was done. Protein was spotted at the center of the grid at amounts 2.4 and 1.2 g deter mined using Bradford assay. The

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58 samples were applied slowly to minimize the area the solution penetrates (3 4 mm diameter). The membrane was left for drying. The membrane blocked in blocking buffer (3 4 ml) at room temperature, on rocker for 30 min. The bl ocked membrane was thoroughly washed with TBST two times for 10 min. Incubation time in primary antibody (1:80) is overnight (10 14 hrs) at 4C on a rocker to ensure even binding. Excess antibody was poured in water containing bleach (30%) followed by wash ing the membrane in TBST two times for 10 min to remove residual antibody. The next step was to incubate the membrane in secondary antibody 1:1000 or 1:3000 for 1 2 hrs at room temperature, with agitation. The membrane was washed with TBST three times for 10 min with agitation to remove excess antibody. To visualize the IgE bound samples the membrane was incubated in ECL reagent for 5 min. After pouring off the excess solution the membrane was placed in the cassette and the air bubbles was gently smoothed o ut and taken to the dark room. In the dark room the autoradiography film was placed on top of the membrane and the cassette was closed. The exposure time was between 30 sec 4min. Membrane was fed into the developer. The X ray film was scanned immediately for analyzing the results with densitometry software. Indirect ELISA The control, PUV HPP and NTP treated samples were diluted to a final concentration of 20 g/ml in PBS (pH 7.4). A volume of 100 l of each sample was coated onto a 96 well microtiter plate (Costar, EIA/RIA, No Lid, 96 Well Easy Wash TM Certified High Binding Non Sterile, Polystyrene, Corning Incorporated, Corning, NY) with a manual repeating pipette (Original Model 4780 Repeating Pipette, Eppendorf, Gremany) The plate was covered with an adhesive plastic (Whatman TM Microplate Devices Uniseal TM Adhesive Clear Ployster Seal Film, 0.05 mm, GE Healthcare UK

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59 Limited, Buckinghamshire, UK) and incubated for 2 hrs at 37C. The samples coated onto the plate were removed and the plate was washe d twice with TBST by filling approximately 200 l in each well and subsequently removing the TBST by flicking the plate over the sink. The remaining drops were removed by patting the plate on a paper towel few times. A volume of 200 l of blocking buffer w as added to each well that contained the samples to ensure that all remaining available binding surfaces of the plastic wells are covered. The plate was covered with plastic adhesive and incubated at 37C for at least 2 hrs. Following blocking, plate was w ashed twice with TBST. A 100 l of primary antibody diluted in PBS at 1:5 (1ml of primary antibody in 4 ml of PBS, pH 7.4) was added to each well using a repeating pipette. The plate was covered with adhesive plastic and incubated for 2 hrs at 37C. After removing the antibody, the plate was cleaned for any residual antibody by washing with TBST at least 2 3 times similar to the first wash. Care was taken to prevent any contamination between the wells. A volume of 100 l of mouse anti human IgE HRP antibody diluted in PBS (1:3000) was added to each well, with multichannel pipette and incubated at 37C for 1 hr. After removing the secondary antibody the same wash procedure was followed. Just before detection a 100 l of freshly prepared substrate solution con taining 10% of Stable Peroxide Substrate Buffe r and OPD (0.5 mg/ml) was added to each well and incubated for 10 30 min. The reaction was stopped with 2.5 N sulfuric acid (50 100 l) and the absorbance was read on microplate reader at 490 nm. For unstopped reaction the absorbance was read at 450 nm. Statistical Analysis Absorbance data were entered into Excel and sorted by treatment time and condition. The experiments were conducted in triplicates with three repetitions. The data

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60 were then analyzed by SAS 9 .2 software (Cary, NC). Data were sorted by treatment time and condition. significant difference (LSD) at performed for the accuracy of the results and to d etermine significant difference. All the samples (PUV, HHP and NTP) were compared with the control (untreated).

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61 A B F i gure 3 1. PUV Equipments A) Xenon Steripulse XL 3000 batch system B) Xenon Steripulse XL 3000 continuous system (Photo courtesy of Dr. Yang, University of Florida)

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62 A B Figure 3 2 HHP equipments A) Lab scale HHP unit B) DASY Lab 7.0 software u sed for HHP treatment (Photo courtesy of Dr. Chen, University of Delaware)

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63 Figure 3 3. NTP T reatment set up (Photo courtesy of Dr. Ruan, University of Minnesota)

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64 CHAPTER 4 RESULTS AND DISCUSSI ON Effect PUV Irradiation on Albumin and G lobulin Tempera ture Measurement D uring PUV treatment and the Effect on Sample Volume The sample temperature increased gradually with increase in treatment time The temperature data is presented in Table 4 1 The final temperature of the sample s were 52.4 C, 64.8 C, 73 .0 C, and 74.7 C when treated for 30, 60, 90, and 120 s, respectively, when a sample volume of 5 ml was kept at a distance of 20.5 cm from the PUV lamp As the treatment time increased the sample volume decreased approximately to about 20% when treated f or 120 s ( Table 4 1) SDS PAGE of PUV T reated Albumin and Globulin Electrophoreis was performed under reducing conditions in the presence of 2 mercaptoethanol to determine the nature of proteins in their non na tive state. The SDS PAGE ( Figure 4 1 ) results illustrate the protein profile of control, boiled, PUV treated (30, 60 and 90 s) and PUV (90 s) followed by boiling samples. For the control samples (lane 1) the bands ranged between 6 kDa 75 kDa. Albumin and globulin samples seem to be affected by PUV tr eatment; the variations in the protein profile were detected in comparison with untreated sampl e. Such variations might be due to PUV treatment, since the instantaneous temperature rise could be much higher than temperature measured. Krishnamurthy and othe rs (2008 ) have reported that there was sign ificant rise in the temperature when the milk sample was treated with PUV at a distance of 8 cm for 180 s. Visually, P UV treatment for 30 s (lane 3) resulted in lesser amount of protein likely due to pr otein fragmentation The fragment proteins could be sm aller than the radius of the SDS PAGE gel hence, were excluded from during electrophoresis.

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65 These results are in agreement with those obtained for beta lactoglobulin treated by UV irradiation (Cho and Koji 2010) However, beta lactoglobulin was treated with continuous UV that has ene rgy from UV regions and comparatively low energy level than PUV. Therefore, proteins fragmentation was observed after 4 to 8 hr of UV exposure. Whereas, in this study merely 30 s of PUV exposure time caused protein fragmentation. As PUV treatment time was increased, aggregated protein bands were observed on top of the SDS PAGE gel. As it has been described earlier that electrophoresis was performed in the presence of 2 mercaptoetahnol therefore, these aggregated proteins give further indication that it is P UV induced cross linking that does involve S S bonds (Cho and Koji 2010; Cooper and Davidson 1965) The PUV treat ed (90 s) sample followed by boiling did not show any apparent difference in protein profile compared to rest of the PUV treated (> 30 s) samples. Western Blotting of Wheat Albumin and Globulin Immunoblotting o f w heat albumin/globulin ( Figure 4 2 ) with poo led human plasma from individuals allergic to wheat recognized 5 bands in the control (untreated) and boiled samples, which ranged between 15 kDa 60 kDa. The lower molecular weight amylase/trypsin inhibitor in pre vious studies (Mittag and others 2004; Pastorello and others 2007; Simonato and others 2001) Other bands might be LTP present in the water/salt soluble fraction and other minor allergens (Pastorello and others 2007) Lower IgE binding to PUV treated sample for 30 s (lane 3) was observed. I n regards to longer PUV treatment time, a decrease in immunoreactivity was apparent ( Yang and others 2010) There was negligible IgE reactivity in PUV (60 s and 90 s) treated samples in bands corresponding to 12 25 kDa, and there was minimal IgE binging to the 35 kDa band. Additionally, PUV

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66 treatment followed by boiling showed no IgE reactivity to any proteins except some minimal rea ctivity to the 35 kDa band. The susceptibility of wheat albumin/globulin antibody binding epitopes to PUV treatment was apparent with increase in treatment time. Effect PUV Irradiation on Gluten SDS PAGE of PUV T reated Gluten The PUV treated gluten extrac ts after treating with PUV light were analyzed by SDS PAGE und er d enaturing conditions ( Figure 4 3 ). The gluten homogenate samples were treated with PUV for 45 s and PUV (45 s) treatment followed by boili ng 100C for 5 min. For gluten supernatant samples o nly PUV (45 s) was used for analysis. The SDS PAGE pattern of the gluten homogenate sample for control (untreated) bands ranged between 14 kDa 110 kDa. On the contrary, the PUV treatment for 45 s was noticeably different compared to control samples. Rap id disappearance of the bands was noted in this sample. Similarly, the PUV 45s followed by boiling also showed identical SDS PAGE results. These results show that PUV treatment is likely effective in reducing the protein solubility and as shown by SDS PAGE The formation in irreversible aggregation might result in high molecular weight proteins that cannot be resolved in SDS PAGE (Chung and others 2008) Control (untreated) gluten supernatant samples showed disappearance of the major proteins corresponding to HMW and LMW glut en subunits whereas, the boiled and PUV 45 showed all the major proteins ranged between 14 kDa 110kDa. Glutenin i s a polymeric protein invlolved in S S interchange, upon reduction it is separated into LMW and HMW glutenin subunits. However, the present study did not indicate distinct band on SDS PAGE. This might due to an error while pipetting or other experimental e rror. A more accurate

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67 characterization of glutenin samples was observed in boiled samples. Therefore, the PUV treated sample was compared with boiled sample. The PUV treated (45 s) showed decrease in band definition. These results show a likelihood of PUV induced fragmentation which did not result in SDS PAGE gel (Chung and others 2008; Yang and others 2010; Krishnamurthy and others 2008 ) Western Blot of PUV Treated Gluten The gluten sample (homogenate and supernatant) were analyze d for the IgE reactivity using the pooled human plasma of individuals with a history of wheat allergy for the control (untreated), boiled and PUV treated samples ( Figure 4 4 ). The antibody against wheat recognized a 15 kDa band in control (untreated), boil ed and PUV (45 s) amylase/trypsin inhibitor protein residue present i n the gluten fractio n (Pastorello and others 2007) However, the IgE no longer recog nized the band in the PUV (45 s ) trea ted sample followed by boiling. These results suggest that gluten fraction are stable to PUV tre atment but are susceptible when it is combined with heating at 100C. The gluten supernatant sample showed IgE reactivity to control and boiled samples at 14 kDa, which is possibly amylase/trypsin inhibitor yet there was no IgE bind ing after PUV treatme nt for 45 s. From these results we can notice that homogenate samples is more stable to PUV due to the presence of other proteins whereas, the supernatant samples contain only glutenin subunits tha t are highly susceptible to PUV treatment.

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68 PUV T reated Tot al Soluble Wheat P rotein SDS PAGE of PUV Treated Wheat Proteins In order to understand the n ature of the proteins after PUV treatment, the total soluble wheat proteins were analyzed by SDS PAGE ( Figure 4 5 ). The treatment conditions included control (unt reated), boiled (100C for 5 min), and PUV treatment for 30 s, 60 s, 90 s and 120 s. An additional treatment condition was PUV (120 s) followed by boiling. Analysis of SDS PAGE results of PUV treated sample indicated apparent distinction between the contr ol and PUV treated sample. Con trol (untreated) sample showed bands ranging from 10 to 70 kDa. At 30 s after PUV exposure, a slight reduction in protein band was observed compared to the control. These modifications are mainly due to UV induced protein frag mentation (Cho and Koji 2010; Cooper and Davidson1965; Krishnamurthy and others 2008 ) With increase of irradiation period, PUV irradiation caused irreversible protein aggregation demonstrated by SDS PAGE. PUV trea tment followed by boiling (lane 7) indicated further increase in aggregation. A smear was observed in PUV treated (> 30 s) samples suggesting PUV induced high molecular weight aggregates (Gennadios and others 1998; Cooper and Davidson 1965; Krishnamurthy and others 2008 ) In the present study, initial exposure to PUV irradiation resulted in fragmentation whereas, prolonged exposures to PUV (>30 s) result ed in irreversible high molecular weight aggregates that are c ovalently cross linked. Dot B lot of PUV T reated Wheat P roteins Dot blot was conducted by loading the proteins on nitrocellulose membrane to analyze and compare the IgE reactivity of control with PUV treated proteins with

PAGE 69

69 d ifferent treatment times ( Figure 4 6 ). Incubation of the spotted membrane with pooled human plasma of wheat allergic patients showed IgE binding in control (1 and 2) and PUV (30 s) (3) The control ( boiled ) sample showed minimal decrease IgE reactivity compared to P UV (30 s ) and control. On the contrary, there was no IgE binding to any of the PUV treated samples with the exposure time above 30 s. Similarly, there was no IgE reactivity to PUV (120 s) followed by boiling. This experiment was repeated 3 times to ensure the cons istency of the results obtained. There was also strong non specific binding to the membrane, which did not contain the proteins. The IgE bound proteins showed dark spots, which could be clearly distinguished from the non specific binding. The pro teins that did not react to IgE in this case PUV treated (> 30 s) showed blank spot (4, 5, 6 and 7) which was noticeably different from the non specific bound membrane sites. The dot blot r esults illustrates that the PUV treatment for 30 s did not alter the wheat al lergen reactivity. The immune reactivity of PUV treated (30 s) allergens was similar to control (untreated) whereas, the boiled samples showed remarkable decrease in IgE binding. Therefore, it can be concluded that wheat proteins susceptibility increases w ith increased PUV exposure time. These effects of PUV have been attributed to alteration of ter tiary and secondary structure primarily caused by UV region (Cho and Koji 2010; Davidson and Cooper1967; Cooper and David son 1965) The effects of PUV has been credited to irreversible aggregation of proteins (Chung and others 2008; Yang and others 2010) and thus, alteration of conformational epitope binding sites. Indirect ELISA of PUV T rea ted Wheat P roteins To further elucidate the contribution of PUV on conformational IgE binding epitope Indirect ELISA was performed ( Figure 4 7 ). The treatment conditions were boiling

PAGE 70

70 (100C for 5 min), PUV treatment (30 s, 60 s, 90 s and 120 s) and PUV (120 s) followed by boiling. The IgE binding capacity was measured a s absorbance at 450 nm. Higher absorbance value indicates higher IgE binding and vice versa. According to the indirect ELISA results, there was no significant (P difference between the control, boiled and PUV treatment for 30 s. On the contrary, th e IgE binding to PUV 60, 90, and 120 s was significantly different. The additional treatment group of PUV (120 s) followed by boiling also showed no significant difference (P .There was approximately 40 50% reduction in IgE binding in PUV treatment for 60, 90 and 120 s compared t o control (untreated) sample. These result s indicate that PUV is effective in reducing IgE binding capacity when the exposure time is above 30 s. However, when the PUV (120 s) is followed by boiling the IgE binding capacity i ncreases considerably. HHP T reated Total Soluble Wheat P roteins SDS PAGE of HHP T reated Wheat P roteins Total soluble wheat protein was treated with HPP at 600 MPa. The treatment was conducted in laboratory scale HHP unit. The treatment condi tions were control (untreated), HHP (21C for 5 min), (21C for 15 min), (21C for 30 min), (70C for 5 min) and (70C for 15 min). Gel electrophoresis was performed in the presence of 2 mercaptoethanol in order to analyze the nature of the proteins follow ing HHP treatment ( Figure 4 8 ). For control (lane 6) sample the band ranged between 1 0 kDa 150 kDa. The boiled sample showed mild smearing in the upper part of the gel but, was very similar to the protein profile of the control sample. The HHP treated samp les showed a remarkable difference in SDS PAGE. The wheat proteins seem to be sensitive to high pressure treatment. High hydrostatic pressure has been speculated to cause

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71 denaturation and/or aggregation. The denatured proteins are involved in extensive hyd rophobic interaction that can include strong covalent interactions or weak di sulfide bonding resulting in aggregation. The SDS PAGE showed similarity in the pro teins profile of HHP amylase/trypsin inhibitor had a distinctive band in all the HHP treatment groups. From the HHP treatment on wheat proteins that there is extensive inter molecular hydrophobic interaction, which led to high molecular weight protein aggregation. Western B lot of HHP T reated Wheat P roteins In immunoblotting experiments all the proteins samples treated with HHP were shown to react with IgE. In particular the IgE reacted to two ba nds a 10 kDa and 14 kDa ( Figure 4 9 amylase/trypsin inhibitor which are mainly considered as major protein responsible for IgE reactivity in wheat allergic patients. There was minimal difference in IgE binding to HHP 21 C for 5 min and 21C for 15 min compared to other HHP treated samples. These data indicated a slight reduction in the IgE reactivity. There was also mild IgE reactivity to 2 other bands corresponding to 35 kDa and 45 kDa which are likely LTP present in LMW glutenin subunits. From the Western blotting results, we found that HHP does not alter IgE binding epitopes and change allergen reactivity. Dot B lot of HHP T reated W heat P roteins The dot blot results ( Figure 4 10 ) indicate IgE reactivity to all the HHP treated samples. However, there was minimal reduction in the IgE binding in HHP treated at 70C for 15 min compared to control (untreated) sample.

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72 Indirect ELISA of HHP T reated W heat P roteins The indirect ELISA results indicate a significant reduction in IgE binding in HHP treatment for 21C for 5 min and 70C for 5 min ( Figure 4 11 ). However there was no significant difference between the control and other HHP treated samples. The results do not display any trend in the reduction in IgE binding either with time or temperature. Nonetheless, the results indicate the susceptibility of wheat proteins to HHP at lower or room temperatu re for short duration. The lower temperature and shorter treatment time has massive effect on wheat all ergen protein structure compared to longer duration at higher temperature. There was approximately 30 40 % reduction in the IgE binding capacity in HHP treatment at 21C for 5 min and 70C for 5 min. NTP T reated Total Soluble Wheat P roteins SDS PAGE of NTP T reated Wheat Proteins SDS PAGE was conducted on NTP treated samples in reducing condition. Analysis of SDS PAGE results of NTP treated samples in reducing conditions showed that NTP treatment for 1 min was similar to the cont rol (untreated) samples ( Figure 4 12 ). After they were exposed for 3 min and 5 min showed marked reduction in the protein profile on the SDS PAGE. There was a noticeable reduction in 14 kDa band amylase/trypsin inhibitor in NTP treatment for 3 min 5 min. During the NTP treatment the free radicals generated can have massive effect on proteins conformation resulting in fragmentation. The frag mented proteins are comparatively smaller in size than the pores of the gel and can easily escape from the gel during the initial electrophoresis.

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73 Western Blot of NTP T reated Wheat P roteins As expected the membrane incubated in the pooled hu man plasma of individual allergic to wheat showed higher IgE reactivity in control and NTP treatment for 1 min but, reduced IgE reactivity in NTP treatm ent for 3 min and 5 min ( Figure 4 13 ). The proteins profile showed reduction in the protein recovered du ring SDS PAGE and therefore there must be less amount of proteins transferred to membrane hence reduced IgE binding. The western blot exhibited IgE reactivity to several bands ranging form10 kDa to 60 kDa. The low molecular weight (around 4 k Da) is presen t in all the NTP treated samples and do not indicate reduction in IgE reactivity. On the contrary, other low and high molecular weight bands (10 kDa, 35 kDa and 60 kDa) exhibited reduction in IgE binding with increase in treatment time. Dot Blot Results o f NTP T reated Wheat P roteins The dot blot results ( Figure 4 14 ) give better understanding of NTP treated proteins in non reducing conditions. The treatment condition included control (untreated), NTP treatment for 1 min, 3 min and 5 min. The results illustrate the IgE binding to control (untreated) and NTP treatment for 1 min. In contrast, the NTP treatment for 3 min and 5 min indicated negligible IgE reactivity. Indirect ELISA Results of NTP T reated Wheat P roteins To further va lidate the results indirect ELISA was conducte d on NTP treated samples ( Figure 4 15 ). Both NTP treated for 1 min and 3 min did not show significant difference in the IgE reactivity but, the IgE binding was lower in both groups compared to control (untreate d). Data showed that NTP treatment for 1 min and 3 min has approximately 25 % reduction in IgE binding expressed as absorbance value. Furthermore, the NTP treatment for 5 min indicated significant reduction in the IgE

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74 binding approximately 37 % reduction in the absorbance value. These results explain that higher exposure to NTP has immense effect on the IgE binding proteins. The free radicals generated during NTP exposure might mask the conformational site and/or disrupt the conformational binding eptiope. Th erefore, the allergen is no longer present or no longer recognized by the IgE to elicit adverse immune response. Discussion Effect of PUV T reatment on Wheat P roteins In the previous study it has been shown that UV and PUV have notable effects on conforma tional and aggregation properties of proteins. Taking these facts into account, the present study evaluated the IgE binding activity of PUV treated samples. Some of the authors (Davidson and Cooper 1967; Cooper and Davidson 1965) have repo rted that UV irradiation disrupt collagen molecules resulting in low molecular weight fragments. The authors also mentions the formation of insoluble precipitates resulting from e xcessive UV treatment. In the present study initial exposure to PUV irradiation (30 s) resulted in fragmentation. On the other hand, increase in the PUV treatment time resulted in the insoluble high molecular weight aggregates demonstrated by SDS PAGE gel. The present data are in good agreement with previously reported studies (Cho and Koji 2010; Chung and others 2008; Ya ng and others 2010; Gennadios and others 1998) Cho and others (2010) h ave also mentioned that UV irradiation cleaves covalent bonds leading to formation of fragments when treatment time was 4 and 8 hr. However, the author observed the irreversible aggregation when after 32 hr irradiation. There are several reports on the effect of gamma irradiation on wheat proteins (Srinivas and others 1972; Leszczynska and others 2003) According to Srinivas and others (1972) gamma irradiation on wheat protein resulted low molecular weight entities

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75 indicating the susceptibility of wheat protein to irradiation treatment. A nother study (Leszczynska and others 2003) on the immunoreactivity of gamma irradiated wheat gliadin indicated increase in the imm unoreactivity with increase in dose level. To elucidate the effect on IgE binding activity Western blot, dot blot and indirect ELISA was performed. The IgE binding activity of PUV treated sample decreased with in crease in treatment time. The maximum reduction in immunoreactivity was observed in 60 s whereas; further increase in PUV irradiation time did not result in reduced IgE binding activity ( Yang and others 2010) The present study illustrated that IgE binding activity of PUV treated sample was lower compared to control sample. Since UV generates free radicals (Cooper and Davidson1965; Krishnamurthy and others 2008 ) particularly hydroxyl radicals, super oxide radicals, and peroxides, which may interact with water molecule present in the samples and modify the proteins structure resulting in aggregation and/or fragmentation. Nonetheless, which is the main mechanism involved in this study that leads to the reduction in allergen potency is unclear. From the results it is postulated t hat PUV treatment caused conformational changes of proteins resulting in fragmentation, denaturation, aggregation, (Chung and others 2008; Yang and others 2010; Krishnamurthy and others 2008 ) and ultimately lost th eir immunogenicity. However, PUV alter the linear protein structure is still needs to be evaluated. Effect of HHP T reat ment on Wheat Proteins High hydrostatic pressure treatment on total soluble wheat proteins at 600 MPa showed reduction in IgE binding to HHP 21C 5 min and 70C 5 min. There was approximately 30 to 40 % reduction in IgE binding expressed as absorbance value at 450 nm. However, HHP treatment for longer duration or higher temperature resulted in

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76 no significant difference. The ultrahigh pressure (200 to 600 lactoglobulin a major milk allergen resulted in increase in antigenicity with increase in the pressure and holding time (Kleber and others 2007) Furthermore, pressure treatment denatured the milk proteins and the proteins unfolding might expose the linear epitope resulting in enhanced IgE binding in the ultr ahigh pressure treated proteins. On the contrary, pressurization treatment on rice grains in the solution released allergenic proteins to its surrounding fluid (Kato and others 2000) The press urized rice grains were better in quality and resulted in reduced allergen reactivity. Based on the present results it is postulated that HHP effect on food proteins varies from the type, pressure, temperature and duration of treatment. The present study h owever did not indicate major difference in the IgE reactivity on western blot and dot blot. But, the indirect ELISA indicates significant reduction in two HHP treated samples at 21C and 70C for 5 min. Therefore, it is difficult to denote which condition is better suited in reducing the wheat allergen potency. Effect of NTP T reat ment on Wheat P roteins NTP treated wheat proteins illustrate a remarkable decrease in allergen reactivity in dot blot. The results indicate a significant reduction in the IgE binding in NTP tre atment for 5 min. The dot blot and ELISA results show similarity in the results whereas, the wetern blot results show IgE binding in NTP samples with minimal reduction at 3 and 5 min. This apparent discrepancy is due to the presence of reducing agent in SD S PAGE. The reducing agent denatures the proteins that might reveal the linear IgE binding epitope on SDS PAGE results. But, in ELSA and dot blot the IgE binds to the conformational and/or linear IgE binding epitope. However, it is unclear as to what is ma in mechanism involved in the decreasing the IgE binding.

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77 Table 4 1. Temperature and time measured during PUV treatment Time Initial Temperature ( C) Final Temperature ( C) Initial Volume (5 mL) Final Volume (5 mL) 30 21.1 52.4 5 4.8 60 22.0 64.8 5 4.6 5 90 21.1 73.0 5 4.4 120 21.1 74.7 5 4.1

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78 Figure 4 1 SDS PAGE analys is of PUV treated wheat albumin and globulin ; Lane s : (1) C ontrol (2 ) B oiled (3) PUV 30 s (4) PUV 60 s (5) PUV 90 s (6) PUV+Boiling (Photo taken by author)

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79 Figure 4 2. Western blot analys is of PUV treated wheat albumin and globulin ; Lane s : (1) Control (2) B oiled (3) PUV 30 s (4) PUV 60 s (5) PUV 90 s (6) PUV+Boiling (Photo taken by author)

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80 Figure 4 3. SDS PAGE analysis of PUV treated wheat gluten ; Lane s: (1) (4) Gluten ho mogenised (1) Control (2) Boiled (3) PUV (45 s) (4) PUV+B oiling. Lane (5) (7) Gluten supernatant (5) Control (6) B oiled (7) PUV (45 s) (Photo taken by author)

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81 Figure 4 4 Western blot analysis of PUV treated wheat gluten ; Lane s : (1 ) (4) Gluten homo genised (1) Control (2) Boiled (3) PUV (45 s) (4) PUV+Boiling. Lane s (5) (7) Gluten supernatant (5) Control (6) B oiled (7) PUV (45 s) (Photo taken by author)

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82 Figure 4 5 SDS PAGE analysis of PUV treated total soluble wheat proteins ; La ne s : M= Marker (1) Control (2) B oiled (3) PUV 30 s (4) PUV 60 s (5) PUV 90 s (6) PUV 120 s (7) PUV 120 s+B oiling (Photo taken by author)

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83 Figure 4 6 Dot blot results of PUV treated total soluble wheat proteins ; (1) Control (2) Boiled (3) PUV 30 s (4) PUV 60 s (5) PUV 90 s (6) PUV 120 s (7) PUV 120 s + boiling (Photo taken by author)

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84 Fig ure 4 7 Immunoreactivity of PUV treated total soluble wheat proteins determined by indirect ELISA ; (1) Control (untreated) (2) Boiled (3) PUV 30 s (4) PUV 60 s (5) PUV 90 s (6) PUV 120 s (7) PUV (120 s) +Boiling Data represents mean of 4 measurements (n=4) and standard error mean (SEM) are represented (bars) Data with same letters are not statistically different from each other (p < 0.05)

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85 Figure 4 8 SDS PAGE anal ysis of HHP treated total soluble wheat protein ; Lane s : M= M arker (1) HPP 21C, 5 min (2) HPP 21C, 15 min (3) HPP 21C, 30 min (4) HPP 70C, 5 min (5) HPP 70C, 15 min (6) control (Photo taken by author)

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86 Figure 4 9 Western blot analysis of HHP tre ated total soluble wheat protein ; Lane s : M= M arker (1) HPP 21C, 5 min (2) HPP 21C, 15 min (3) HPP 21C, 30 min (4) HPP 70C, 5 min (5) HPP 70C, 15 min (6) control (Photo taken by author)

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87 Figure 4 10 Dot blot results of HHP treated total soluble wheat protein ; (1) Control (2) HPP 21C, 5 min (3) HPP 70C, 5 min (4) HPP 21C, 15 min (5) HPP 70C, 15 min (Photo taken by author)

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88 Figure 4 11 Immunoreactivity of HHP treated total soluble wheat proteins determined by indirect ELISA ; (1) Control (untreated) (2) HHP 21C, 5 min (3) HHP 70C, 5 min (4) HHP 21C, 15 min (5) HHP 70C, 15 mi n. Data represents mean of 12 measurements (n=12) and standard error mean (SEM) are represented (bars) Data with same letters are not statistically different from each other (p < 0.05)

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89 Figure 4 12 SDS PAGE analysis of NTP treated total soluble wheat protein ; Lane s : M= M arker (1) control (2) NTP 1 min (3) NTP 3 min (4) NTP 5 min (Photo taken by author)

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90 Figure 4 13 Western blot analysis of NTP treat ed total soluble wheat protein ; Lane: M= Marker (1) C ontrol (2) NTP 1 min (3) NTP 3 min (4) NTP 5 min (Photo taken by author)

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91 Figure 4 1 4 Dot blot results of total soluble wheat proteins treated with HPP ; (1) Control (2) NTP 1 min (3) NTP 3 min (4 ) NTP 5 min (Photo taken by author)

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92 Figure 4 1 5 Immunoreactivity of NTP treated total soluble wheat proteins determined by indirect ELISA ; (1) Control (2) NTP 1 min (2) NTP 3 min (4) NTP 5 min Data represents mean of 12 measurements (n=12) and st andard error mean (SEM) are represented (bars) Data with same letters are not statistically different from each other (p < 0.05)

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9 3 CHAPTER 5 SUMMARY AND CONCLUSI ON PUV, HHP and NTP technology were applied to wheat proteins. The effect of PUV in dicated a significant reduction in the IgE reactivity at 60 and 90 s. Increasing the exposure time did not result in further reduction decrease. The optimal treatment condition were 60 and 90 s at 20.5 cm from the PU V lamp at 3 pulses/sec. From HHP results it can be conclude that HHP treated wheat proteins demonstrated a reduction in IgE at 21 C and 70 C with treamtment time being 5 mi n for both the temperature. NTP treatment on wheat proteins resulted in remarkable decreace in IgE binding demonstrated by dot blot. The highest percentage reduction in IgE binding was o btained by PUV approximately 46 % followed by HHP and NTP treatment approximately 42% and 37%. Non thermal treatment may produce conformational and chemical changes in major wheat allergen st ructure that have influence on their immunoreactivity. The results obtained show that non thermal technology may be employed for the reduction of major wheat allergen potency. However, future experiments such as invivo studies are essential in order to ex amine the actual nature of non thermally treated products in the human digestive tract. The future work should also include sensory and microbial analysi s.

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94 APPENDIX THE ANOVA PROCEDURE The ANOVA Procedure Class Level Information Class Levels Values PUV_treated_sample 7 Boil Control PUV sixty PUV thirty PUV ninety PUV one twenty PUV+Boil Number of observations 28 The ANOVA Procedure Dependent Variable: Result Result Sum of Source DF Squares Mean Square F Value Pr > F Model 6 0.04105236 0.00684206 17.08 <.0001 Error 21 0.00841250 0.00040060 Corrected Total 27 0.04946486 R Square Coeff Var Root MSE Result Mean 0.829930 12.02611 0.020015 0.166429 Source DF Anova SS Mean Square F Value Pr > F PUV_treated_sample 6 0.04105236 0.00684206 17.08 <.0001 The ANOVA Procedure Duncan's Multiple Range Test fo r Result NOTE: This test controls the Type I compar isonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 21 Error Mean Square 0.000401 Number of Means 2 3 4 5 6 7 Critical Range .02943 .03090 .03183 .03249 .03297 .03 335 Means with the same letter are not significantly different. PUV_treated_ Duncan Grouping Mean N sample A 0.20700 4 Control A 0.20475 4 PUV thirty A 0.20225 4 Boil

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95 A 0.17775 4 PUV+Boil B 0.14025 4 PUV ninety B 0.12225 4 PUV one twenty B 0.11075 4 PUV sixty Means and Descriptive Statistics 1 PUV treated Mean of Std. Dev. Std. Error Variance sample RESULT of RESULT of RESULT of RESULT Boil 0.20225 0.009811 0.004905 .000096250 Control 0.20700 0.015748 0.007874 .000248000 PUV sixty 0.11075 0.016070 0.008035 .000258250 PUV thirty 0.20475 0.024391 0.012195 .000594917 PUV ninety 0.14025 0.029960 0.014980 .000897583 PUV one twenty 0.12225 0.005123 0.002562 .000026250 PUV+Boil 0.17775 0.026133 0.013066 .000682917 The ANOVA Procedure Class Level I nformation Class Levels Values HHP_treated_sa mple 5 Control HP2115m HP21C5m HP70C15m HP70C5m Number of observations 60 The ANOVA Procedure Dependent Variable: Resul t Result Sum of Source DF Squares Mean Square F Value Pr > F Model 4 0.06403710 0.01600928 4.27 0.0044 Error 55 0.20628343 0.00375061 Corrected Total 59 0.27032053 R Square Coeff Var Root MSE Result Mean 0.236893 34.54807 0.061242 0.177267 Source DF Anova SS Mean Square F Value Pr > F HHP_treated_sample 4 0.06403710 0.01600928 4.27 0.0044 The ANOVA Procedure Duncan's Multiple Range Test for Result NOT E: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 55 Error Mean Square 0.003751

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96 Number of Means 2 3 4 5 Critical Range .05011 .05270 .05442 .05566 Means with the same letter are not significantly different. HHP_treated_ Duncan Grouping Mean N sample A 0.22557 12 Control B A 0.19207 12 HP2115m B A 0.18457 12 HP70C15m B C 0.15332 1 2 HP70C5m C 0.13082 12 HP21C5m HHP treated Mean of St d. Dev. Std. Error Variance sample RESULT of RESULT of RESULT of RESULT Control 0.22557 0.053751 0.015517 .002889155 HP2115m 0.19207 0.068034 0.019640 .004628628 HP21C5m 0.13082 0.060701 0.017523 .003684651 HP70C15m 0.18457 0.052116 0.015045 .002716101 HP70C5m 0.15332 0.069531 0.020072 .004834505 1 The ANOVA Procedure Class Level Information Class Levels Values NTP_treated_samples 4 C ontrol NTP 1 m NTP 3 m NTP 5 m Number of observations 48 The ANOVA Procedure Dependent Variable: Result Result Sum of Source DF Squares Mean Square F Value Pr > F Model 3 0.04570773 0.01523591 2.71 0.0565 Error 44 0.24739561 0.00562263 Corrected Total 47 0.29310334 R Square Coeff Var Root MSE Result Mean 0.155944 42.78596 0.074984 0.175254 Source DF Anova SS Mean Square F Value Pr > F

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97 NTP_treated_samples 3 0.04570773 0.01523591 2.71 0.0565 The ANOVA Procedure Duncan's Multiple Range Test for Result NOTE: This test controls the Type I comparisonwise error rate, not the exp erimentwise error rate. Alpha 0.05 Error Degrees of Freedom 44 Error Mean Square 0.005623 Number of Means 2 3 4 Critical Range .06170 .06488 .06697 Means with the same letter are not significantly different. NTP_treat ed_ Duncan Grouping Mean N samples A 0.22557 12 Control B A 0.16740 12 NTP 1 m B A 0.16657 12 NTP 3 m B 0.14148 12 NTP 5 m Means and Descriptive Statistics 1 NTP Variance treated Mean of Std. Dev. Std. Error of samples RESULT of RESULT of RESULT RESULT Control 0.22557 0.05375 0.015517 0.002889 NTP 1 m 0.16740 0.05495 0.015864 0.003020 NTP 3 m 0.16657 0.05338 0.015411 0.002850 NTP 5 m 0.14148 0.11718 0.033827 0.013731 The ANOVA Procedure Class Level Information Class Levels Values PUV_treated_samples 4 Control PUV ninety PUV sixty PUV thirty Number of observations 48 The ANOVA Procedure Dependent Variable: Results Results Sum of Source DF Squares Mean Square F Value Pr > F

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98 Model 3 0.06330673 0.02110224 3.50 0.0230 Error 44 0.26507021 0.00602432 Corrected Total 47 0.32837694 R Square Coeff Var Root MSE Results Mean 0.19 2787 38.24965 0.077617 0.202921 Source DF Anova SS Mean Square F Value Pr > F PUV_treated_samples 3 0.06330673 0.02110224 3.50 0.0230 The ANOVA Procedure Duncan's Multiple Range Test for Results NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 44 Error Mean Square 0.006024 Number of Means 2 3 4 Critical Range .06386 .06716 .06932 Means with the same letter are not significantly different. PUV_treated_ Duncan Grouping Mean N samples A 0.23773 12 Control A 0.23732 12 PUV thirty B A 0.18390 12 PUV sixty B 0.15273 12 PUV ninety Means and Descriptive Statisti cs 1 PUV treated Mean of Std. Dev. Std. Error Variance samples RESULTS of RESULTS of RESULTS of RESULTS Control 0.23773 0.04192 0.012102 0.001758 PUV ninety 0.15273 0.04641 0.013399 0.002154 PUV sixty 0.18390 0.11013 0.031791 0.012128 PUV thirty 0.23732 0.0897 6 0.025912 0.008057

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99 REFERENCES Ahmedna M, Prinyawiwatkul W Rao R M. 1999. Solubilized wheat protein isolate: Functional properties and potential food applications. J Agric Food Chem 47(4):1340 5. Asero R Ballmer Weber BK, Beyer K, Conti A, Dubakiene R, Fernandez Riv as M Hoffmann Som mergruber K, Lidholm J Mustakov T, Elberink JNGO Pumphrey RS H Skov PS, Ree R vee Vlieg Boerstra BJ, Hiller R, Hourihane JO, Kowalski M, Papadopoulos N G, Wal JM, Mills EN C Vieths S 2007. Ige mediated food allergy diagnosis: Current s tatus and new perspectives. Mol Nutr Food Res. 51(1):135 47. Bannon G A. 2004. What makes a food protein an allergen? Curr Allergy Asthma Rep 4(1):43 6. Battais F, Pineau F, Popineau Y, Aparicio C, Kanny G, Guerin L, Moneret Vautrin DA, Denery Papini S. 2 003. Food allergy to wheat: Identification of immunogloglin e and immunoglobulin g binding proteins with sequential extracts and purified proteins from wheat flour. Clin Exp Allergy 33(7):962 70. Battais F, Courcoux P, Popineau Y, Kanny G, Moneret Vautrin D A, Denery Papini S 2005. Food allergy to wheat: Differences in immunoglobulin e binding proteins as a function of age or symptoms. J Cereal Sci 42(1):109 17. Branum AM, Lukacs S L. 2008. U.S. Department of Health and Human Services. Centers for Disease Control and Prevention (CDC) National Centers f or Health Statistics (NCHS) Available from: http://www.cdc.gov/nchs/data/databriefs/db10.pdf Accessed Dec 20, 2010. Byun MW, Lee JW Yook H S Jo C Kim HY 2002. Application of gamma irradiation for inhibition of food allergy. Radiat Phys Chem 63(3 6):369 70. Carver BF 2009. Wheat: Science and trade. Ames: Wiley Blackwell Publishing. 557 p. Cho YS, Song KB, Yamda K 2010. Effect of ultraviol et irradiation on molecular properties and immunoglobulin production regulating activit lactoglobulin. Food Sci Biotechnol 19(3):595 602. Chung SY, Yang W, Krishnamurthy K 2008. Effects of pulsed uv light on peanut allergens in extracts and liquid peanut butter. J Food Sci 73(5):C400 4. Cooper DR, Davidson R J 1965. The effect of ultraviolet irradiation on soluble collagen. Biochem. J 97:139 47.

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106 BI OGRAPHICAL SKETCH Jyotsna K Nooji was born and raised in Karnataka, India. She is the sec ond of two children born to Jaikrishna and Vidya Nooji. She graduated from University of Mysore, India an in Food Science and Human Nutrition in June 2006. After that i n s Science and Human Nutrition Department at University of Florida. Jyotsna is an active member of Institute of Food Technologists (IFT), and has presented her thesis IFT Chicago, 2010. She has also presented an oral presentation at IFT In her free time, J yots na enjoys pain ting sketch ing cook ing and trave l l ing She has a strong int erest in baking, wine industry and food product development, and intends to pursue her career in that discipline.