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Effect of Cleaning Protocols on the Removal of Milk, Egg, and Peanut Allergens from Abraded and Unabraded Stainless Stee...

Permanent Link: http://ufdc.ufl.edu/UFE0041276/00001

Material Information

Title: Effect of Cleaning Protocols on the Removal of Milk, Egg, and Peanut Allergens from Abraded and Unabraded Stainless Steel Surfaces
Physical Description: 1 online resource (128 p.)
Language: english
Creator: Spektor, Yael
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: abraded, allergens, cleaning, egg, milk, peanut, protocols, stainless, steel, unabraded, validation
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: Food allergens represent a major threat for allergic consumers. Although a law is in place to protect individuals that suffer from allergies, the Food Allergen Labeling and Consumer Protection Act (FALCPA), the presence of undeclared allergens is still a huge concern for the food industry since it is known to have caused severe reactions in sensitive people. It has been proven that one of the critical control points for successful food allergen management is thorough cleaning of shared equipment and processing lines, as well as adherence to Good Manufacturing Practices (GMPs). However, present techniques are not 100% effective in preventing allergen cross-contact, and there is a lack of consensus on the validation of cleaning protocols within the food processing industry. The objectives of this study were: 1) To determine the effectiveness of four cleaning protocols for the removal of egg, peanut and milk residues from abraded and unabraded stainless steel surfaces; and 2) To validate the technique that proves to be most efficient. Potentially allergenic food products (peanut butter, pasteurized liquid egg, and milk) were applied in a controlled manner to abraded and unabraded stainless steel coupons (type 304, 2B finish). The food contact surfaces were subjected to four cleaning protocols that encompassed a water rinse, and the application of a Juice Products Association (JPA) Type 4 wash, a chlorinated alkaline detergent (CAD) and food degreaser wash, an acid detergent (AD) and food degreaser wash, and a water only treatment, all applied at 63degreeC (145degree F). Allergen residues were tested with commercial test kits (Veratox Allergen Test Kits, Neogen Corporation, Lansing, MI.) in conjunction with the development of a standard curve. SAS 9.2 software was used to compare treatments and surfaces (? = 0.05). This experiment was conducted two times. For all three allergens, JPA and CAD resulted in the highest % reductions (99.6% on average for all surfaces), while AD resulted in the least allergen % reduction (91.6% on average for all surfaces). The average reduction for water was 96.5% for all allergens and surfaces. According to the statistical analysis, there were no significant differences between CAD, JPA type 4 wash, and water protocols for egg and milk allergens, but these were significantly different from AD wash in all three allergens. For peanut allergens, there were no significant differences between CAD, JPA type 4 wash, and water protocols in the first study, but the second study showed that JPA and CAD were different from water and AD, and the latter two were different from each other. For peanut allergens, water was the least successful cleaning protocol with a combined average reduction of 92.6% in both types of surfaces. CAD and JPA were equally effective. For milk allergens, the acid detergent (AD) reduced a combined average of 88% in both surfaces. CAD was the most successful method with over 99% reduction. For egg allergens, JAP, CAD, and water achieved 100% allergen reductions in all samples. AD wash achieved a combined average reduction of 93.4% for abraded and unabraded surfaces, respectively.
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 Yael Spektor.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Goodrich, Renee M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041276:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041276/00001

Material Information

Title: Effect of Cleaning Protocols on the Removal of Milk, Egg, and Peanut Allergens from Abraded and Unabraded Stainless Steel Surfaces
Physical Description: 1 online resource (128 p.)
Language: english
Creator: Spektor, Yael
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: abraded, allergens, cleaning, egg, milk, peanut, protocols, stainless, steel, unabraded, validation
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: Food allergens represent a major threat for allergic consumers. Although a law is in place to protect individuals that suffer from allergies, the Food Allergen Labeling and Consumer Protection Act (FALCPA), the presence of undeclared allergens is still a huge concern for the food industry since it is known to have caused severe reactions in sensitive people. It has been proven that one of the critical control points for successful food allergen management is thorough cleaning of shared equipment and processing lines, as well as adherence to Good Manufacturing Practices (GMPs). However, present techniques are not 100% effective in preventing allergen cross-contact, and there is a lack of consensus on the validation of cleaning protocols within the food processing industry. The objectives of this study were: 1) To determine the effectiveness of four cleaning protocols for the removal of egg, peanut and milk residues from abraded and unabraded stainless steel surfaces; and 2) To validate the technique that proves to be most efficient. Potentially allergenic food products (peanut butter, pasteurized liquid egg, and milk) were applied in a controlled manner to abraded and unabraded stainless steel coupons (type 304, 2B finish). The food contact surfaces were subjected to four cleaning protocols that encompassed a water rinse, and the application of a Juice Products Association (JPA) Type 4 wash, a chlorinated alkaline detergent (CAD) and food degreaser wash, an acid detergent (AD) and food degreaser wash, and a water only treatment, all applied at 63degreeC (145degree F). Allergen residues were tested with commercial test kits (Veratox Allergen Test Kits, Neogen Corporation, Lansing, MI.) in conjunction with the development of a standard curve. SAS 9.2 software was used to compare treatments and surfaces (? = 0.05). This experiment was conducted two times. For all three allergens, JPA and CAD resulted in the highest % reductions (99.6% on average for all surfaces), while AD resulted in the least allergen % reduction (91.6% on average for all surfaces). The average reduction for water was 96.5% for all allergens and surfaces. According to the statistical analysis, there were no significant differences between CAD, JPA type 4 wash, and water protocols for egg and milk allergens, but these were significantly different from AD wash in all three allergens. For peanut allergens, there were no significant differences between CAD, JPA type 4 wash, and water protocols in the first study, but the second study showed that JPA and CAD were different from water and AD, and the latter two were different from each other. For peanut allergens, water was the least successful cleaning protocol with a combined average reduction of 92.6% in both types of surfaces. CAD and JPA were equally effective. For milk allergens, the acid detergent (AD) reduced a combined average of 88% in both surfaces. CAD was the most successful method with over 99% reduction. For egg allergens, JAP, CAD, and water achieved 100% allergen reductions in all samples. AD wash achieved a combined average reduction of 93.4% for abraded and unabraded surfaces, respectively.
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 Yael Spektor.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Goodrich, Renee M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041276:00001


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1 EFFECT OF CLEANING PROTOCOLS ON THE REMOVAL OF MILK, EGG, AND PEANUT ALLERGENS FROM ABRADED AND UNABRADED STAINLESS STEEL SURFACES By YAEL SPEKTOR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN P ARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Yael Spektor

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3 To my loving family

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4 ACKNOWLEDGMENTS I would like to thank my major advisor, Dr. R ene Goodrich, for her continuous support and guidance and for being a mentor I would also like to thank my other committee members, Dr. Keith R. Schneider and Dr. Bruce A. Welt, for their assistance and time dedicated to my project. Dr. Charles Sims for his help with the data analysis, Dr. Paul Winniczuk for taking extra time and going out of his way to help me with my research, and Dr. Ronald Schmidt for his endless advice. The present study was supported in part by USDANIFSI Grant #2004 5111002171. I would like to thank my parents, Tania and Alejandro, for their unconditional love and support throughout my life. They are an example of great parents, for whom I am proud of being the person I have become. To my grandparents, who have always motivated m e to keep going and to be successful in all my endeavors Finally, I would like to give my dearest thanks to Juan David, for encouraging me every step of the way and for the happiness and joy he has brought into my life.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .......................................................................................................................11 ABSTRACT ...................................................................................................................................12 CHAPTER 1 INTRODUCTION ..................................................................................................................14 2 LITERATURE REVIEW .......................................................................................................16 2.1 Food Allergies Overview ..............................................................................................16 2.2 Egg Allergens ................................................................................................................19 2.3 Milk Allergens ..............................................................................................................20 2.4 Peanut Allergens ...........................................................................................................22 2.5 Detection Methods ........................................................................................................23 2.5.1 Factors Affecting Detection Methods of Allergenic Residues .........................24 2.5.2 Factors Affecting Detection of Allergens on Equipment and Processed Foods .................................................................................................................25 2.6 Cleaning Protocols and Prevention Programs ...............................................................26 2.7 Cleaning Studies ...........................................................................................................31 2.8 Objectives ......................................................................................................................35 3 MAT ERIALS AND METHODS ...........................................................................................37 3.1 Stainless Steel Coupons Preparation .............................................................................37 3.2 Samples Preparation and Inoculation ............................................................................37 3.3 Juice Products Association (JPA) Type 4 Wash ...........................................................38 3.4 Chlorinated Alkali Detergent (CAD) Wash ..................................................................39 3.5 Acid Detergent (AD) Wash ...........................................................................................39 3.6 Water Only Wash ..........................................................................................................39 3.7 Food Allergens Quantification and Analysis ................................................................40 3.7.1 Sample Preparation and Extraction ...................................................................40 3.7.2 Statistical Analysis ............................................................................................41

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6 4 RESULTS AND DISCUSSION .............................................................................................42 4.1 Results and Discussion ..................................................................................................42 4.2 Allergen Removal Study 1 ............................................................................................43 4.2.1 Juice Products Association (JPA) Type 4 Wash ...............................................43 4.2.1.1 Peanut allergens ..................................................................................43 4.2.1.2 Milk allerg ens .....................................................................................44 4.2.1.3 Egg allergens ......................................................................................45 4.2.2 Chlorinated Alkali Detergent (CAD) Wash ......................................................47 4.2.2.1 Peanut allergens ..................................................................................47 4.2.2.2 Milk allergens .....................................................................................48 4.2.2.3 Egg allergens ......................................................................................48 4.2.3 Acid Detergent (AD) Wash ...............................................................................49 4.2.3.1 Peanut allergens ..................................................................................49 4.2.3.2 Milk allergens .....................................................................................50 4.2.3.3 Egg allergens ......................................................................................51 4.2.4 Water Treatment ................................................................................................52 4.2.4.1 Peanut aller gens ..................................................................................52 4.2.4.2 Milk allergens .....................................................................................52 4.2.4.3 Egg allergens ......................................................................................53 4.2.5 Statistical Analysis ............................................................................................53 4.1 Allergen Removal Study 2 ............................................................................................54 4.3.1 Juice Products Association (JPA) Type 4 Wash ...............................................54 4.3.1.1 Peanut allergens ..................................................................................54 4.3.1.2 Milk allergens .....................................................................................54 4.3.1.3 Egg allergens ......................................................................................55 4.3.2 Chlorinated Alkali Detergent (CAD) Wash ......................................................55 4.3.2.1 Peanut allergens ..................................................................................55 4.3.2.2 Milk allergens .....................................................................................55 4.3.2.3 Egg allergens ......................................................................................56 4.3.3 Acid Detergent (AD) Wash ...............................................................................56 4.3.3.1 Peanut allergens ..................................................................................56 4.3.3.2 Milk allergens .....................................................................................57 4.3.3.3 Egg allergens ......................................................................................58 4.3.4 Water Treatment ................................................................................................59 4.3.4.1 Peanut allergens ..................................................................................59 4.3.4.2 Milk allergens .....................................................................................59 4.3.4.3 Egg allergens ......................................................................................60 4.3.5 Statistical Analysis ............................................................................................60 4.4 Overal l Results ..............................................................................................................60 5 CONCLUSION .....................................................................................................................113 APPENDIX: STATISTICAL ANALYSIS ...............................................................................115

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7 LIST OF REFERENCES .............................................................................................................124 BIOGRAPHICAL SKETCH .......................................................................................................128

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8 LIST OF TABLES Table page 41 JPA t ype 4 wash removal results for peanut allergens on unabraded surfaces ..................62 42 JPA type 4 wash removal results for peanut allergens on abraded surfaces ......................63 43 JPA type 4 wash removal results for milk allergens on unabraded surfaces .....................64 44 JPA type 4 wash removal results for milk allergens on abraded surfaces .........................65 45 JPA type 4 wash removal results for egg allergens on unabraded surfaces .......................66 46 JPA type 4 wash removal results for egg allergens on a braded surfaces ...........................67 47 CAD wash removal results for peanut allergens on unabraded surfaces ...........................68 48 CAD wash removal results for p eanut allergens on abraded surfaces ...............................69 49 CAD wash removal results for milk allergens on unabraded surfaces ..............................70 410 CAD wash re moval results for milk allergens on abraded surfaces ..................................71 411 CAD wash removal results for egg allergens on unabraded surfaces ................................72 412 CAD wash removal results for egg allergens on abraded surfaces ....................................73 413 AD wash removal results for peanut allergens on unabraded surfaces .............................74 414 AD wash removal results for peanut allergens on abraded surfaces .................................75 415 AD wash removal results for milk allergens on unabraded surfaces .................................76 416 AD wash removal results for milk allergens on abraded surfaces .....................................77 417 AD wash removal results for egg allergens on unabraded surfaces ..................................78 418 AD wash removal results for egg allergens on abraded surfaces ......................................79 419 Water wash removal results for peanut allergens on unabraded surf aces .........................80 420 Water wash removal results for peanut allergens on abraded surfaces .............................81 421 Water wash removal results for milk allergens on unabraded surfaces .............................82 422 Water wash removal results for milk allergens on abraded surfaces .................................83 423 Water wash remova l results for egg allergens on unabraded surfaces ..............................84

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9 424 Water wash removal results for egg allergens on abraded surfaces ..................................85 425 JPA type 4 wash removal results for peanut allergens on unabraded surfaces ..................86 426 JPA type 4 wash removal results for peanut allergens on abraded surfaces ......................87 427 JPA type 4 wash removal results for milk allergens on unabraded surfaces .....................88 428 JPA type 4 wash removal results for milk allergens on abraded surfaces .........................89 429 JPA type 4 wash removal results for egg allergens on unabraded surfaces .......................90 430 JPA type 4 wash removal results for egg a llergens on abraded surfaces ...........................91 431 CAD wash removal results for peanut allergens on unabraded surfaces ...........................92 432 CAD wash remova l results for peanut allergens on abraded surfaces ...............................93 433 CAD wash removal results for milk allergens on unabraded surfaces ..............................94 434 CAD wash removal results for milk allergens on abraded surfaces ..................................95 435 CAD wash removal results for egg allergens on unabraded surfaces ................................96 436 CAD wash removal results for egg allergens on abraded surfaces ....................................97 437 AD wash removal results for peanut allergens on unabraded surfaces .............................98 438 AD wash removal results for peanut allergens on abraded surfaces .................................99 439 AD wash removal results for milk allergens on unabraded surfaces ...............................100 440 AD wash removal results for milk allergens on abraded surfaces ...................................101 441 AD wash removal results for eggallergens on unabraded surfaces .................................102 442 AD wash removal results for egg allergens on abraded surfaces ....................................103 443 Water wash removal results for peanut allergens on unabraded surfaces .......................104 444 Water wash removal results for peanut allergens on abraded surfaces ...........................105 445 Water wash removal results for milk allergens on unabraded surfaces ...........................106 446 Water wash removal results for milk allergens on abraded surfaces ...............................107 447 Water wash removal results for egg allergens on unabraded surfaces ............................108 448 Water wash removal results for egg allergens on abraded surfaces ................................109

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10 A 1 Statistical analysis of peanut allergen reduction across methods. ...................................115 A 2 Statistical analysis of milk allergen reduction across methods. .......................................116 A 3 Statistical analysis of egg allergen reduction across methods. ........................................118 A 4 Statistical analysis of peanut allergen reduction across methods. ...................................119 A 5 Statistical analysis of milk allergen reduction across methods. .......................................121 A 6 Statistical analysis of egg allergen reduction across metho ds. ........................................122

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11 LIST OF FIGURES Figure page 41 Standard curve for peanut allergen quantification test 1 ...................................................63 42 Standard curve for milk allergen quantification test 1 .......................................................65 43 Standard curve for egg allergen quantification test 1 ........................................................67 44 Standard curve for peanut allergen quantification test 2 ...................................................81 45 Standard curve for milk allergen quantification test 2 .......................................................83 46 Standard curve for egg allergen quantification test 2 ........................................................85 47 Standard curve for peanut allergen quantification for water treatment .............................87 48 Standard curve for milk allergen quantification for water treatment .................................89 49 Standard curve for egg allergen quantification for water treatment ..................................91 410 Peanut allergen reduction across methods in study 1 ......................................................110 411 Peanut allergen reduction across methods in study 2 ......................................................110 412 Milk allergen reduction across methods in study 1 .........................................................111 413 Milk allergen reduction across methods in study 2 .........................................................111 414 Egg allergen reduction across methods in study 1 ...........................................................112 415 Egg allergen reduction across methods in study 2 ...........................................................112

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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 the Degree of Master of Science EFFECT OF CLEANING PROTOCOLS ON THE REMOVAL OF MILK, EGG, AND PEANUT ALLERGENS FROM ABRADED AND UNABRADED STAINLESS STEEL SURFACES By Yael Spektor December 2009 Chair: Ren e M. Goodrich Major: Food Science and Human Nutrition Food allergens represent a major threat for allergic consumers. Although a law is in place to protect individuals tha t suffer from allergies the Food Allergen Labeling and Consumer Protection Act (FALCPA), the presence of undeclared allergens is still a huge concern for the food industry since it is known to have caused severe reacti ons in sensitive people. It has been proven that one of the critical control points for successful food allergen management is thorough cleaning of shared equipment and processing lines, as well as adherence to Good Manufacturing Practices (GMPs). However, present techniques are not 100% effective in preventing allergen cross contact, and there is a lack of consensus on the validation of cleaning protocols with in the food processing industry. The objectives of this study were: 1) T o determine the effectiveness of four cleaning protocols for th e removal of egg, peanut and milk residues from abraded and una braded stainless steel surfaces; and 2) T o validate the technique that proves to be most efficient. Potentially allergenic food products (peanut butter, pasteurized liquid egg, and milk) were applied in a controlled manner to abraded and unabraded stainless steel coupons (type 304, 2B finish). The food contact surfaces were subjected to four cleaning protocols that encompassed

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13 a water rinse, and the application of a Juice Products Association (JPA) Type 4 wash, a chlorinated alkaline detergent (CAD) and food degreaser wash, an acid detergent (AD) and food degreaser wash, and a water only treatment, all applied at 63C (145 F). Allergen residues were tested with commercial test kits (Veratox Alle rgen Test Kits, Neogen Corporation, Lansing, MI.) in conjunction with the development of a standard curve. SAS 9.2 software was used to compare This experiment was conducted two times. For all three allergens, JPA and CA D resulted in the highest % reductions (99.6% on average for all surfaces), while AD resulted in th e least allergen % reduction (91.6 % on average for all surfaces). The average reduction for water was 96.5% for all allergens and surfaces. According to the statistical analysis, there were no significant differences between CAD JPA type 4 wash, and water protocols for egg and milk allergens but these were significantly different from AD wash in all three allergens For peanut allergens, there were no signif icant differences between CAD, JPA type 4 wash, and water protocols in the first study, but the second study showed that JPA and CAD were different from water and AD, and the latter two were different from each other. For peanut allergens, water was the le ast successful cleaning protocol with a combined average reduction of 92.6 % in both types of surfaces CAD and JPA were equally effective. For milk allergens, the acid detergent (AD) reduced a combined average of 88% in both surfaces CAD was the most successful method with over 99% reduction. For egg allergens, JAP, CAD, and water achieved 100% allergen reductions in all samples. AD wash achieved a combined average reduction of 93.4% for abraded and unabraded surfaces, respectively.

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14 CHAPTER 1 INTRODUCTION Food allergies have been a major concern for sensitive individuals for a very long time. It is estimated that about 3% of adult Americans and 7% of infants are affected by food allergies, and these numbers are continuously increasing (NIAID 2008). According to a recent study by the Centers for Disease Control and Prevention (CDC), the prevalence of food allergies amongst children has increased by 18% between 1997 and 2007, which translates to 1 in 26 from that age group (Branum, CDC 2008). Food allergens, proteins in the food that cause adverse immune reactions, represent a serious problem in the food processing industry. Allergens known as the big eight are the cause of approximately 90% of all allergic reactions, and these are soy, eggs, milk, fish, wheat, shellfish, tree nuts, and peanuts. However, there are other numerous products capable of eliciting allergic reactions, as well as food additives and ingredients that can act as sensitizing agents (Jackson 2008). There are no present treatments for foo d allergies, thus allergic consumers rely solely on labels to avoid potential allergens. Avoidance is not always easy, since allergens can be unintentionally introduced to a food product by cross contact during processing. Food processors are responsible f or developing and implementing procedures to address allergen control. Currently, each processor has its own allergen removal techniques, but cross contamination can result in serious health risks, hence there is an immediate need to develop standard protocols that will ensure complete removal. Four cleaning methods will be tested on abraded and unabraded stainless steel: a basic Juice Products Association (JPA) Type 4 wash, a c hlorinated a lkali detergent (CAD) wash, an a cid detergent (AD) wash and a water only treatment Abraded and unabraded stainless steel

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15 coupons (type 304, 2B finish) will be soiled with milk, egg, and peanut butter and the experimental cleaning protocols will be applied. The benefits of an effective allergen removal protocol are countl ess, including prevention of cross contamination, which leads to safer products on the shelves, and ultimately the well being of the end user by avoiding dangerous allergic reactions. The food industry will benefit from these techniques by ensuring allergi c consumers that their products do not accidentally contain allergens. At the same time, individuals suffering from allergies will have the peace of mind that they will not get sick by the presence of undeclared allergens in their food. The main objective of the present study is to determine which protocol is most effective at removing one or more of the allergens. Statistical analyses of each protocol will decide which one(s) is/are more successful at achieving the highest allergen reduction. A secondary objective is to validate the technique that proves to be most efficient. The strategy towards approaching this study was to choose one common cleaning method for the removal of allergens currently used in the food industry. This method was chosen as the ba se or control from where the other three methods originated. The hypothesis is that the spin off methods will actually be more effective than the control. If that was the case, verification would have to follow in order to validate the best method to ensur e its effectiveness for the industry. The allergens chosen for this study are the three most prevalent for reactions in the U .S

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16 CHAPTER 2 LITERATURE REVIEW 2.1 Food Allergies O verview Food allergies are a major health problem around the world, especially in developed countries where consumption of processed foods is highly common. In the U.S. an estimated 10 to 12 million people are affected by food allergies, constituting about 3% of adults and about 7% of infants and children. Every year, about 30,000 people are hospitalized due to foodrelated allergic reactions and about 150 individuals die from them. Individuals with a family history of food allergies are more predisposed to develop allergies themselves (NIAID 2008). Studies suggest that the incidenc e of food allergies has been on the rise in recent years, and one study has found that the prevalence of peanut allergy has doubled in the past five years (van Hengel 2007). There are more than 160 foods known to produce allergic reactions. However, only eight are accounted for more than 90% of the reactions in the U.S. The eight most common foods, known as the big eight, are: milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat, and soybeans (Jackson 2008). Adults are mostly affected by consumption of peanuts, tree nuts, fish, and shellfish, while children have most reactions after consuming milk, eggs, and peanuts (Sampson 2004). These foods contain what is known as food allergens which are proteins that cause immunological reactions in the body ( Poms 2004). Once they enter the bloodstream, they are transported to specific organs such as the nose, skin, or lungs (NIAID 2008). A food allergy is defined as an adverse reaction of the immune system to a protein (antigen) in a food or food ingre dient. More specifically, when in contact with these antigens the body produces allergen specific antibodies called immunoglobin (Ig) E. IgEs are a type of protein that interact with specific allergens in food (NIAID 2008). Individuals can become sensitiv e to food allergens in a process called sensitization. When the immune system is exposed

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17 to a food allergen, a stimulus is triggered to produce IgE specific antibodies. Sensitization occurs when these antibodies bind to the surfaces of mediator cells, mast cells in the tissues or basophils in the blood, and activate them. This process is known to be symptomless and it does not always result in hypersensitivity. However, when sensitization is effective, a subsequent exposure to the same allergen can produce an allergic reaction by releasing inflammatory molecules such as leukotrienes and histamine. In this case, the food allergen cross links two IgE molecules on a mast cell or basophil, triggering allergic symptoms (Taylor 2006). An allergic reaction can oc cur within minutes of ingesting the food or up to one hour after ingestion. Factors such as eating and digestion time can determine the onset and location of symptoms (NIAID 2008). The severity and duration of these symptoms depends on the amount of allerg en consumed, the route of exposure, and the organs involved (FDA/CFSAN 2006). Symptoms range from mild to severe and can occur in the skin (eczema, urticaria), eyes (conjunctivitis), nose (sneezing, rhinitis), oral cavity (swelling and itching), and gastro intestinal tract (vomiting, diarrhea, nausea). A severe case of a food allergic reaction can cause anaphylaxis, a condition involving multiple organs in the body that requires immediate medical attention (FDA/CFSAN 2006). Anaphylaxis is a very serious alle rgic reaction that occurs rapidly and may lead to death. It may start with a tingling sensation, itching, or a metallic taste in the mouth. Other common symptoms include hives, a sensation of warmth, wheezing or other difficulty breathing, coughing, swelli ng of the mouth and throat area, vomiting, diarrhea, cramping, a drop in blood pressure, and loss of consciousness. The onset of these symptoms may begin right away or take up to two hours after exposure to the allergen, but life threatening reactions may worsen after several hours upon ingestion (FAAN 2009).

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18 Once an individual is diagnosed with a food allergy or allergies the only prevention based treatment available to date is to eliminate the food or foods that trigger the reaction. Sometimes allergy p roducing ingredients are used in foods that people would not normally associate with them. For example, peanuts could be used as a protein source or a dressing could contain eggs or milk. There have been cases where food allergy reactions occurred due to undeclared ingredients in labels. For this reason, the Food and Drug Administration (FDA) passed a law in 2004 requiring to state food allergens in the labels called the Food Allergen Labeling and Consumer Protection Act (FALCPA), which became effective Jan uary 1, 2006 (FDA/CFSAN 2004) Under this law, food manufacturers are required to clearly label foods that are or contain one or more of the big eight food allergens (FDA/CFSAN 2006). This law is extremely important because food allergic individuals do not have any preventive approaches to avoid food allergies other than avoiding the food altogether. However, it does not require FDA to establish a threshold level for any food allergen, nor does it require advisory labeling such as may contain even if allergens may be present as a result of crosscontamination (FDA/CFSAN 2006). Currently, it is not clear what the threshold doses of certain allergens are, so it is very difficult to determine if a person will suffer a reaction even if minimal consumption occurs. In some cases, traces amount of a food allergen will elicit a severe reaction; however, the food matrix and the sensitivity of the individual play a big role in determining the outcome (Taylor 2006). To date, the scientific community has not been a ble to reach a consensus on the minimum level of allergens required to cause a reaction, and the ranges observed for the different types of proteins change drastically from person to person (Jackson 2008). Current FDA regulation

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19 requires manufacturers to remove all detectable allergenic residues from food processing surfaces in order to prevent cross contamination (FDA/CFSAN 2006). A clinical study was conducted to determine the thresholds of reactivity to milk, egg, and peanut. Results indicated a threshol d equal or below to 65 mg of solid food or 0.8 mL of milk, resulted in 16% of egg allergies, 18% of peanut allergies, and 8% of milk allergies. A threshold equal or below to 15 mg of solid food or 0.3 mL of milk was seen in 5.6% of egg allergies, 3.9% of p eanut allergies, and 1.7% of milk allergies. The lowest reactive doses were < 2 mg of crude egg white, 5 mg of peanut, and 0.1 mL of milk. These results suggest that the risk doubles for peanut compared to milk and egg. The findings in this study suggest t hat sensitivity limits of tests should be at least 10 ppm for egg, 24 ppm for peanut, and 30 ppm for milk, to guarantee a safety of 99%, 96%, and 98%, respectively, for individuals affected to these foods ( Morisset 2003). 2.2 Egg A lle rgens Eggs are one o f the most common sources of food allergies, affecting an estimated 35% of children and 12% of adults ( Poms 2004). Studies have shown that individuals have reacted to doses between 1 and 200 mg of egg, with a second study showing a reaction with as little as 0.03 mg of spray dried whole egg ( Poms 2004). The main allergens in egg white are ovomucoid, ovalbumin, ovotransferring, and lysozyme making up 80% of the total protein content in the white (Hildebrandt 2008). T livetin. Most reactions are caused by egg white compared to the yolk and the major agent identified is ovomucoid, the dominant allergen in hens eggs ( Poms 2004). According to Matsuda et al. (1993), individuals suffer ing from egg allergies are especially sensitive to ovomucoid in hardboiled eggs which is a heat stable glycoprotein.

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20 Egg allergen stability has been extensively studied. One study suggested that in cooked eggs the yolk and the white coagulated, thus decr easing the eggs allergenicity. In the same study, researchers discovered that by heating the egg white for ten min at 90C and feeding it to patients they decreased adverse reactions by 50% compared to the consumption of raw eggs (Demeulemester and Giovan acci 2006). Another study of similar nature was able to demonstrate that 21 out of 38 people who have had a positive allergic reaction to freeze dried egg white did not have a reaction to heated egg white (Urisu 1997). However, that same study showed that ovomucoid was a heat stable allergen compared to other egg proteins that were previously labeled as heat labile. It has been shown that thermal processing and enzymatic hydrolysis can influence the hens egg allergenicity. Enzymatic hydrolysis can efficie ntly reduce allergenic potential, especially if proteins are partially or fully denatured since this makes the proteins more available for enzymatic digestion. Pasteurization, or any other thermal process, will cause the enzymes to cleave the egg proteins more efficiently (Hildebrandt 2008). Eggs are used as major ingredients in numerous foods and consumers generally can identify them in an ingredient list. However, issues arise when egg derivatives, such as lecithin, provitamin A, or other products are integrated in food systems, since they can cause allergic reactions to very sensitive individuals. When listed by their function, for example binder, coagulant, or emulsifier, people have a hard time recognizing they are present ( Poms 2004). There are variou s detection methods for the presence of egg residues in foods, especially ELISA test kits and techniques based on gel electrophoresis. 2.3 Milk A llergens Milk allergens account for a pproximately 2% of food allergies in children ages 2 and under, but most of them (over 50%) become tolerant past 6 years old. However, in some cases

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21 severe allergic reactions can occur in adults ( Poms 2004). The major allergens present in cows lactalbumin, bovine serum albumin (BSA) and bovine immunoglobulins (Igs) ( Poms 2004). These proteins are known to be heat resistant and stable after industrial processes (Demeulemester and Giovanacci 2006 ). Generally, hydrolysis of milk significantly reduces its allergenicity (Wal 20 01). Studies have found that cows milk contains between 30 and 35g of protein per liter (Wal 1998). When milk is acidified to pH 4.6 two fractions of milk can be obtained: whey and curd. The whey fraction contains a pproximately 20% of the proteins, which are essentially globular. The main ones are lactalbumin which are synthesized in the mammary gland, while bovine serum albumin comes from the blood. Initially, it was believed that lactoglobulin was responsible for the majority of allergic reactions. However, recent studies on large populations suggest that the majority are sensitive to a wide variety of milk proteins besides lactoglobulin. In addition, proteins present in small quantities in the milk, such as bovine serum albumin and lactoferrin, seem to affect 35 50% of allergic patients. Besides, sensitivity to caseins and frequency of cases has been on the rise in recent years (Wal 2001). Conversely, t he curd contains the caseins that S1S2, caseins and these are defined as phos phorylated proteins (Wal 1998). Caseins are known to be resistant to severe heat treatments but are affected by proteinases and exopeptidases. Cows milk allergens are present in a variety of sources, ran ging from breast milk (due to the mothers consumption) and infant formulas, to other dairy products like cheese and butter. Other products classified as nondairy may also contain these allergens, either accidentally or as additives not mentioned in the l abel. Extracted milk proteins are used as emulsifiers or protein sources ( Poms 2004).

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22 Similar to other allergens, there is very little information regarding the minimum quantities required to elicit severe reactions. One such example is the case of a 3 ye ar old boy who experienced a severe anaphylactic reaction after consumption of lemon sorbet containing trace amounts of milk (Laoprasert 1998). Some of the symptoms included angioadema, vomiting, and urticaria, within 20 min of ingesting 4 to 6 oz of the s orbet. The sample analyzed was found to contain minimal amounts of whey proteins (<200 g), and it was traced to a plant that also produced and packaged ice cream. This is a prime example of individuals who are extremely sensitive to trace levels of allerg en containing substances and it highlights the importance of proper and effective allergen removal practices. As with egg residues, there are a number of detection methods for milk residues in the form of readily available test kits (ELISA). These have ver y low detection limits, as low as 1 mg kg -1 for lactoglobulin, or whey proteins, with detection levels as low as 2.5 mg kg -1 in food products. 2.4 Peanut A llergens Peanuts are probably the most analyzed food when i t comes to allergenic reactions. Special attention has been paid to peanut allergens, since they are responsible for more than 50% of allergy related deaths and have a prevalence rate of approximately 0.6% in the U.S general population ( Poms 2004). They ar e also responsible for 1047% of foodinduced anaphylactic reactions. It has been reported that doses as low as 100 g of peanut have caused mild to moderate reactions ( Poms 2004). There are two major storage proteins in peanuts that belong to two globuli n families, arachin and conarachin. The main allergens are defined as Ara h 1 and Ara h 2, while Ara h 3 and Ara h 4, which are glycinin proteins, are classified as somewhat relevant allergens ( Poms 2004). It has been reported that peanut seeds contain a pp roximately 29% protein, with 20% of

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23 the total protein belonging to the major allergen Ara h 1and 10% to Ara h 2 ( Hefle 2006). Ara h 2 has been linked to the most severe cases of allergic reactions in people who are sensitive to it (Hefle 1996). Studies ha ve shown that the major peanut allergens are resistant to food processing methods and stable to digestion ( Burks 1998) O ther studies have come to the conclusion that roasting did not affect the allergenicity of Ara h 1, while one study claims that roasting actually enhanced it (Hefle 1996). Most of the global peanut production is marketed as salted peanuts, peanut butter, in confectionary, or as oil. The majority of the time it is clearly stated in labels that peanuts are present; however, cross contamin ation during processing can result in trace amounts of peanuts in products supposed to be peanut free. Mislabeling is also an issue and can result in severe reactions ( Poms 2004). 2.5 Detection Methods There are several detection methods for allergens in food products, and they either target the protein itself or have a marker that indicates the presence of the offending food. Some of the protein based methods are radioallergosorbent test (RAST), enzyme allergosorbent test (EAST), rocket immune electropho resis (RIE), immunoblotting, and enzyme linked immunosorbent assay (ELISA), among others. Of all these techniques, ELISA is the method of choice in routine food analysis since it is quantitative and very accurate, simple to use, and has a high potential fo r standardization ( Poms 2004). ELISA test kits work based on the detection of allergens or specific marker proteins by colorimetric reaction after they bind with a specific enzyme labeled antibody. By applying a standard curve the concentration of the ant igen/antibody complex can be estimated. ELISA tests come in two formats, competitive and sandwich, with competitive ELISA mostly used for the

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24 detection of small proteins. Sandwich ELISA tests are mostly used for the detection of food allergens ( Poms 2004). Competitive ELISAs are based on an indirect ratio: the more allergenic residue in sample, fewer antibodies are bound and less color is developed. Sandwich ELISAs are based on direct ratios since the amount of color developed is directly proportional to th e amount of allergens present in the sample (Immer 2006). 2.5.1 Factors A ffecting D etection M ethods of A llergenic R esidues Immunoassay effectiveness is highly dependent on the quality of the antigen being measured and the antibody used to do it, as well a s the test itself being used. Generally there are two main factors affecting assay efficacy: the extraction of the allergen and the assay (Immer 2006). There are several factors that should be taken into account to avoid false or faulty results. 1) Conta mination: it is imperative that contamination be avoided. Clean containers and solutions should be used to avoid either bacterial or allergenic contamination. A study showed that there were differences in the standard curves obtained between a fresh prepar ed buffer and a contaminated one (Immer 2006). 2) Extraction: this step should ideally extract the analyte 100% from the food. However, storage or even processing conditions of the food can significantly affect extraction effectiveness. Therefore, it is im portant to take into consideration the type of sample being analyzed to obtain better results. 3) Handling: pipetting, washing of plates, and precision, among others, all contribute to the end results. It is important that pipetting is performed correctly and that pipettes are calibrated. Proteins adsorb onto surfaces so pipette tips should be rinsed repeatedly before being used. Incubation time is also a factor in the number of samples run at once. If the incubation period is long then more samples can be run at the same time, but if it i s short (~ 5 min) fewer samples should be analyzed because of the difference in time between the first and last sample pipetted (Immer 2006).

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25 ELISA tests are mostly reliable, but since detection is achieved through binding of target proteins any changes to these proteins can affect results. For example, thermal processing, hydrolysis, and even oxidizing agents have an effect on the reactivity of the antibodies. Therefore, if a surface, piece of equipment, or a finished product were in contact with high temperatures or sanitizing agents, ELISAs may not be able to detect allergenic residues (Jackson 2008). It has also been shown that exposure to oxidizing chemicals, such as those present in cleaning solutions, may affect the solubility and immunoreactivity of proteins preventing ELISA to detect residues on surfaces exposed to cleaning. This is known as background noise and is a disadvantage of these types of analytical methods. 2.5.2 Factors A ffecting D etection of Allergens on E quipment and P rocessed F oods There are various limitations that affect the detection of allergens, such as extraction of the analyte from the food (as mentioned above), matrix interference, and changes in protein configuration due to processing (Crevel 2006). Extraction of analyte from food is by far the most significant limitation. The efficacy of the extraction depends on the solubility of the protein, since most immunoassays like the ELISA method operate under aqueous conditions. Many products contai n lipids as part of their formulation which can make it harder to extract. One study of edible oils found that total residual protein content recovery was only 50% by extraction in phosphate buffered saline ( Crevel 2006). Another study was only able to rec over 2 3% of peanut protein added to chocolate (Crevel 2006) These findings show the importance of familiarizing with the matrix of the food as well as the protein being analyzed. Matrix interference can be a problem when analyzing for allergens. High sugar content lactoglobulin. Other materials commonly added to foods, such as colors, can also hinder the results obtained (Crevel 2006).

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26 As previously mentioned, protein configuration changes can have a large effect on allergen detection. During processing, protein allergenicity may change or its ability to be detected in a particular food due to immunoreactivity alteration, reactivity between the protein and the food matrix, or a combination of both (Cr evel 2006 ). 2.6 Cleaning P rotocols and Prevention Programs Although consumers rely on food labels to determine if a product is acceptable or not, allergens could still be unintentionally present in that product. A food allergen can be accidentally ingest ed in several ways, including incorrect labeling of the food product, improper handling, cross contamination, and ineffective equipment cleaning and sanitation. Cross contamination is a huge concern in the food industry and can occur at any time. One of the most common ways cross contact can occur is through the transfer of allergens during processing or handling, especially if the same equipment or processing line is used for various foods, both allergenic and nonallergenic. It has been shown that cross c ontamination has resulted in allergic reactions in several occasions (Jackson 2008). Cleaning is an invaluable technique in the process of food allergen cross contact and its effectiveness is crucial in combating adverse reactions. Adequate sanitation in shared equipment is pivotal, and when successful, it can cut costs in machinery and equipment. Rarely do food manufacturers have two sets of equipment for allergenic and non allergenic food materials. A study conducted by the FDA from 2002 to 2004 shows t hat around 80% of the facilities visited that shared equipment had one or more specific control measures to avoid cross contamination. However, the same study determined that 96% of the inspected plants used at least one of the big eight allergenic foods as an ingredient. As a result, FDA inspectors estimated a 25% chance of producing cross contaminated products during manufacture (Jackson 2008).

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27 Food soils are defined as unwanted matter on foodcontact surfaces (Schmidt 2003). Soils are usually classified depending on the method of removal from the surface cleaned. The two main categories are water soluble and non water soluble. Water soluble soils are those that can dissolve in water that contains no cleaning agent, and these are inorganic salts, sugar, starches, and minerals (Marriot 2006). Among the water insoluble soils are those that can dissolve in alkaline detergents solutions with a pH above 7.0, such as fatty acids and proteins, and those that dissolve in acid media (pH below 7.0) like mineral de posits (Marriot 2006). T here are four types of food soils commonly found on equipment and these are carbohydrates, proteins, fats, and minerals. Of the four, proteins (including allergenic proteins) are the most difficult to remove, and it is even harder i f they have been heat denatured and adhered to surfaces (Schmidt 2003) Cleaning and sanitation are very important aspects when it comes to the development of a successful sanitation program (Schmidt 2003). Cleaning is defined as the complete removal of f ood soils using detergents under manufacturers specifications (Schmidt 2003). The appropriate sequence should be 1) Rinse, 2) Clean, 3) Rinse, and 4) Sanitize. The last step is performed to reduce microorganisms to safe levels. Detergents are commonly used to aid in cleaning of food soils by lowering the surface tension of the water and loosening the mater (Marriot 2006). T hey can be classified according to the effect they produce upon contact with the soil. Two main classes of detergents are alkaline and acid cleaners. Alkaline detergents are those with a pH higher than 7.0 and they are subdivided as the alkalinity (pH) increases. In general, fats, oils, and proteins require a pH of 11 or higher for effective removal. Conversely, acid detergents are those with a pH lower than 7.0. They are useful for removal of mineral deposits (Schmidt 2003). In addition to the soil, surface characteristics should be taken into account when choosing a

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28 detergent. Other factors that affect detergent performance are time, temperature, and quality of the water (Marriot 2006). Alkali based detergents are commonly used to remove proteins and oxidizing agents can be added to solutions to further solubilize the soil (Jackson 2008). Removal of protein films requires alkaline deter gents in conjunction with chlorine (usually hypochlorite) (Schmidt 2003). Fat based soils are present as emulsions and generally can be removed with hot water above the melting point or through alkaline detergents (Schmidt 2003). The condition of the soil also affects the ability to be removed. Fresh soils are easier to remove in cold solutions compared to old, dried, or bakedon deposits (Schmidt 2003). In addition, soils that fall into cracks or crevices or on uneven surfaces are harder to remove, thus soil removal also depends on surface qualities like smoothness, porosity, and wettability (Marriot 2006). Allergen removal protocols can be divided into wet and dry cleaning. Wet cleaning is performed in facilities where high water activity foods are proces sed; therefore, they have accommodations to have water available everywhere as well as equipment that is resistant to constant moisture. The existing four types of wet cleaning methods are 1) clean in place, where equipment is not disassembled, 2) clean out of place, where equipment is partially disassembled, 3) foam or gel cleaning, and 4) manual cleaning, where fully dismantled equipment is cleaned by hand. Most of the facilities choose clean in place systems because they can be automated and constantly a pplied. The efficiency of a cleaning procedure depends on the cleaning time, the temperature and composition of the cleaning solution, and the force used to apply such solution (Jackson 2008).

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29 Conversely, dry allergen cleaning is much more difficult to su ccessfully achieve since it is applied in facilities where dry goods are produced and no water contact is desired. Usually, these are plants designed so that water is not readily available, if at all, to avoid contact with the product. Some of the most com mon used methods are vacuuming, sweeping, scraping, wiping, and using compressed air. According to an Institute of Food Technologists ( IFT) report of allergen control practices in the food industry, more than 50% of the companies use these dry cleaning tec hniques for allergen removal (Jackson 2008). As part of a safe allergen control program, many food plants are establishing several programs to tackle this ever increasing issue. There are various ways to control allergens in processing plants. Some factor s that should be taken into consideration are manufacturing, separation of operations, sanitary design, hygienic practices, as well as verification and validation techniques (Newcomer 1999). Separation of operations is extremely important, especially when allergenic and nonallergenic products are produced on the same line. Production of allergenic foods should be done before cleanup and within several hours of processing nonallergenic products. If possible, workers handling products containing allergens should not come in contact with nonallergen containing ones (Newcomer 1999) Good sanitary designs should be in place, including selection of materials and utensils that are easy to clean and examine d for food residues. Proper system design and cleaning should be available t o minimize allergen cross contamination. Visual v erification should be easy to perform by providing clear access to all pieces of equipment. Besides, thorough cleaning between product runs is imperative to avoid contamination (Huggett and Hischenhuber 1998).

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30 Having an effective Hazards Analysis Critical Control Points (HACCP) approach in place is a good way to establish an allergen control prevention plan. Current Good Manufacturing Practices ( c GMPs) should also be part of an aggressiv e approach for dealing with allergens ( FDA/CFSAN 2004 ). The GMP approach includes implementing a preventive plan to avoid the unwanted presence of allergens in products they are not supposed to be a part of ( Huggett and Hischenhuber 1998). The HAACP approa ch requires a team of personnel that needs to have a thorough understanding of the manufacturing process and the ingredients used, and that is close to the dayto day operations (Clark 2005, Huggett and Hischenhuber 1998). The objective of HAACP is to identify the critical control points during production where steps can be taken towards minimizing the risk of having hidden allergens in finished products, and to establish a continuous monitoring plan for each critical control point ( Huggett and Hischenhuber 1998). Hygienic practices are fundamental and all plant workers should be taught about their importance (Newcomer 1999). Workers should be fully aware of the risks associated with cross contamination and the consequences attached to it (Clark 2005). Tagging, color coding, and other identification procedures can help employees quickly and correctly identify allergen sensitive ingredients or finished goods (Newcomer 1999). Labeling is a key step as sometimes is the only source of information about the presence of allergens that alerts consumers. Having the correct label is important but verifying that the final formula matches what is written on the package is also imperative (Huggett and Hischenhuber 1998). In some cases, formulas are modified at the last m inute but labels are not updated with the correct information that can cause distress for consumers.

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31 2.7 Cleaning S tudies Stainless steel has been one of the preferred materials for food contact surfaces due to easy cleanability, mechanical strength and c orrosion resistance, and cost benefit advantages (Milledge and Jowitt, 1980). Several finishes are used in the food industry depending on the application. One of the most used types for food applications is 304, polished to a #4 finish, or milled or rolled (2B) finish (Frank 1997, 2001). A study by Jackson et al. (2005) was performed to determine the efficacy of several cleaning methods for the removal of hot milk soils and cold milk soils from a stainless steel surface. Plates (304A, 2B finish) were prepa red by adding either 1) hot milk soils (0.75 mL whole milk, stored for 1.5hrs in 88C oven) or 2) cold milk soils (0.75 mL whole milk, stored for 48hrs in 6C refrigerator). Plates were washed with water or chlorinated alkali detergent (CAD) at several tem peratures (ambient, 62.8C, 73.8C) for 30 min. Residues we re determined using ELISA milk test kits Results indicate d that water alone removed all cold milk residues at 62.8C and at 73.8C, but was not able to remove hot milk residues from stainless stee l. Chlorinated alkali cleaner removed hot milk soils at all temperatures. As demonstrated by these findings, it is imperative for food manufacturers to correctly determine the type of cleaning conditions that suits them best for each kind of soil. In 2004, Jackson et al. performed a study to determine the removal of peanut allergens from several food contact surfaces. Various food contact materials (stainless steel, Teflon, polyethylene, urethane, and polycarbonate) were contaminated with peanut butter, and washed for 30 min with either water, chlorinated alkali detergent (CAD), or acid detergent (AD), each at room temperature and at 62.8C. Results indicated that CAD and AD solutions removed residues from all foodcontact surfaces at 62.8C, but room temperature CAD was not effective

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32 in some plates. Room temperature water did not remove residues from any of the materials, and hot water (62.8C) was not effective in urethane and Teflon plates. Frank and Chmielewski (2001) determined the effect of several fi nishes on the cleanability of stainless steel. A total of nine type 304 samples of stainless steel were used, and several finishes were tested, including no finish, #4 finish, 2B mechanical polished, and electropolished. Coupon samples were soiled with eit her cultured milk inoculated with Bacillus stearothermophilus or a Pseudomonas spp. biofilm. Cleaning was performed by immersion in a turbulent bath containing 1.28% NaOH at 66C for 3 min followed by a sterile water rinse, neutralization in 0.1% phosphori c acid for 30 s ec, rinsing on phosphate buffer, sanitizing in 100 ppm hypochlorite neutralization in sodium thiosulfate, and drying. Results indicate d that removal of milk residues from soiled coupons depends on the surface defects rather than finish type Also, biofilm residue was harder to remove than milk soil. In a study conducted by Guzel Seydim et al. (2000), the removal of dairy soil from heated stainless steel surfaces using ozonated water as a prerinse was investigated. Stainless steel coupons w ere cleaned, passivated, and soiled by autoclaving (121C at 15 psi for 15 min) with reconstituted nonfat dry milk (20% solids). Plates were subjected to a 15 min treatment of either warm water (40C) or ozonated cold water (10C) to compare the pre rinse cleanability. Results showed that the ozone treatment removed 84% of soil versus 51% by the warm water treatment. Using electron microscopy (at 200x and 2000x magnification), it was determined that the amount of soil present in the plates washed with ozona ted water was significantly less than in those washed with warm water. Frank and Chmielewski (1997) compared the effectiveness of sanitation of several materials used as food contact surfaces. Mechanically polished (type 304, #4 finish) and

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33 electropolished stainless steel, polycarbonate, and mineral resin were tested. Samples were prepared by contamination with Staphylococcus aureus for 4 hr s until a population of 104 to 105 CFU/cm2 was obtained. The cleaning procedure consisted on immersing the samples in either 1) sanitizer solution (200 mg of N alkyl dimethyl benzyl alkonium chloride/L distilled water) for 10 s ec at room temperature, followed by wiping with a Quaternary Ammonium Compound (QAC) saturated cloth for 5 s ec, or 2 sec) chlorine sanitizer (200 m g/L) for 20 s ec, followed by wiping with a chlorine saturated cloth for 5 s ec. According to the results, the stainless steel and polycarbonate surfaces were better sanitized by QAC than the mineral resin surfaces. Chlorine was effective on the mechanically polished and electr opolished stainless steel, as well as the polycarbonate samples. However, it was not as effective on the abraded electropolished stainless steel and mineral resin surfaces. Overall, the QAC and the chlorine were successful in reducing Staphylococcus aureus by 1000fold on all surfaces except on unabraded mineral resin. A study by Jensen (1970 ), determined the cleanability of milksoiled stainless steel plates by using chlorinateddetergent solution. Stainless steel plates were either pretreated with 100 ppm chlorine or no chlorine, soiled with cold raw milk, and washed by 16 detergents at 0.35% concentration, ranging from 0.016 to 0.102% active alkalinity, and 0 to 100 ppm available chlorine. Nonpretreated plates soiled with milk were s uccessfully cleaned by all nonchlorinated detergents, but soil build up occurred when washed in alkaline solutions with 25 ppm available chlorine. Less build up was noted from 5054 ppm chlorine, and none from 75100 ppm chlorine. Pre treated plates had a high build up when washed by alkaline solutions. The buildup was a result of adhesive nonsoluble chloroprotein, which occurs at low concentration of chlorine ions. At chlorine concentrations between 75 and 100 ppm, the chloro protein was solubilized and nonadhesive.

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34 Schlegel et al. (2007) tested a direct sampling method for verifying the cleanliness of equipment shared with peanut products. The peanut protein was prepared by mixing 20 g of ground peanuts with 100 mL of 20 mM bis Tris propane (pH 7.2) for 2 hrs The aqueous phase was separated and centrifuged several times to remove fats and insoluble particles. The peanut protein residues were placed directly onto stainless steel plates in 3 x 3 cm2 areas. The coupons had a base or acid cleaning cycle, and quantitation of residues was achieved with a non specific assay with SDS PAGE verification. Results indicated between a 90 95% of peanut residue recovery. According to Reinemann et al. (2000), cleaning of milk handling equipment should involve chemical, t hermal, and physical action. They found that a water rinse, usually between 3855 C, should be performed right away after the completion of milking. A chlorinated alkaline detergent should be used to remove organic soils, such as fat and protein, within a temperature range of 4377 C These detergents contain alkalies, phosphates, and other agents that help dissolve fat, protein, and carbohydrates (Jones 2001). Katsuyama (1993) had found that cleaning detergents should be applied at a temperature between 54 71 C. Reinemann et al. (2000) also discusses that an acid rinse should be applied to remove mineral deposits, such as those caused by salts like calcium following the alkaline cleaner (Jones 2001) In addition, Jones (2001) has proposed that is necessary to know the water hardness for effective cleaning of milking equipment He suggested that as the hardness increases, the detergent concentration should also increase. Heat aids in the removal of soils because it can soften them and they also allow for better chemical action in detergents. Heat also affects proteins because it denatures them, but some proteins retain their allergenic properties even after heat is applied. Therefore, the addition of

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35 chlorine to alkaline detergents is recommended to aid in the protein removal process since it aids in peptizing or breakage of protein into smaller units for easier removal (Katsuyama 1999) Since temperature is a big factor in the removal of soils it has been extensively studied. Watrous (1975) showed that a 10C increase in temperature between 32 42C doubles the cleaning efficiency. However, he determined that at temperatures above 85C the milk proteins bind more tightly to the surface due to heat induced interactions which ultimately decreases the cleaning efficiency and can leave residues In 1982, Bradley found that 60C (140F) appeared to be an optimum temperature to remove all milk residues with respect to the detergent. As discussed above, food allergies represent a big threat to sensitive individuals, so more reliable detection and cleaning methods are necessary to allow consumers to feel safe about the products they purchase. Validation of cleaning techniques plays a huge factor in trying to combat food allergens. Validation is defined as the process of assuring that a defined cleaning procedure is able to effectively and reproducibly remove the allergenic food from the specific food processing line or equipment (Jackson 2008). Some validation practices include visual inspection of equipment, inspect ion of finished goods, final rinse water, and diagnostic swab samples, or any combination thereof. Even though research has made great progress regarding the control of food allergens, undeclared allergens are still a concern in the food industry. More research is needed in the area of cleaning protocols for several allergen contaminated soils from different food contact surfaces (Jackson 2008). 2.8 Objectives The main objective of this stu dy was to evaluate and compare four different cleaning protocols fo r the removal of peanut, egg, and milk allergens from stainless steel surfaces commonly used in the food industry. A polished and an abraded type of surface were chosen to compare and contrast the removal differences between the two. The abraded stainless steel

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36 surface was chosen to mimic the abrasion that occurs in food plants over the years. The allergens were chosen due to their high impact in the U.S. population, since they are the cause of most adverse reactions. The first protocol chosen was a modified Juice Products Association (JPA) type 4 wash, which is currently used to remove allergens, especially in tankers. The chlorinated alkali detergent (CAD) and acid detergent (AD) methods were modified versions of the JPA type 4 wash. The water only treatment was applied to see the effects of the cleaning chemicals on the removal of the allergens. A secondary objective was to validate the method most efficient for each type of allergen for further use in manufacturing plants.

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37 CHAPTER 3 MATERIALS AND METHO DS 3.1 Stainless Steel Coupons Preparation For this study, two types of stainless steel plates were used. Unabraded coupons were t ype 3042B stainless steel panels ( 0.037 x 3 x 6 ), and abraded coupons were obtained by grinding one of the sides of the 3042B stainless steel panels ( Q Lab Corporation, Westlake, Ohio). The plates were first submerged in a solution of 10 mL Fisherbran d Sparkleen 1 detergent solution (Fisher Scientific, Pittsburgh, PA ) for 24 hrs to remove impurities and grease, foll owed by a r inse with distilled water, and allowed to air dry. Plates were wrapped in aluminum foil (Publix brand) and sterilized by autoclaving for 15 min at 121C and 15 psi. After being autoclaved, the plates were allowed t o cool down at room temperature and stored in closed containers until further use. 3.2 Samples Preparation and Inoculation The food samples were purchased from a local supermarket. The peanut butter used was Publix brand (creamy type), the eggs were Publix brand pasteurized whole liquid eggs, and the milk was shelf stable pasteurized whole milk (Borden brand). The peanut butter samples had to be diluted in water before being applied to the stainless steel coupons. The solution was a 50% peanut butter and 50% de ionized water mixture. One gram of this mixture was applied to a 15 in2 area of the stainless steel plates in a uniform manner and spread out with a plastic hockey stick (Fisher Scientific, Pittsburgh, PA ). After the application t he plates were allowed to dry for 15 min The pasteurized liquid eggs were directly applied to the plates. One gram of product was applied to a 15 in2 area of the stainless steel plates in a uniform manner with a spraying bottle,

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38 and spread out using plastic hockey stick (Fisher Scientific, Pittsburgh, PA ). After the application t he plates were allowed to dry for 15 min The shelf stable milk was directly applied to the plates. One gram of product was applied to a 15 in2 area of the stainless steel plates in a uniform manner with a spraying bottle, a nd spread out using plastic hockey stick (Fisher Scientific, Pittsburgh, PA ). After the application the plates were allowed to dry for 15 min For the three samples, the initial concentration of protein applied was calculated from the nutritional information provided on the label of the products. From this, it was determined that approximately 2259.8 ppm peanut allergen/g peanut butter/15 in2, 1016.53 ppm egg allergen/g liquid egg/15 in2, and 354.17 ppm milk allergen/ g milk/15 in2 were the initial concentrations applie d. For this study, there were two controls to see the effects of the different types of washes and allergens. The negative control consisted on an empty stainless steel plate for each type of wash. The positive control consisted on a coupon soiled with the food allergen but no cleaning protocol was applied. The latter control was the baseline for each allergen. 3.3 Juice Pro ducts Association (JPA) Type 4 Wash The Type 4 wash was designed to be applied to food tankers for the removal of food allergens as p art of the Model Tanker Wash Guidelines for the Fruit Industry ( JPA 2008). For this study, the Type 4 wash was modified to accommodate the needs of this research. The pH of the solution was 11.5. The protocol used for removing the soils from the coupons wa s as follows: 1. Rinse plates with room temperature water. 2. Wash stainless steel plates by immersing them in a hot (63C) potable water and food grade degreaser solution ( Dawn Liquid Detergent, P&G, Cincinnati, OH) (3fl oz/1 gallon water) for 15 min 3. Rinse pla tes thoroughly with room temperature water.

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39 4. Apply hot cleaning solution (63C) utilizing a USDA A1 rated cleaner (ZEP, Atlanta, GA) (3fl oz/1 gallon water) for 15 min 5. Rinse plates thoroughly with room temperature water 3.4 Chlo rinated Alkali Detergent (C AD) W ash The Chlorinated Alkali Detergent wash is a modified version of the JPA Type 4 wash and was adapted to fit the study requirements. The pH of the solution was 12.2. The protocol used for removing the soils from the coupons was as follows: 1. Rinse plat es with room temperature water. 2. Wash plates by immersing them in hot (63C) potable water with chlorinated alkali detergent (ZEP, Atlanta, GA) (3fl oz/1 gallon water) for 15 min 3. Rinse plates with room temperature water. 4. Immerse plates in hot (63C) potabl e water and food grade degreaser solution ( Dawn Liquid Detergent, P&G, Cincinnati, OH) (3fl oz/1 gallon water) for 15 min 5. Rinse plates thoroughly with room temperature water. 3.5 Acid Detergent (AD) W ash The Acid Detergent wash is a modified version of th e JPA Type 4 wash and was adapted to fit the study requirements. The pH of the solution was 1.35. The protocol used for removing the soils from the coupons was as follows: 1. Rinse plates with room temperature water. 2. Wash plates by immersing them in hot (63C) potable water with acid detergent (ZEP, Atlanta, GA) (1:5 ratio) for 15 min 3. Rinse plates with room temperature water. 4. Immerse plates in hot (63C) potable water and food grade degreaser solution ( Dawn Liquid Detergent, P&G, Cincinnati, OH) (3fl oz/1 ga llon water) for 15 min 5. Rinse plates thoroughly with room temperature water. 3.6 Water Only Wash The protocol used for removing soils from the plates was as follows:

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40 1. Rinse plates with room temperature water. 2. Wash plates by immersing them in hot (63C) pota ble water for 15 min 3. Rinse plates with room temperature water. 4. Immerse plates in hot (63C) potable water for 15 min 5. Rinse plates thoroughly with room temperature water. 3.7 Food Allergens Quantification and Analysis After the wash was applied, a visual assessment was performed on the plates to determine if there were visible residues left. The residues from the plates were collected using a Spongesicle (International BioProducts St. Paul, MN ) containing 10 mL of neutralizing buffer and returned to the sample bag. The collection was done using both sides of the sponge on a 5 x 3 in2 surface area. To the bags 90 mL of a 0.1% solution of sodium phosphate dibasic was added (pH ~ 7) The bags were thoroughly massaged and stored under refrigerated condition s for further testing. 3.7.1 Sample Preparation and E xtraction Prior to testing, the sample bags were taken out of the refrigerator and brought to room temperature. Once at ambient temperature, modified test kit procedures were followed for all three allergens using commercially available test kits (Veratox Quantitative Allergen Test Kits, Neogen Corporation, Lansing, MI, Peanut No. 8430, Total Milk No. 8470, and Egg No. 8450). For all allergens, 5 mL were taken from the sample bag and transferred to a st erile 18 oz WhirlPak bag (Fisher Scientific, Pittsburgh, PA ). One level scoop of extraction additive and 125 mL of the 60C extraction solution were added to the sterile bag. The extraction was performed by stomaching each bag at high speed for 2 min ( Sew ard Stomacher 80 Biomaster Lab System, Brinkmann Instruments, Mississauga, ON ). The extracts were allowed to cool down to room temperature before beginning the analysis. After the modified extraction, test procedures were

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41 followed as indicated in the manual for each allergen. A ll wells were read using a micro well reader ( Stat Fax Model 321 Plus, Neogen Corporation, Lansing, MI) at 650 nm per manufacturers specifications. All readings were done in triplicates for each well and then averaged. Standard curv es were constructed for each kind of allergen in the form of part per million (ppm) of allergen present (0, 2.5, 5, 10, and 25 ppm) at an absorbance of 650 nm to calculate the residue left on the plates. 3.7.2 Statistical A nalysis Data was entered into Exc el and sorted by surface type, treatment type, sample number, and peanut, milk, and egg concentrations. The data were then transferred onto SAS 9.2 software (Cary, NC) for analysis and comparison between surfaces, treatments, and allergens Data were sorte d into seven classifications for analysis of variance comparison (AOV): surface, treatment s ample rep egg concentration, m ilk concentration, and peanut conc entration. Surface referred to either an abraded or unabraded coupon, treatment was the type of w ash applied, sample was the negative, positive, or the sample number a nd rep was the reading number on the samples. In addition, a separation of means was conducted on the surface and treatment AOV results to see if there were any differences between them. The two tests performed were Duncan's Multiple Range Test and Least Significant Difference (LSD) Test for more accurate results.

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42 CHAPTER 4 RESULTS AND DISCUSSION 4.1 Resu lts and Discussion To test and compare the four different washes on the removal of allergens, abraded and unabraded stainless steel plates were soiled with approximately 1 g of each food allergen. The contaminated area was 15 in2. After applying the food, the plates were allowed to air dry for 15 min to mimic what would happen in a food plant. After the 15 min were up, the plates were subjected to their corresponding washes. For each wash, kind of allergen, and type of coupon, there was a negative control, a positive control, and three coupons soiled with the same food allergen to obtain triplicate samples. Each sample was independent of each other. The plates were submerged in beakers containing the cleaning solution that corresponded to the wash being tes ted. The beakers were contained in a water bath set to a temperature of 63C at all times. Following the wash, the coupons were swabbed with a Spongesicle and test kit procedures were applied as described in Chapter 3. Standard curves were constructed fo e each allergen and with each new test kit. Each standard curve contains three readings per data point (absorbance) and a linear regression equation was calculated using the mean of the three data points. A statistical analysis was performed to evaluate th e performance of each cleaning protocol based on the surface type. As can be seen in the results below, some of the positive controls showed some reduction. This was due to the fact that the positive control coupons were swabbed like the samples, thus not all of the food residues were removed by the swab. However, all the positive controls show little or no reduction, indicating the presence of allergens that was the goal. In addition, very few of the negative controls showed some allergen residue, which ma y have been caused by accidental crosscontamination. However, the most probable reason behind the apparent allergen

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43 residue on the negative controls may have been due to background noise caused by the chemicals on the test kits. As previously mentioned, E LISA test kits may not be able to detect residues on surfaces that have been exposed to cleaning, since the oxidizing chemicals can affect the solubility and immunoreactivity of the proteins. The tables of results show the initial allergen concentration, c alculated from the amount of protein indicated on the labels, the absorbance for each sample (read at 650 nm), the final allergen concentration before dilution (calculated from the standard curve equation), the final allergen concentration after dilution, the average final concentration after dilution, and the % reduction. 4.2 Allergen Removal Study 1 4.2.1 Juice Products Association (JPA) Type 4 Wash 4.2.1.1 Peanut allergens The source of peanut allergens was commercial peanut butter, which had to be dilu ted in water in a 1:1 ratio for easier application onto the plates. The initial content of peanut allergens before the dilution was calculated to be 2259.8 ppm allergen in 1 gram of peanut butter. The results for the removal of peanut allergens from the u nabraded stainless steel coupons are shown in Table 4 1, and they have taken the dilution into account According to these, the JPA Type 4 protocol achieved over a 99% reduction of peanut allergens in two of the samples and 100% in the remaining one The a verage combined reduction for the triplicates was 99.9%. These positive results were expected because the Type 4 wash is currently in use in the food industry so a high reduction was anticipated A standard curve was constructed for allergen quantification, as can be seen in Figure 1. The equation on the graph was used to determine the peanut concentration (ppm) left on the coupons by substituting the yvalue with the absorbance obtained for each sample. The %

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44 reduction was calculated by subtracting the f inal concentration from the initial concentration and dividing it by the initial concentration. The result was expressed as a percentage. The ideal was 100% reduction. The correlation obtained was 0.999 that is almost ideal. The results from the peanut all ergen reduction from abraded stainless steel plates are presented in Table 4 2. As with the unabraded stainless steel plates the overall reduction was over 99% and the average combined reduction was 99.5%. A standard curve was used to quantitate the remov al of allergens (Figure 4 1). No food residue was visually observed in any if the plates. Even though the results from the abraded plates are extremely favorable, they were surprising due to the type of surface used. Our hypothesis was that there were goi ng to be significant differences among the two types of surfaces when in reality there were none. Therefore, we can conclude that the JPA Type 4 wash was very successful at removing peanut allergens from both abraded and unabraded stainless steel surfaces. 4.2.1.2 Milk allergens One gram of milk was directly applied to the coupons and spread evenly in a controlled manner. The estimated content of allergens present in a gram of milk was calculated to be 354.17 ppm (per 152 in) The milk allergens removal results from the unabraded stainless steel coupons are presented in Table 4 3. These have taken into account the 1:10 dilution after the sodium phosphate was added. From the table it is possible to see that the reduction ranges from 87% to 100% in some of the plates, with a combined average of 96.1% showing a high variability. No food residues were observed after the wash was applied. From visual observation it was possible to see that the milk formed a film on the coupons after 15 min

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45 A standard curve was constructed to determine the final allergen content using the Neogen test kits, as can be seen in Figure 4 2. The correlation for the standards was 0.945. According to the manufacturers specifications, a high correlation is important for better quantifica tion of allergens because it is more reliable. Table 4 4 presents the results for the removal of milk allergens from the abraded stainless steel coupons. Allergen reduction ranged from 97% to 100% with a combined average of 98.2% for all coupons. These r esults were obtained by using the standard curve used for the unabraded results (Figure 4 2). No food residues were present. When comparing the results from the unabraded plates to the abraded plates it is possible to see that the abraded ones were cleane r overall. This was not expected because the abraded surfaces were supposed to capture more food than the smooth surfaces due to the ridges in them. In addition, one would assume that a smooth surface is much easier to clean than a rough one. Overall, th e JPA Type 4 wash was found to be a good option for removing milk allergens from stainless steel surfaces, more so from abraded ones. The average reduction for both types of surfaces was 97.2%, making this kind of wash a suitable option for cleaning milk a llergen residues. 4.2.1.3 Egg allergens One gram of pasteurized liquid egg was directly applied to the coupons and spread evenly in a controlled manner. The estimated content of allergens present in a gram of egg was calculated to be 1016.53 ppm (per 152 i n). The results for the unabraded stainless steel coupons are presented in Table 4 5. According to the results, the JPA Type 4 wash was extremely successful are removing egg allergens since 100% reduction was obtained for all the samples. A standard curve was constructed for the quantification of egg allergens, as shown in Figure 43. The correlation between the absorbance

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46 at 650 nm and the allergen concentration was 0.89 that is still considered a good correlation for quantitation. From the visual assessm ent it was concluded that no residues were present after the wash The positive controls showed some reduction, but as mentioned before, it just means that when the coupons were swabbed not all the food was collected. However, allergens were collected fro m the swab that was the reason for the positive control. Some of the positive controls showed an absorbance higher than 3.000 which is the highest the instrument can read. This means that the % reduction calculated is the maximum amount of residue that could be present, but most likely that number is lower than shown in the table (63.61% reduction). It was observed that a film was formed on the positive control plates after 15 min of exposure. The results for the removal of egg allergens from the abraded pl ates are very similar to those from the unabraded stainless steel plates since there was 100% reduction across all samples (Table 4 6) The standard curve presented in Figure 43 was used to quantitate allergen residues. The visual assessment did not rende r any food residues. The recorded absorbance for the positive controls was 3.000, which means that most likely the actual readings were higher than this value. Ther efore, the maximum egg allergen reduction was 51.5% which only confirms the presence of egg allergens in the food. From the results obtained, it is possible to conclude that the Juice Products Association Type 4 wash was a huge success for the removal of egg allergens. The egg allergens were removed 100% from both abraded and unabraded surfaces, proving that this method could be applied in the food industry on a regular basis.

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47 4.2.2 Chlorinated Alkali Detergent (CAD) Wash 4.2.2.1 Peanut allergens Approximately one gram of a solution of peanut butter and water (1:1) was applied in a controlled manner to 15 in2 of abraded or unabraded stainless steel plates. The food was spread evenly throughout the surface. Results for the removal of peanut allergen residues from unabraded coupons are shown in Table 4 7. These have already taken into account bot h dilutions, the water dilution and the sodium phosphate dilution. All the samples show a similar reduction value, between 98% and 99%, with a com bined average reduction of 99.3 %. These results were quantitated using the standard curve from Figure 41. The positive control values (54.6%) represent the maximum reduction at an absorbance of 3.000. As in previous cases, this absorbance is probably higher than 3.000 but it could not be read by the machine. However, it shows the presence of peanut allergens in the food. After the wash was applied, no food residues could be seen with the naked eye. Table 4 8 shows the removal results of peanut allergens from the abraded coupons. These were obtained using the standard curve from Figure 4 1. From the table we can co nclude that for most samples the reduction was over 99% or absolute (100%). The average reduction was 99.9%, slightly higher than that obtained from the unabraded plates (99.3%). These results are surprising since expectations were to have higher reducti on for the smooth surfaces. The same was seen for the removal of peanut allergens using the JPA Type 4 wash. This indicates that the surface type did not have a big effect on the effective removal as previously thought. Similar findings were reported in a study where it was shown that CAD at 62.8C was able to successfully reduce peanut allergens from stainless steel surfaces (Jackson 2004)

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48 4.2.2.2 Milk allergens Results for the reduction of milk allergens from unabraded and a braded stainless steel are shown in Table s 49 and 410, respectively. As with the JPA Type 4 wash, the standard curve in Figure 42 was used to calculate the concentration of allergens present. The reduction from the unabraded surfaces was between 97% and 100%, with a combined average of 98.6% for all samples. Reduction for positive controls was negative which means that there was no reduction at all. This is possible because the absorbance readings were 3.000. Table 4 10 indicates that the allergen removal from the abraded plates was 100% for all samples except for one resulting in a combined average of 99.8% for all samples. Once again, the results for the abraded surfaces were better that for the unabraded ones, although not by a huge extent. The same was observed with the JPA Type 4 wash, where the milk allergens were better removed from the rough surfaces Overall, the CAD wash reduced 99.2% of milk allergens among all samples from both types of surfaces, making it a valuable alternative to other washes for this type of allergens. These results are similar to those obtained by a study conducted by Jackson et al. (2005), where it was found that milk soils were completely removed from stainless steel surfaces using a CAD at 62.8C. Visual assessments from all the coupons did not reve al any food residues left and, as previously mentioned, it was observed that the milk formed a film on the coupons after 15 min of exposure. 4.2.2.3 Egg allergens The removal results for egg allergens for unabraded and a braded surfaces are presented in Ta bles 4 11 and 4 12. The standard curve shown in Figure 4 3 was used to determine the allergen reduction for all samples. Results show that 100% reduction was achieved for all plates and surfaces. The presence of allergens was confirmed by obtaining some re duction from the positive control plates. It is

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49 important to mention that there were some food residues left on two out of the three abraded plates, but none was observed on the unabraded coupons. It is possible that even though food was left on the surfaces, the allergenic components of the egg were deactivated (denatured) by the wash, thus leading to 100% reduction. These results compare to those obtained with the JPA Type 4 wash where 100% reduction was observed for all the samples as well. In conclusion the CAD wash was a success for removing egg allergens and with further studies it could be validated for regular use in the food industry. 4.2.3 Acid Detergent (AD) Wash 4.2.3.1 Peanut allergens The results for the removal of peanut allergens from unabr aded stainless steel are presented in Table 4 13. As in previous cases, the removal was calculated by using the peanut allergens controls standard curve presented in Figure 4 1. The reduction of the allergens varied from 85.7% to 96.9% for all plates, with a combined average of 90.1% reduction. The positive controls had some reduction, approximately 65.5%, confirming the presence of allergens. Results from t he abraded plates are displayed in Table 4 14. The removal of peanut allergens was lower than that fo r the abraded coupons, as expected. Reduction varied from 81.3% to 89.1%, with a combined average of 85.6%. The absorbance for the positive control plates was 3.000 for the triplicates, with a maximum reduction of 46.3%, once again confirming the presence of the allergens. In both types of surfaces, there was reduction variability between plates. From visual observation it was possible to see large quantities of food residue left on the plates, thus the large variability in reduction can be explained from t he amount of food that was picked up by the Spongesicle. In some plates, more food may have been picked up compared to other, which

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50 can account from the differences in reduction. Additionally some protein may have been denatured by the heat, thus the food residues collected may have had deactivated proteins in them which were not recognized by the allergen test kit. The acid detergent wash was less successful at removing the peanut allergens compared to the other methods. The objective is to obtain 100% reduction in most cases, and this protocol felt short at achieving optimum results. For both abraded and unabraded plates, the average reduction was 87.8%, which is not sufficient to ensure allergenfree surfaces. Therefore, it is possible to conclude tha t this cleaning method would not be suitable for peanut allergen removal, since a small part of the population could still be at risk for allergenic reactions resulting from cross contamination. These results contradict findings in a study where it was observed complete removal of peanut allergens using an AD at 62.8C for the removal of milk allergens from stainless steel plates (Jackson 2004). 4.2.3.2 Milk allergens The results for milk allergen removal for unabraded and abraded surfaces are presented in Table s 415 and 4 16, respectively. As in previous cases, the standard curve from Figure 42 was used for allergen quantification. From Table 415, we can conclude that reduction ranged from 82.8% to 100%, which is a huge variability. The average reduction for all plates was 94.6%. As in the case of the peanut allergens, the variability can be attributed to the amount of food picked up for allergen quantitation. It is possible that for some plates more food was swabbed with the Spongesicle compared to othe rs or that proteins were denatured The reduction results for milk alle rgens from the abraded coupons showed variability ranging from 81% to almost 100% reduction, with a combined reduction average of 91.3% (Table 4 16) It was observed that some residue was left after the acid detergent was applied, but

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51 some of it was removed after the detergent was applied, suggesting that the first part of the protocol was not as effective as the second portion. As expected, less reduction was seen in the abraded surfaces compared to the not abraded ones. The overall reduction for all surfaces was 92.9% which is still not as effective as the other methods. From the visual assessment it is evident that the acid wash alone, without the detergent, does not remove food resi dues or reduce allergenicity as much. 4.2.3.3 Egg allergens The results for the removal of egg allergens are presented in Tables 4 17 and 4 18. The standard curve in Figure 43 was used for the quantitation of egg allergens. In the case of the unabraded s urfaces, the reduction varied from 97% to 100%, with an average of 98.7% reduction. These results were surprising because large quantities of residues were left on the plates after the wash was applied. However, the allergenicity of the eggs may have been reduced by the acid detergent, the soap detergent, or a combination of both. Due to some reduction of allergens in the positive controls it was possible to confirm that allergens were in the food. Table 4 18 contains the results for the removal of egg alle rgens from the abraded plates. Reduction ranged from 85% to 88.7%, with an average of 86.6% removal. There were also large quantities of egg residue left and reduction was expected to be lower. However, as mentioned above, the allergenici ty may have been r educed by a combination of the detergents and high heat. From these results, we could conclude that the acid detergent may be an effective method for the removal of egg allergens from smooth surfaces, but not rough ones. The combined average reduction for both types of surfaces was 92.3% which would not qualify as being very effective. As expected, the reduction was higher for the polished plates but more testing is

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52 needed to render it an efficient method, especially due to higher efficacy of the other methods discussed above. 4.2.4 Water Treatment 4.2.4.1 Peanut allergens According to the results, the peanut allergen reduction varied from 88.6% to 96.5% for unabraded plates, with an average of 93.6% (Table 419). The results for the abraded coupons are show n in Table 420. The percent reduction ranged from 93.6 to 98.4, with a combined average of 96.0% removal. The results were quantitated using the standard curve in Figure 4 4. After the first 15 min the plate s had most of the residue left on them, but it partially came out after the first water rinse. They also seemed to have grease in them, which can be attributed to the fat of the peanut butter that could not be removed since no degreaser detergent was used as in previous cases. Unexpectedly, the result s for the abraded plates were better than those for the unabraded ones, as was the case with the JPA and CAD washes. A study by Jackson et al. (2004) had found that water at 62.8C was able to completely remove peanut butter allergens from stainless steel plates that had the treatment applied for 30 min 4.2.4.2 Milk a llergens The results for the removal of milk allergens from unabraded and abraded surfaces are presented in Tables 4 21 and 4 22, respectively. These were quantified using the standard curve presented in Figure 45. For the unabraded plates, the reduction ranged from 91.1% to 98.0%, with an average of 94.8%. For the abraded coupons, removal ranged from 95.5% to 99.7% reduction, with a combined average of 97.6% for all samples. As in previous ca ses, the removal from abraded plates was a little more effective than the unabraded ones. There were no food residues visible by the naked eye.

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53 These results were similar to those presented in a study by Jackson et al. (2005) where it was demonstrated tha t water alone was able to remove cold milk residues from stainless steel plates at 62.8C and 73.8C. In our study, most of the residues were removed by the water but the remaining present may have been a result of some proteins in the milk not being able to fully solubilize into solution. 4.2.4.3 Egg allergens According to the results, there was 100% egg allergen reduction for all surfaces and samples as can be seen in Tables 4 23 and 424. The standard curve in Figure 46 was used to quantify allergen reduction. These results were surprising because it means that water alone can be applied to clean egg residues, proving it is as efficient as either the JPA Type 4 or CAD washes. They were even more unexpected due to the fact that much residue was left on th e plates after the first half of the wash, but some of it came off with the first water rinse (as was the case of the peanut residues). 4.2.5 Statistical A nalysis From the statistical analysis performed with SAS 9.2 software, there were no differences betw een the JPA Type 4, the CAD, and the water wash in the reduction of milk and egg allergens (Tables A 2 and A 3) The acid wash was different from these three washes for both egg and milk allergens In the case of the peanut allergens, there were no differences between the JPA Type 4 wash and the CAD wash. However, these two were different from the AD and the water. The water treatment was also different from the acid wash (Table A 1) For egg allergens, there were s ignificant differences between the removal from abraded and unabraded stainless steel surfaces (Table A 3) Conversely, there were no differences observed for the type of surfaces for either peanut or milk allergens (Tables A 1 and A 2).

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54 4.1 Allergen Removal Study 2 4.3.1 Juice Products A ssociation (JPA) Type 4 Wash 4.3.1.1 Peanut a llergens The results for the removal of peanut allergens from unabraded and abraded coupons are presented in Tables 425 and 426, respectively. The results were quantitated using the standard curve presented in Figure 4 7. According to the results, the JPA Type 4 wash achieved an average allergen reduction of 99.9% from the unabraded surfaces. These results exactly compare to those obtained in the first study, since then the average reduction was also 99.9%. The average removal from the abraded surfaces was 100% for all samples. These results are slightly better than those obtained for the polished surfaces, which is somewhat surprising. However, they also compare to those seen in s tudy 1, where the reduction was 99.5%, on average. The visual assessment did not render any food residues. According to the observed results, it is safe to say that the JPA Type 4 wash was very effective for the removal of peanut allergens from both types of surfaces. The second study validated the previous results obtained for this wash, since in both studies the results were practically the same. 4.3.1.2 Milk allergens Tables 427 and 4 28 show the reduction results for the removal of milk allergens from the smooth and rough surfaces, respectively. These were quantified using the standard curve from Figure 48. In both cases, the reduction was 100% for all samples and surfaces. These results were better than those obtained in the first study, where the averages were 96.1% and 98.2% for unabraded and abraded coupons respectively and more variability was observed. As in the case of the peanut allergens, it is possible to conclude that this wash is very effective at removing milk allergens from both types of surfaces. The average reduct ion between

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55 studies 1 and 2 was 98.1% for unabraded plates and 99.1% for abraded surfaces. Therefore, the JPA Type 4 wash is an excellent cleaning method to get rid of milk allergens. 4.3.1.3 Egg allergens The results for the removal of egg allergens are s hown in T able 4 29 and Table 430. Allergen quantification was calculated using the standard curve in Figure 4 9. For the unabraded and a braded surfaces the protocol achieved 100 % reduction across all samples. These results measure up to those obtained in study 1, where 100% reduction was also obtained for all samples and surfaces. From these results we can conclude that the JPA Type 4 wash can be safely applied for the removal of egg from either abraded or unabraded stainless steel surfaces. 4.3.2 Chlorin ated Alkali Detergent (CAD) Wash 4.3.2.1 Peanut allergens The results from the removal of peanut allergens for unabraded and abraded plates are presented in Tables 4 31 and 432, respectively These results were quantified using the standard curve from Figure 47. Results show that there was 100% reduction for all samples and both surfaces. In study 1, the average removal was 99.3% and 99.9% for unabraded and abraded plates. Overall, the average between both studies was 99.7% for unabraded surfaces and almost 100% for abraded ones indicating that the CAD protocol was very effective for the removal of peanut allergens. As previously mentioned, these results match those found by Jackson et al. (2004). Hence, even more studies are necessary to completely valid ate these results. 4.3.2.2 Milk allergens Tables 4 33 and 434 show the results obtained for the removal of milk allergens from the unabraded and abraded plates, respectively. These were calculated using the standard curve from Figure 48. The results show that there was 100% reduction across all samples and surfaces. In the first study, the average reductions were 98.6% and 99.8% for unabraded and a braded plates.

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56 Thus, the average reduction between the two studies was 99.3% and 99.9%, respectively. These f indings compare to those observed by Jackson et al. (2005), were CAD was able to completely remove milk allergens from stainless steel surfaces. 4.3.2.3 Egg allergens The results for the removal of egg allergens are presented in Tables 4 35 and 436, for unabraded and abraded respectively. They were quantified by using the standard curve from Figure 49. According to the results, there was complete egg allergen removal (100%) from both types of surfaces. These exactly match the results from the first study where 100% was also achieved across the board. Since in both trials the findings were the same, it is possible to conclude that the CAD method can be safely applied to clean egg allergen residues from stainless steel surfaces. 4.3.3 Acid Detergent (AD) Was h 4.3.3.1 Peanut allergens Results for the removal of peanut allergens from unabraded and abraded surfaces are shown in Tables 4 37 and 4 38. These were quantified using the standard curve from Figure 47. The average reductions were 99.0% and 99.6% for those surfaces, respectively. These results were surprising due to the high amount of food residue that was visible on the plates, thus less reduction was expected. We could speculate that the food residues left on the plates contained denatured proteins, and therefore were not recognized by the allergen test kits. In addition, less reduction was also expected in the abraded plates due to the type of surface, but this was not the case. In study 1, the average reductions were 90.1% and 85.6% for unabraded and abraded plates These were the kind of results expected in the second study. As previously mentioned, a study by Jackson et al. (2004) had reported complete peanut protein reduction from the plates.

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57 This was observed in the second study of this research bu t not in the first one. Between the two trials, the average reductions were 94.5% and 92.6% (unabraded and abraded). 4.3.3.2 Milk allergens The results for the milk allergens removal utilizing the AD wash are presented in Tables 439 and 440. Allergen re duction was quantified using the standard curve from Figure 48. I t is possible to see a large variation in reduction of milk allergens from the unabraded plates, ranging from 41.7% to 98.1%. The combined average was 66.7%. These results are very different from the ones obtained in the first study, where the average reduction was 94.6% and the removal ranged from 82.8% to 100%. Visually, the coupons had extensive food residue left on them. One possible explanation for the second study results is to take into account the amount of food residue that was swabbed from the plates. One theory is that more residues were collected from some plates compared to others. However, in study 1 there was a significant amount of food left on the coupons and there was also t he issue of the amount swabbed with the Spongesicle. Therefore, since in both studies the amount of residue was the same and the food picked up by the swab was variable, the results from the second study could mean that the protein present in the food was not completely, or partially, denatured as was the case in study 1. Caseins are known to be heat resistant due to the random folding of their structure (Gregory 2009). In addition, whey proteins are stable to acid but are sensitive to heat. In general, mi lk solubility increases with increasing pH, and it is at its lowest around the isoelectric point of milk at pH = 4 .6 (Zayas 1997) Besides, it has been shown that at high incubation temperatures, between 60 and 70C, and at low pH the solubility of milk pr oteins is not very high (Zayas 1997). Therefore, since the pH of the AD was very low (pH = 1.35) and the temperature of the wash was 63C the proteins that stayed on the coupon that were collected were not denatured.

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58 Conversely, the removal of milk allerg ens from the abraded plates was much higher than the unabraded, with an average of 95.1% reduction. As in previous AD washes, there was a significant amount of food residues left on the plates. The large difference between the two surfaces can be attribute d to the amount of food that was rinsed off with ambient water in between solutions of the AD wash. Additionally, the quantity collected with the swab could have impacted the amount of protein residue present. 4.3.3.3 Egg allergens The results for the remo val of egg allergens from unabraded and abraded surfaces are shown in Tables 441 and 4 42. Allergen reduction was calculated using the standard curve depicted in Figure 49. Results show a combined reduction of 92.5% for both types of surfaces. A gain, thi s is surprising since in theory it would be harder to remove allergens from a rough surface rather than a smooth one. In addition, the variability range for reduction was very similar for the two kinds of coupons, ranging from approximately 91% to 94%. In study 1, the average reduction for unabraded plates was 98.7% while that for abraded was 86.7%, which was expected for the second study. Once more, the variability can be partly attributed to the quantity of food that was swabbed. It can also be attributed to the fact that egg prote ins are heat sensitive and the solubility increases with increasing pH (Zayas 1997). In fact, it has been shown that at 70C there was a significant increase in solubility when the pH varied from 2, 3, 4, and 5 (Zayas 1997). Thus some of the egg proteins may have been denatured by the heat, but the portions that were not soluble in the solution may have still had undenatured proteins in them, which were captured by the allergen test kit.

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59 4.3.4 Water Treatment 4.3.4.1 Peanut aller gens Reduction for peanut allergens from unabraded surfaces varied from 89.2% to 99.6% for all samples, with an average of 93.5% (Table 443). Results for the removal from abraded surfaces show a range between 84.7 to 91.5%, with an average of 87.1% reduct ion (Table 4 44). Allergen reduction was quantified using the standard curve from Figure 44. As expected, reduction was lower for the rough surfaces, but these results differed from those in study 1. In the first study, the combined average for the unabr aded surfaces was lower than for the abraded. However, the average removal for the unabraded plates in study 1 compared almost exactly to that in study 2 (93.6% vs. 93.5%), indicating good reproducibility. As for the abraded plates, the second study had a lower average in relation to the first study (87.1% vs. 96.0%). The average between both studies was 91.5% reduction. Like in the first study, most of the initial food was left on the plates but was partially removed by the water rinse. 4.3.4.2 Milk allerg ens The reduction of the unabraded samples ranged from 94.4% to 97.7%, and 95.5% reduction on average (Table 445). These results compare to those obtained in the first study where the average was 94.8%. The overall removal between the 2 studies was 95.1%. Once again no food residues were observed. Milk allergen reduction for the abraded plates ranged from 98.3% to 100% removal, and 99.6% reduction on average (Table 4 46). These numbers were slightly better than those in the first study (97.6%), and betwee n the two the reduction was 98.6%, which is still very effective. The results for both types of surfaces were quantified using the equation from Figure 45.

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60 4.3.4.3 Egg allergens Results for the removal of egg allergens are presented in Tables 4 47 and 448, for unabraded and abraded surfaces. These were quantitated using the equation presented in Figure 46. Reduction was 100% for all samples and surfaces, as was the case in study 1, which shows that hot water (62.8C) is a very effective tool for removi ng egg residues. This can be explained by the high solubility of the proteins present in the egg at the neutral pH of water At pH ~ 7 the proteins present in the egg are still far from their isoelectric point (pH ~ 4.8) which is the point of lowest solub ility (Gregory 2009). There were residues left after the first half of the wash, but they were mostly removed after the first water rinse (as was the case in study 1). 4.3.5 Statistical Analysis For all three allergens, the acid wash was different from th e other 3 washes, but these were not different from each other (Tables A 4 to A 6). In terms of surfaces, there were no differences among abraded and unabraded plates for peanut allergens (Table A 4). However, surfaces showed differences for milk and egg a llergens (Tables A 5 and A 6). 4.4 Overall Results Side by side comparison of all treatments and surfaces for studies 1 and 2 are presented in Figures 410 through 415 Each set of data is colored differently from the others for better understanding. The bars in each set represent the samp les for a total of 9 observations per set. The standard deviation is also presented in the graph. For each pair of bars, the bar on the left represents the unabraded coupons and the one on the right corresponds to the abr aded surfaces. T he order of the treatments is JPA Type 4, CAD, AD, and Water. In general, r esults are similar for both studies, especially for the JPA and CAD washes. For the peanut allergens, there was more variability for the water and AD washes between studies 1 and 2, and the results from the second study appear to be more consistent (Figures 4 10 and 4-

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61 11). For the milk allergens, there was more consistency in the second study once again, but there was a large variability between AD washed samples in unabraded plates (Figures 4 12 and 413). Additionally, the standard deviation among samples of the first study was larger compared to those of the second one. In the case of egg allergens, there was no variability for JPA, CAD, and water washes in either study because they all achieved 100% reduction for all surfaces (Figures 414 and 415). In study 1, reduction for unabraded plates was higher than study 2. Conversely, reduction for abraded plates was lower in the first study.

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62 Table 4 1. JPA type 4 wa sh removal results for peanut allergens on unabraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label a bsorbance for each sample was read at 650 nm final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc .] Sample Trial # Initial w eight (g) Initial c onc (ppm) Abso rbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc a fter Dilution (ppm) %Reduction 1 1.8 4067.6 2.068 43.24 864.8 825.01 78.7 + 2 1.8 4067.6 1.950 40.28 805. 8 80.2 3 1.8 4067.6 1.948 40.23 804.5 80.2 1 0.0 0 .0 0.341 <2 .5 0.00 0.18 2 0.0 0 .0 0.346 <2.5 0.00 3 0.0 0 .0 0.348 <2.5 0.55 1 1.3 2937.7 0.336 <2.5 0.00 0.54 100 1 2 1.3 2937.7 0.347 <2.5 0.05 100 3 1.3 2937.7 0.350 <2.5 1.56 99.9 1 1.1 2485.7 0.349 <2.5 1.06 4.57 99.9 2 2 1.1 2485.7 0.360 < 2.5 6.58 99.7 3 1.1 2485.7 0.359 <2.5 6.08 99.7 1 1.6 3615.6 0.350 <2.5 1.56 3.40 99.9 3 2 1.6 3615.6 0.357 <2.5 5.08 99.8 3 1.6 3615.6 0.354 <2.5 3.57 99.9 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

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63 Table 4 2. J PA type 4 wash removal results for peanut allergens on abraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample wa s read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initi al conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 1807.8 2.318 49.52 990.5 1001.89 45.2 + 2 0.8 1807.8 2.357 50.50 101 0.1 44.1 3 0.8 1807.8 2.347 50.25 1005.0 44.4 1 0.0 0 .0 0.365 <2.5 9.10 13.79 2 0.0 0 .0 0.375 <2.5 14.12 3 0.0 0 .0 0.383 <2.5 18.14 1 0.9 2033.8 0.361 <2.5 7.09 4.41 99.7 1 2 0.9 2033.8 0.356 <2.5 4.57 99.8 3 0.9 2033.8 0.350 <2.5 1.56 99.9 1 1.2 2711.8 0.364 <2.5 8.59 6.25 99.7 2 2 1.2 2711.8 0.355 <2.5 4.07 99.9 3 1.2 2711.8 0.359 <2.5 6.08 99.8 1 1.0 2259.8 0.387 <2.5 20.15 20.99 99.1 3 2 1.0 2259.8 0.39 0 <2.5 21.66 99.0 3 1.0 2259.8 0.389 <2.5 21.16 99. 1 1Allerge n concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm Figure 41. Standard curve for peanut allergen quantification test 1 Peanut controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concent ration was read 3 times at an absorbance of 650 nm and plotted in the graph. The linear regression equation was calculated using the average of the 3 readings.

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64 Table 4 3. JPA type 4 wash removal results for milk allergens on unabraded surfaces Initial al lergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Av g c onc after dilution (ppm) %Reduction 1 0.9 318. 8 2.987 57.21 572.1 573.53 79.5 + 2 0.9 318.8 2.998 57. 46 574.6 80. 3 3 0.9 318.8 2.995 57.39 573.9 80. 1 1 0.0 0 .0 0.496 <2.5 6.0 1.98 2 0.0 0 .0 0.442 <2.5 0.0 3 0.0 0 .0 0.46 0 <2.5 0.0 1 1.2 425.0 0.711 5.482 54.82 40.50 87.1 1 2 1.2 425. 0 0.616 3.323 33.23 92.2 3 1.2 425. 0 0.617 3.345 33.45 92.1 1 1.0 354. 2 0.525 <2.5 12.55 4.18 96.5 2 2 1.0 354.2 0.445 <2.5 0.0 100 3 1.0 354. 2 0.444 <2.5 0.0 100 1 1.0 354.2 0.518 <2.5 10.95 3.65 96.9 3 2 1.0 354.2 0.447 <2.5 0.0 100 3 1.0 354.2 0.453 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

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65 Table 4 4. J PA type 4 wash removal re sults for milk allergens on abraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from t he standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) F inal c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 318.8 2.961 56.62 566.2 565.8 77.6 + 2 0.9 318.8 2.944 56.23 562.3 76.4 3 0.9 318.8 2.973 56.89 568.9 78. 5 1 0.0 0 .0 0.560 <2.5 20.5 6.83 2 0.0 0 .0 0.466 <2.5 0.0 3 0.0 0 .0 0.467 <2.5 0.0 1 0.8 283.3 0.502 <2.5 7.318 5.50 97.4 1 2 0.8 283.3 0.484 <2.5 3.227 98.9 3 0.8 283.3 0.496 <2.5 5.955 97.9 1 1.0 354.2 0.578 <2.5 24.59 11.86 93.1 2 2 1.0 354. 2 0.498 <2.5 6.409 98.2 3 1.0 354.2 0.490 <2.5 4.591 98.7 1 1.0 354.2 0.432 <2.5 0.0 0.00 100 3 2 1.0 354.2 0.437 <2.5 0.0 100 3 1.0 354.2 0.453 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm Figure 42. Standard curve for milk allergen quantification test 1 Milk controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concentration was read 3 times at an absorbance of 650 nm and plotted in the graph. The linear regression equation w as calculated using the average of the 3 readings.

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66 Table 4 5. JPA type 4 wash removal results for egg allergens on unabraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sam ple was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 813.2 2.932 28.66 286.6 292.8 64.8 + 2 0.8 813.2 3.000 29.59 2 95.9 63.6 3 0.8 813.2 3.000 29.59 295.9 63.6 1 0.0 0 .0 0.479 <2.5 0.0 0.00 2 0.0 0 .0 0.469 <2.5 0.0 3 0.0 0 .0 0.466 <2.5 0.0 1 1.1 1118.2 0.520 <2.5 0.0 0.00 100 1 2 1.1 1118.2 0.459 <2.5 0.0 100 3 1.1 1118.2 0.448 <2.5 0.0 100 1 1.0 1016.5 0.423 <2.5 0.0 0.00 100 2 2 1.0 1016.5 0.421 <2.5 0.0 100 3 1.0 1016.5 0.415 <2.5 0.0 100 1 1.0 1016.5 0.472 <2.5 0.0 0.00 100 3 2 1.0 1016.5 0.458 <2.5 0.0 100 3 1.0 1016.5 0.451 <2.5 0.0 100 1Allergen concentrations lower than t he limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absor bance reading equal to 3.000 indicates the highest value read by the machine.

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67 Table 4 6. JPA type 4 wash removal results for egg allergens on abraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and mul tiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after diluti on (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.6 609.9 3.000 29.59 295.9 295.9 51.5 + 2 0.6 609.9 3.000 29.59 295.9 51.5 3 0.6 609.9 3.000 29.59 295.9 51.5 1 0.0 0 .0 0.569 <2.5 0.0 0.00 2 0.0 0 .0 0.557 <2.5 0.0 3 0.0 0 .0 0.568 <2. 5 0.0 1 1.2 1219.8 0.622 <2.5 0.0 0.00 100 1 2 1.2 1219.8 0.616 <2.5 0.0 100 3 1.2 1219.8 0.631 <2.5 0.0 100 1 1.1 1118.2 0.472 <2.5 0.0 0.00 100 2 2 1.1 1118.2 0.483 <2.5 0.0 100 3 1.1 1118. 2 0.508 <2.5 0.0 100 1 0.7 711.6 0.478 <2.5 0. 0 0.00 100 3 2 0.7 711.6 0.496 <2.5 0.0 100 3 0.7 711.6 0.496 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine. Figure 43. Standard curve for egg allergen quantification test 1 Egg controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concentration was read 3 times at an absorbance of 650 nm and plotted in the graph. The linea r regression equation was calculated using the average of the 3 readings.

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68 Table 4 7. CAD wash removal results for peanut allergens on unabraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, abso rbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(init ial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.3 2937.7 3.000 66.66 1333.2 1333.2 54.6 + 2 1 .3 2937.7 3.000 66.66 1333. 2 54.6 3 1.3 2937.7 3.000 66.6 6 1333. 2 54.6 1 0.0 0 .0 0.378 <2.5 15.63 19.48 2 0.0 0 .0 0.388 <2.5 20.65 3 0.0 0 .0 0.391 <2.5 22.16 1 0.6 1355.9 0.380 <2.5 16.63 15.80 98.8 1 2 0.6 1355.9 0.377 <2.5 15.13 98 .9 3 0.6 1355.9 0.378 <2.5 15.63 98.8 1 1.1 2485.8 0.355 <2.5 4.07 4.07 99.8 2 2 1.1 2485.8 0.355 <2.5 4.07 99.8 3 1.1 2485. 8 0.355 <2.5 4.07 99.8 1 1.3 2937.7 0.375 <2.5 14.12 11.27 99.5 3 2 1.3 2937.7 0.368 <2.5 10.60 99.6 3 1.3 2937.7 0. 365 <2.5 9.10 99.7 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2An absorbance reading equal to 3.000 indicates the highest value read by the machine.

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69 Table 4 8. CAD wash remo val results for peanut allergens on abraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculate d from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (6 50 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 2485. 8 2.579 56.08 1121.7 1131.5 54.9 + 2 1.1 2485. 8 2.615 56.99 1139.6 54.2 3 1.1 2485. 8 2.602 56.66 1133.2 54.4 1 0.0 0 .0 0.356 <2.5 4.5 7 3.57 2 0.0 0 .0 0.354 <2.5 3.57 3 0.0 0 .0 0.352 <2.5 2.56 1 1.6 3615.7 0.343 <2.5 0.00 0.00 100 1 2 1.6 3615.7 0.342 <2.5 0.00 100 3 1.6 3615.7 0.337 <2.5 0.00 10 0 1 1.1 2485. 8 0.36 <2.5 6.58 3.40 99.7 2 2 1.1 2485. 8 0.350 <2.5 1.56 99.9 3 1.1 2485. 8 0.351 <2.5 2.06 99.9 1 1.0 2259.8 0.361 <2.5 7.09 6.42 99.7 3 2 1.0 2259.8 0.359 <2.5 6.08 99.7 3 1.0 2259.8 0.359 <2.5 6.08 99.7 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are exp ressed as <2.5 ppm

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70 Table 4 9. CAD wash removal results for milk allergens on unabraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, fi nal allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Tri al # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.0 354.2 3.000 57.51 575.1 575. 1 62.4 + 2 1.0 354. 2 3.000 57.51 575.1 62. 4 3 1.0 354. 2 3.000 57.51 575.1 62. 4 1 0.0 0 .0 0.402 <2.5 0.0 0.00 2 0.0 0 .0 0.417 <2.5 0.0 3 0.0 0 .0 0.418 <2.5 0.0 1 1.0 354.2 0.485 <2.5 3.455 3.83 99.0 1 2 1.0 354.2 0.488 <2.5 4.136 98.8 3 1.0 354.2 0.487 <2.5 3.909 98.9 1 1.1 389. 6 0.522 <2.5 11.86 3.95 96.9 2 2 1.1 389.6 0.435 <2.5 0.0 100 3 1.1 389.6 0.439 <2.5 0.0 100 1 1.1 389.6 0.507 <2.5 8.455 8.45 97.8 3 2 1.1 389.6 0.512 <2.5 9.591 97.5 3 1.1 389.6 0.502 <2.5 7.318 98.1 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

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71 Table 4 10. CAD wash removal results for milk allergens on abraded surfaces. Initial allergen conc entration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution fa ctor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc afte r dilution (ppm) %Reduction 1 0.8 283.3 3.000 57.51 575.1 575.1 103 + 2 0.8 283. 3 3.000 57.51 575.1 103 3 0.8 283.3 3.000 57.51 575.1 103 1 0.0 0 .0 0.515 <2.5 10.27 9.59 2 0.0 0 .0 0.51 <2.5 9.136 3 0.0 0 .0 0.511 <2.5 9.364 1 1.0 354.2 0.494 <2.5 5.500 1.83 98.4 1 2 1.0 354.2 0.436 <2.5 0.0 100 3 1.0 354.2 0.442 <2.5 0.0 100 1 1.1 389.6 0.457 <2.5 0.0 0.00 100 2 2 1.1 389.6 0.453 <2.5 0.0 100 3 1.1 389.6 0.459 <2.5 0.0 100 1 1.0 354.2 0.455 <2.5 0.0 0.00 100 3 2 1.0 354.2 0.449 <2.5 0.0 100 3 1.0 354.2 0.453 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the ma chine

PAGE 72

72 Table 4 11. CAD wash removal results for egg allergens on unabraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentrati on before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 1118 .2 3.000 29.59 295.9 295.9 73.5 + 2 1.1 1118.2 3.000 29.59 295.9 73.5 3 1.1 1118.2 3.000 29.59 295.9 73. 5 1 0.0 0 .0 0.491 <2.5 0.0 0.00 2 0.0 0 .0 0.520 <2.5 0.0 3 0.0 0 .0 0.513 <2.5 0.0 1 0.8 813.2 0.480 <2.5 0.0 0.00 100 1 2 0.8 813. 2 0.525 <2.5 0.0 100 3 0.8 813. 2 0.502 <2.5 0.0 100 1 0.8 813.2 0.533 <2.5 0.0 0.00 100 2 2 0.8 813.2 0.533 <2.5 0.0 100 3 0.8 813.2 0.537 <2.5 0.0 100 1 1.1 1118.2 0.533 <2.5 0.0 0.00 100 3 2 1.1 1118.2 0.489 <2.5 0.0 100 3 1.1 1118.2 0.488 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) ar e expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 73

73 Table 4 12. CAD wash removal results for egg allergens on abraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 813.2 3.00 0 29.59 295 .9 295.9 63.6 + 2 0.8 813.2 3.000 29.59 295.9 63.6 3 0.8 813.2 3.000 29.59 295.9 63.6 1 0.0 0 .0 0.610 <2.5 0.0 0.00 2 0.0 0 .0 0.621 <2.5 0.0 3 0.0 0 .0 0.621 <2.5 0.0 1 0.7 711.6 0.582 <2.5 0.0 0.00 100 1 2 0.7 711.6 0.600 <2.5 0.0 100 3 0.7 711.6 0.583 <2.5 0.0 100 1 0.7 711.6 0.495 <2.5 0.0 0.00 100 2 2 0.7 711.6 0.510 <2.5 0.0 100 3 0.7 711.6 0.540 <2.5 0.0 100 1 0.9 914.9 0.638 <2.5 0.0 0.00 100 3 2 0.9 914.9 0.674 <2.5 0.0 100 3 0.9 914.9 0.667 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 74

74 Table 4 13. AD wash removal results for peanut allergens on unabraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.4 3163.7 2.492 53.90 1077.9 1084.8 65.9 + 2 1.4 3163.7 2.519 54.58 1091.5 65.5 3 1.4 3163.7 2.506 54.25 1085.0 65.7 1 0.0 0 .0 0.351 <2.5 2.06 0.69 2 0.0 0 0 0.342 <2.5 0.00 3 0.0 0 .0 0.344 <2.5 0.00 1 1.3 2937.7 0.554 5.204 104.1 98.71 96.5 1 2 1.3 2937.7 0.551 5.128 102.6 96.5 3 1.3 2937.7 0.525 4.475 89.50 96.9 1 1.2 2711.8 1.014 16.76 335.2 328.53 87.6 2 2 1.2 2711.8 0.995 16.28 325.7 8 8.0 3 1.2 2711.8 0.993 16.23 324.7 88.0 1 1.0 2259.8 0.986 16.05 321.2 321.49 85.8 3 2 1.0 2259.8 0.985 16.03 320.6 85.8 3 1.0 2259.8 0.989 16.13 322.6 85.7 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 75

75 Table 4 14. AD wash removal results for peanut allergens on abraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 2485. 8 3.000 66.66 1333.2 1333.2 46.4 + 2 1.1 2485.8 3.000 66.66 1 333. 2 46.4 3 1.1 2485. 8 3.000 66.66 1333.2 46.4 1 0.0 0 .0 0.350 <2.5 1.56 1.04 2 0.0 0 .0 0.345 <2.5 0.00 3 0.0 0 .0 0.35 <2.5 1.56 1 1.3 2937.7 1.156 20.33 406.6 405.9 86.2 1 2 1.3 2937.7 1.153 20.25 405.1 86.2 3 1.3 2937.7 1.155 20.30 406.1 86.2 1 1.3 29 37.7 1.437 27.39 547.8 543.4 81.3 2 2 1.3 2937.7 1.418 26.91 538.2 81.7 3 1.3 2937.7 1.430 27.21 544.3 81.5 1 1.1 2485.8 0.886 13.54 270.9 270.9 89.1 3 2 1.1 2485.8 0.889 13.62 272.4 89.0 3 1.1 2485.8 0.883 13.47 269.4 89.2 1Allergen concentra tions lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 76

76 Table 4 15. AD w ash removal results for milk allergens on unabraded surfa ces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after di lution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 283.3 2.970 56.82 568.2 555.9 100 + 2 0.8 283.3 2.885 54.89 548.9 93.7 3 0.8 283. 4 2.892 55.05 550.5 94.3 1 0.0 0 .0 0.532 <2.5 14.1 4.71 2 0.0 0 .0 0.433 <2.5 0.0 3 0.0 0 .0 0. 428 <2.5 0.0 1 0.9 318.6 0.711 5.482 54.82 44.59 82.8 1 2 0.9 318. 6 0.644 3.959 39.59 87.6 3 0.9 318. 6 0.643 3.936 39.36 87.6 1 1 .0 354. 2 0.537 <2.5 15.27 5.09 95.7 2 2 1 .0 354. 2 0.465 <2.5 0.0 100 3 1 .0 354. 2 0.469 <2.5 0.0 100 1 1.2 42 5.0 0.507 <2.5 8.455 2.82 98.0 3 2 1.2 425.0 0.422 <2.5 0.0 100 3 1.2 425.0 0.423 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 77

77 Table 4 16. AD wash removal results for milk allergens on abraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standa rd curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 389.6 2.985 57.16 571.6 571.3 46.7 + 2 1.1 389.6 2.980 57.05 570.5 46. 4 3 1.1 389.6 2.985 57.16 571.6 46.7 1 0.0 0 .0 0.485 <2.5 3.455 1.15 2 0.0 0 0 0.458 <2.5 0.0 3 0.0 0 .0 0.443 <2.5 0.0 1 1.2 425.0 0.482 <2.5 2.773 1.86 99.3 1 2 1.2 425.0 0.481 <2.5 2.545 99.4 3 1.2 425.0 0.471 <2.5 0.273 99.9 1 1.1 389.6 0.815 7.845 78.45 74.21 79.9 2 2 1.1 389.6 0.794 7.368 73.68 81. 1 3 1.1 389.6 0.780 7.050 70.50 81.9 1 1.1 389.6 0.582 2.550 25.50 25.05 93.5 3 2 1.1 389.6 0.577 <2.5 24.36 93.8 3 1.1 389.6 0.581 2.527 25.27 93.5 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as < 2.5 ppm

PAGE 78

78 Table 4 17. AD w ash removal results for egg allergens on una braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.3 1321.5 2.947 28.86 288.6 286.9 78.2 + 2 1.3 1321.5 2.937 28.73 287.3 78.3 3 1.3 1321.5 2.921 28.51 285.1 78.4 1 0.0 0 .0 0.468 <2.5 0.0 0.00 2 0.0 0 .0 0.473 <2.5 0.0 3 0.0 0 .0 0.476 <2.5 0.0 1 0.9 914.9 1.042 2.695 26.95 22.92 97.1 1 2 0.9 914.9 0.995 <2.5 20.49 97.8 3 0.9 914.9 1.001 <2.5 21.32 97.7 1 1.1 1118.2 0.958 <2.5 15.4 1 14.45 98.6 2 2 1.1 1118.2 0.954 <2.5 14.86 98.7 3 1.1 1118.2 0.941 <2.5 13.08 98.8 1 1.1 1118.2 0.674 <2.5 0.0 0.00 100 3 2 1.1 1118.2 0.672 <2.5 0.0 100 3 1.1 1118.2 0.683 <2.5 0.0 100 1Allergen concentrations lower than the limit of quanti tation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 79

79 Table 4 18. AD wash removal results for egg allergens on a braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each samp le was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 914.9 3.000 29.59 295.9 295.9 67.7 + 2 0.9 914.9 3.000 29.59 29 5.9 67.7 3 0.9 914.9 3.000 29.59 295.9 67.7 1 0.0 0 .0 0.539 <2.5 0.0 0.00 2 0.0 0 .0 0.550 <2.5 0.0 3 0.0 0 .0 0.552 <2.5 0.0 1 0.9 914.9 1.637 10.86 108.7 107.4 88.1 1 2 0.9 914.9 1.651 11.06 110.6 87.9 3 0.9 914.9 1.595 10.29 102.9 88.8 1 0.9 914.9 1.735 12.21 122.1 123.8 86.7 2 2 0.9 914.9 1.760 12.56 125.6 86.3 3 0.9 914.9 1.746 12.36 123.7 86.5 1 0.9 914.9 1.840 13.66 136.6 134.5 85.1 3 2 0.9 914.9 1.829 13.51 135.1 85.2 3 0.9 914.9 1.807 13.20 132.0 85.6 1Allerge n concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 80

80 Table 4 19. Water wash removal results for peanut allergens on unabraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regre ssion equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (p pm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.5 3389.7 2.086 16.6 8 333.6 335.0 90.2 + 2 1.5 3389.7 2.095 16.78 335.6 90.1 3 1.5 3389.7 2.096 16.79 335.9 90.1 1 0.0 0.0 0.540 <2.5 0.0 0.00 2 0.0 0.0 0.531 <2.5 0.0 3 0.0 0.0 0.521 <2.5 0.0 1 1.0 2259.8 1.752 12.8 8 257.6 257. 6 88.6 1 2 1.0 2259.8 1.786 13.2 7 265.3 88.3 3 1.0 2259.8 1.718 12.49 249.9 88.9 1 1.0 2259.8 1.034 4.711 94.22 94.22 95.8 2 2 1.0 2259.8 1.041 4.791 95.81 95.8 3 1.0 2259.8 1.027 4.631 92.63 95.9 1 1.2 2711.8 1.040 4.779 95.59 94.60 96.5 3 2 1.2 2711.8 1.039 4.768 95.36 96.5 3 1.2 2711.8 1.028 4.643 92.86 96.6 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2. 5 ppm

PAGE 81

81 Table 4 20. Water wash removal results for peanut allergens on abraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g ) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.5 3389.7 2.429 20.58 411.6 412.3 87.9 + 2 1.5 3389.7 2.420 20.48 409.6 87.9 3 1.5 3389.7 2.447 20.79 415.7 8 7.7 1 0.0 0.0 0.432 <2.5 0.0 0.00 2 0.0 0.0 0.435 <2.5 0.0 3 0.0 0.0 0.429 <2.5 0.0 1 1.3 2937.7 1.130 5.803 116.1 113.0 96.0 1 2 1.3 2937.7 1.121 5.701 114.0 96.1 3 1.3 2937.7 1.099 5.451 109.0 96.3 1 1.1 2485.8 0.820 2.276 116.1 4 0.83 98.2 2 2 1.1 2485.8 0.811 2.174 114.0 98.3 3 1.1 2485.8 0.767 1.673 109.0 98.7 1 1.1 2485.8 1.323 7.999 45.53 159.4 93.6 3 2 1.1 2485.8 1.323 7.999 43.48 93.6 3 1.1 2485.8 1.316 7.919 33.47 93.6 1Allergen concentrations lower than the lim it of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2An absorbance reading equal to 3.000 indicates the highest value read by the machine. Figure 44. Standard c urve for peanut allergen quantification test 2 Peanut controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concentration was read 3 times at an absorbance of 650 nm and plotted in the graph. The linear regression equation was calculated using the average of the 3 readings.

PAGE 82

82 Table 4 21. Water wash removal results for m ilk allergens on unabraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standa rd curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 318.8 2.945 33.30 333.0 555. 9 101 + 2 0.9 318.8 2.959 33.48 334.8 93.7 3 0.9 318.8 2.923 33.01 330.1 94.3 1 0.0 0.0 0.431 <2.5 6.93 4.71 2 0.0 0.0 0.437 <2.5 7.70 3 0.0 0.0 0.429 <2.5 6.67 1 0.9 354.2 0.443 <2.5 8.48 44.59 82.8 1 2 0.9 354.2 0.437 <2.5 7.70 87.6 3 0.9 354.2 0.419 <2.5 5.37 87.7 1 1.0 354.2 0.620 3.144 31.44 5.09 95.7 2 2 1.0 354.2 0.624 3.196 31.96 100 3 1.0 354. 2 0.621 3.157 31.57 100 1 1.0 354.2 0.508 <2.5 16.91 2.82 98.0 3 2 1.0 354.2 0.510 <2.5 17.17 100 3 1.0 354.2 0.495 <2.5 15.23 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 83

83 Table 4 22. Water w ash removal results for milk allergens on abraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 318.8 3.000 34.01 340.1 336.5 6.7 1 + 2 0.9 318.8 2.993 33.92 339.2 6.4 2 3 0.9 318.8 2.923 33.01 330.1 3. 57 1 0 .0 0.0 0.393 <2.5 1.997 2.39 2 0.0 0.0 0.415 <2.5 4.851 3 0.0 0.0 0.380 <2.5 0.311 1 1.0 354.2 0.439 <2.5 7.964 8.53 97.8 1 2 1.0 354.2 0.456 <2.5 10.17 97.1 3 1.0 354.2 0.435 <2.5 7.445 97.9 1 1.0 354.2 0.494 <2.5 15.10 15.96 95.7 2 2 1.0 354.2 0.513 <2.5 17.56 95.0 3 1.0 354.2 0.495 <2.5 15.23 95.7 1 1.0 354.2 0.387 <2.5 1.219 0.83 99.7 3 2 1.0 354.2 0.385 <2.5 0.960 99.7 3 1.0 354.2 0.380 <2.5 0.311 99.9 1Allergen concentrations lower than the limit of quantitation of t he test kit (2.5 ppm) are expressed as <2.5 ppm 2An absorbance reading equal to 3.000 indicates the highest value read by the machine. Figure 45. Standard curve for milk allergen quantification test 2 Milk controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concentration was read 3 times at an absorbance of 650 nm and plotted in the graph. The linear regression equation was calculated using the average of the 3 readings.

PAGE 84

84 Table 4 23. Water w ash removal results for egg allergens on una braded s urfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc afte r dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 1118.2 2.775 27.70 277.0 273.9 75.2 + 2 1.1 1118.2 2.747 27.26 272.6 75.6 3 1.1 1118.2 2.744 27.22 272.2 75.7 1 0.0 0.0 0.621 <2.5 0.0 0.00 2 0.0 0.0 0.625 <2.5 0.0 3 0.0 0. 0 0.625 <2.5 0.0 1 0.9 914.9 0.640 <2.5 0.0 0.00 100 1 2 0.9 914.9 0.653 <2.5 0.0 100 3 0.9 914.9 0.641 <2.5 0.0 100 1 0.9 914.9 0.706 <2.5 0.0 0.00 100 2 2 0.9 914.9 0.698 <2.5 0.0 100 3 0.9 914.9 0.703 <2.5 0.0 100 1 0.8 813.2 0.828 <2 .5 0.0 0.00 100 3 2 0.8 813.2 0.815 <2.5 0.0 100 3 0.8 813.2 0.83 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 85

85 Table 4 24. Water wash removal results for egg allerge ns on a braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 ( ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 1118.2 2.696 26.46 264.6 254.7 76.3 + 2 1.1 1118.2 2.585 24.72 247.2 77.9 3 1.1 1118.2 2.618 25.24 252.4 77.4 1 0.0 0.0 0.730 <2.5 0.0 0.00 2 0.0 0.0 0.711 <2.5 0. 0 3 0.0 0.0 0.700 <2.5 0.0 1 1.1 1118.2 0.703 <2.5 0.0 0.00 100 1 2 1.1 1118.2 0.709 <2.5 0.0 100 3 1.1 1118.2 0.703 <2.5 0.0 100 1 1.2 1219.8 0.661 <2.5 0.0 0.00 100 2 2 1.2 1219.8 0.639 <2.5 0.0 100 3 1.2 1219.8 0.636 <2.5 0.0 100 1 1.0 1016.5 0.513 <2.5 0.0 0.00 100 3 2 1.0 1016.5 0.512 <2.5 0.0 100 3 1.0 1016.5 0.512 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm Figure 46. Standard c urve fo r egg allergen quantification test 2 Egg controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concentration was read 3 times at an absorbance of 650 nm and plotted in the graph. The linear regression equation was calculated using the average of the 3 readings.

PAGE 86

86 Table 4 25. JPA type 4 wash removal results for peanut allergens on unabraded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.8 4067.6 2.068 43.24 864.9 825.0 78.7 + 2 1.8 4067.6 1.950 40.28 805.6 80.2 3 1.8 4067.6 1.9 48 40.23 804.6 80.2 1 0.0 0 .0 0.340 <2.5 0.0 0.02 2 0.0 0 .0 0.347 <2.5 0.1 3 0.0 0 .0 0.341 <2.5 0.0 1 1.0 2259.8 0.338 <2.5 0.0 1.21 100 1 2 1.0 2259.8 0.351 <2.5 2.1 99.9 3 1.0 2259.8 0.350 <2.5 1.6 99.9 1 1.2 2711.8 0.358 <2.5 5. 6 7.09 99.8 2 2 1.2 2711.8 0.367 <2.5 10.1 99.6 3 1.2 2711.8 0.358 <2.5 5.6 99.8 1 0.9 2033.8 0.332 <2.5 0.0 0.00 100 3 2 0.9 2033.8 0.340 <2.5 0.0 100 3 0.9 2033.8 0.337 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitati on of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 87

87 Table 4 26. JPA type 4 wash removal results for peanut allergens on a braded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 1807.8 2.318 49.52 990.5 1001.9 45.2 + 2 0.8 1807.8 2. 357 50.50 1010.1 44.1 3 0.8 1807.8 2.347 50.25 1005. 1 44.4 1 0.0 0 .0 0.339 <2.5 0.0 0.00 2 0.0 0 .0 0.339 <2.5 0.0 3 0.0 0 .0 0.337 <2.5 0.0 1 0.9 2033.8 0.339 <2.5 0.0 0.00 100 1 2 0.9 2033.8 0.344 <2.5 0.0 10 0 3 0.9 2033.8 0.346 <2 .5 0.0 100 1 1.0 2259.8 0.333 <2.5 0.0 0.00 100 2 2 1.0 2259.8 0.334 <2.5 0.0 100 3 1.0 2259.8 0.332 <2.5 0.0 100 1 1.2 2711.8 0.355 <2.5 0.0 0.00 100 3 2 1.2 2711.8 0.360 <2.5 0.0 100 3 1.2 2711.8 0.356 <2.5 0.0 100 1Allergen concentration s lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm Figure 47. Standard curve for peanut allergen quantification for water treatment Peanut controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concentra tion was read 3 times at an absorbance of 650 nm and plotted in the graph. The linear regression equation was calculated using the average of the 3 readings.

PAGE 88

88 Table 4 27. JPA t ype 4 wash removal results for milk allergens on una braded surfaces Initial all ergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 318. 6 2.987 57.21 57 2.1 573.5 79.5 + 2 0.9 318. 6 2.998 57.46 574.6 80.3 3 0.9 318.6 2.995 57.39 573.9 80. 1 1 0.0 0 .0 0.444 <2.5 0.0 0.00 2 0.0 0 .0 0.443 <2.5 0.0 3 0.0 0 .0 0.439 <2.5 0.0 1 1.1 389.6 0.497 <2.5 0.0 0.00 100 1 2 1.1 389. 6 0.493 <2.5 0.0 100 3 1.1 389.6 0.485 <2.5 0.0 100 1 1.2 425.0 0.456 <2.5 0.0 0.00 100 2 2 1.2 425.0 0.453 <2.5 0.0 100 3 1.2 425.0 0.452 <2.5 0.0 100 1 1.2 425.0 0.444 <2.5 0.0 0.00 100 3 2 1.2 425.0 0.447 <2.5 0.0 100 3 1.2 425.0 0.439 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 89

89 Table 4 28. JPA type 4 wash removal results for milk allergens on a bra ded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 318.6 2.961 56.62 566.2 565.8 77.6 + 2 0.9 318.6 2.944 56.23 562.3 76.4 3 0.9 318. 6 2.973 56.89 568.9 78. 5 1 0.0 0 .0 0.432 <2.5 0.0 0.00 2 0.0 0 .0 0.435 <2.5 0.0 3 0 .0 0 .0 0.428 <2.5 0.0 1 1.1 389.6 0.448 <2.5 0.0 0.00 100 1 2 1.1 389.6 0.456 <2.5 0.0 100 3 1.1 389.6 0.438 <2.5 0.0 100 1 1.0 354.2 0.393 <2.5 0.0 0.00 100 2 2 1.0 354.2 0.397 <2.5 0.0 100 3 1.0 354.2 0.392 <2.5 0.0 100 1 1.0 354.2 0.4 12 <2.5 0.0 0.00 100 3 2 1.0 354.2 0.416 <2.5 0.0 100 3 1.0 354.2 0.409 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm Figure 48. Standard curve for milk allergen qua ntification for water treatment Milk controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concentration was read 3 times at an absorbance of 650 nm and plotted in the graph. The linear regression equation was calculated using the average of the 3 readings.

PAGE 90

90 Table 4 29. JPA type 4 wash removal results for egg allergens on una braded surfaces Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 813.2 2.932 28.66 286.6 292.8 64.8 + 2 0.8 813. 2 3.000 29.59 295.9 63.6 3 0.8 813. 2 3.000 29.59 2 95.9 63.6 1 0.0 0 .0 0.335 <2.5 0.0 0.00 2 0.0 0 .0 0.329 <2.5 0.0 3 0.0 0 .0 0.320 <2.5 0.0 1 1.0 1016.5 0.325 <2.5 0.0 0.00 100 1 2 1.0 1016.5 0.323 <2.5 0.0 100 3 1.0 1016.5 0.318 <2.5 0.0 100 1 1.1 1118.2 0.317 <2.5 0.0 0.00 100 2 2 1.1 1118.2 0.315 <2.5 0.0 100 3 1.1 1118.2 0.309 <2.5 0.0 100 1 1.0 1016.5 0.363 <2.5 0.0 0.00 100 3 2 1.0 10 16.5 0.356 <2.5 0.0 100 3 1.0 1016.5 0.320 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test k it (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 91

91 Table 4 30. JPA t ype 4 wash removal results for egg allergens on a braded surface s Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentratio ns for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduct ion 1 0.6 609.9 3.000 29.59 295.9 295.9 51.5 + 2 0.6 609.9 3.000 29.59 295.9 51.5 3 0.6 609.9 3.000 29.59 295.9 51.5 1 0.0 0 .0 0.285 <2.5 0.0 0.00 2 0.0 0 .0 0.285 <2.5 0.0 3 0.0 0 .0 0.286 <2.5 0.0 1 1.0 1016.5 0.332 <2.5 0.0 0.00 10 0 1 2 1.0 1016.5 0.320 <2.5 0.0 100 3 1.0 1016.5 0.317 <2.5 0.0 100 1 1.1 1118.2 0.306 <2.5 0.0 0.00 100 2 2 1.1 1118.2 0.307 <2.5 0.0 100 3 1.1 1118.2 0.307 <2.5 0.0 100 1 1.0 1016.5 0.312 <2.5 0.0 0.00 100 3 2 1.0 1016.5 0.314 <2.5 0.0 10 0 3 1.0 1016.5 0.308 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine. Figure 49. St andard curve for egg allergen quantification for water treatment Egg controls contained 0, 2.5, 5, 10, and 25 ppm of allergen. Each concentration was read 3 times at an absorbance of 650 nm and plotted in the graph. The linear regression equation was calculated using the average of the 3 readings.

PAGE 92

92 Table 4 31. CAD wash removal results for peanut allergens on una braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial c onc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.3 2937.7 3.000 66.66 1333.2 1333.2 54.6 + 2 1.3 2937.7 3.000 66.66 1333. 2 54.6 3 1.3 2937.7 3.000 66.66 1333.2 54.6 1 0.0 0 .0 0.411 <2.5 0.0 0.00 2 0.0 0 .0 0.361 <2.5 0.0 3 0.0 0 .0 0.354 <2.5 0.0 1 1.0 2259.8 0.337 <2.5 0.0 0.00 100 1 2 1.0 2259.8 0.342 <2.5 0.0 100 3 1.0 2259.8 0.336 <2.5 0.0 100 1 1 .2 2711.8 0.340 <2.5 0.0 0.00 100 2 2 1.2 2711.8 0.346 <2.5 0.0 100 3 1.2 2711.8 0.339 <2.5 0.0 100 1 1.1 2485.78 0.337 <2.5 0.0 0.00 100 3 2 1.1 2485.78 0.342 <2.5 0.0 100 3 1.1 2485.78 0.336 <2.5 0.0 100 1Allergen concentrations lower than t he limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 93

93 Table 4 32. CAD wash removal results for peanut allergens on a braded surfaces. Initial a llergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial weight (g) Initial conc (ppm) Absorbance (650 nm) Final concentration1 (ppm) Conc after dilution (ppm) A vg conc after dilution (ppm) %Reduction 1 1.1 2485. 8 2.579 56.08 1121.7 1131.5 54.9 + 2 1.1 2485.8 2.615 56.98 1139.8 54.2 3 1.1 2485.8 2.602 56.66 1133.2 54.4 1 0.0 0 .0 0.359 <2.5 0.0 0.00 2 0.0 0 .0 0.352 <2.5 0.0 3 0.0 0 .0 0.355 <2.5 0 .0 1 1.0 2259.8 0.352 <2.5 0.0 0.00 100 1 2 1.0 2259.8 0.335 <2.5 0.0 100 3 1.0 2259.8 0.343 <2.5 0.0 100 1 0.9 2033.8 0.338 <2.5 0.0 0.00 100 2 2 0.9 2033.8 0.335 <2.5 0.0 100 3 0.9 2033.8 0.335 <2.5 0.0 100 1 1.2 27 11.8 0.339 <2.5 0.0 0.00 100 3 2 1.2 2711.8 0.331 <2.5 0.0 100 3 1.2 2711.8 0.334 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 94

94 Table 4 33. CAD wash removal results for milk allergens on una braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regressi on equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.0 354.2 3.000 57.51 575.1 575.1 6 2.4 + 2 1.0 354.2 3.000 57.51 575.1 62.4 3 1.0 354.2 3.000 57.51 575.1 62.4 1 0.0 0 .0 0.448 <2.5 0.0 0.00 2 0.0 0 .0 0.449 <2.5 0.0 3 0.0 0 .0 0.447 <2.5 0.0 1 0.9 318.7 0.413 <2.5 0.0 0.00 100 1 2 0.9 318.7 0.419 <2.5 0.0 100 3 0.9 318.7 0.417 <2.5 0.0 100 1 1.0 354.2 0.425 <2.5 0.0 0.00 100 2 2 1.0 354.2 0.402 <2.5 0.0 100 3 1.0 354.2 0.401 <2.5 0.0 100 1 1.0 354 .2 0.495 <2.5 0.0 0.00 100 3 2 1.0 354.2 0.431 <2.5 0.0 100 3 1.0 354.2 0.426 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indica tes the highest value read by the machine.

PAGE 95

95 Table 4 34. CAD wash removal results for milk allergens on a braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 283.3 3.000 57.51 575.1 575.1 103 + 2 0.8 283.3 3.000 57.51 575.1 103 3 0.8 283.3 3.000 57.51 575.1 103 1 0.0 0 .0 0.495 <2.5 0.0 0.00 2 0.0 0 .0 0.497 <2.5 0.0 3 0.0 0 .0 0.490 <2.5 0.0 1 1.0 354.2 0.407 <2.5 0.0 0.00 100 1 2 1.0 354.2 0.408 <2.5 0.0 100 3 1.0 354.2 0.406 <2.5 0.0 100 1 0.9 318.8 0.407 <2.5 0.0 0.00 100 2 2 0.9 318.8 0.404 <2.5 0.0 100 3 0.9 318. 0.408 <2.5 0.0 100 1 1.2 425.0 0.537 <2.5 0.0 0.00 100 3 2 1.2 425.0 0.536 <2.5 0.0 100 3 1.2 425.0 0.535 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 96

96 Table 4 35. CAD wash removal results for egg allergens on una braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final conc entrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 1118.2 3.000 29.59 295.9 295.9 73.5 + 2 1.1 1118.2 3.000 29.59 2 95.9 73.5 3 1.1 1118.2 3.000 29.59 295.9 73.5 1 0.0 0 .0 0.337 <2.5 0.0 0.00 2 0.0 0 .0 0.335 <2.5 0.0 3 0.0 0 .0 0.329 <2.5 0.0 1 1.0 1016.5 0.424 <2.5 0.0 0.00 100 1 2 1.0 1016.5 0.412 <2.5 0.0 100 3 1.0 1016.5 0.451 <2.5 0.0 100 1 1.1 1118.2 0.314 <2.5 0.0 0.00 100 2 2 1.1 1118.2 0.313 <2.5 0.0 100 3 1.1 1118.2 0.306 <2.5 0.0 100 1 1.0 1016.5 0.327 <2.5 0.0 0.00 100 3 2 1.0 1016.5 0.322 < 2.5 0.0 100 3 1.0 1016.5 0.314 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 97

97 Tabl e 4 36. CAD wash removal results for egg allergens on a braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilu tion was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc ( ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 813.2 3.000 29.59 295.9 295.9 63.6 + 2 0.8 813. 2 3.000 29.59 295.9 6 3.6 3 0.8 813.2 3.000 29.59 295.9 63.6 1 0.0 0 .0 0.3 22 <2.5 0.0 0.00 2 0.0 0 .0 0.314 <2.5 0.0 3 0.0 0 .0 0.319 <2.5 0.0 1 0.9 914.9 0.326 <2.5 0.0 0.00 100 1 2 0.9 914.9 0.320 <2.5 0.0 100 3 0.9 914.9 0.321 <2.5 0.0 100 1 1.1 1118.2 0.311 <2.5 0.0 0.00 100 2 2 1.1 1118.2 0.314 <2.5 0.0 100 3 1.1 1118.2 0.313 <2.5 0.0 100 1 1.2 1219.8 0.349 <2.5 0.0 0.00 100 3 2 1.2 1219.8 0.322 <2.5 0.0 100 3 1.2 1219.8 0.330 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

PAGE 98

98 Table 4 37. AD wash removal results for peanut allergens on una braded surfaces. Initial allergen concentration was calculated from the amount of protein indi cated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.4 3163.7 2.492 53.90 1 077.9 1084.8 65.9 + 2 1.4 3163.7 2.519 54.58 1091.5 65.5 3 1.4 3163.7 2.506 54.25 1085.0 65.7 1 0.0 0 .0 0.362 <2.5 0.0 0.00 2 0.0 0 .0 0.370 <2.5 0.00 3 0.0 0 .0 0.370 <2.5 0.00 1 1.0 2259.8 0.578 3.522 70.43 69.86 96.9 1 2 1.0 2259.8 0.574 3.435 68.70 97.0 3 1.0 2259.8 0.578 3.522 70.43 96.9 1 1.1 2485. 8 0.397 <2.5 0.0 0.00 100 2 2 1.1 2485. 8 0.398 <2.5 0.0 100 3 1.1 2485. 8 0.397 <2.5 0.0 100 1 1.0 2259.8 0.364 <2.5 0.0 0.00 100 3 2 1.0 2259.8 0.369 <2.5 0.0 100 3 1.0 2259.8 0.370 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 99

99 Table 4 38. AD wash removal results for peanut allergens on a braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final c oncentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (p pm) %Reduction 1 1.1 2485. 8 3.000 66.66 1333.2 1333.2 46.4 + 2 1.1 2485. 8 3.000 66.66 1333. 2 46.4 3 1.1 2485. 8 3.000 66.66 1333.2 46.4 1 0.0 0 .0 0.357 <2.5 0.0 0.00 2 0.0 0 .0 0.344 <2.5 0.0 3 0.0 0 .0 0.339 <2.5 0.0 1 1.0 2259.8 0.45 8 0.913 18.26 15.22 99.2 1 2 1.0 2259.8 0.447 0.674 13.48 99.4 3 1.0 2259.8 0.448 0.696 13.91 99.4 1 1.0 2259.8 0.441 0.543 10.87 9.42 99.5 2 2 1.0 2259.8 0.437 0.457 9.13 99.6 3 1.0 2259.8 0.435 0.413 8.26 99.6 1 1.1 2485. 8 0.372 <2.5 0.0 0. 00 100 3 2 1.1 2485. 8 0.369 <2.5 0.0 100 3 1.1 2485. 8 0.366 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest va lue read by the machine.

PAGE 100

100 Table 4 39. AD wash removal results for milk allergens on una braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final al lergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # I nitial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.8 283.3 2.970 56.82 568.2 555.9 100 + 2 0.8 283.3 2.885 54.89 548.9 93.7 3 0.8 283.3 2.892 55.05 550.5 94.3 1 0.0 0 .0 0.431 <2.5 0.0 0.00 2 0.0 0 .0 0.433 <2.5 0.0 3 0.0 0 .0 0.424 <2.5 0.0 1 0.9 318.8 1.380 12.64 126.4 124.8 60.3 1 2 0.9 318.8 1.381 12.66 126.6 60.3 3 0.9 318.8 1.349 12.16 121.6 61.9 1 1 354.2 0.623 <2.5 8.13 8.02 97.7 2 2 1 354.2 0.629 <2.5 9.06 97.4 3 1 354.2 0.615 <2.5 6.88 98.1 1 1.2 425.0 2.156 24.77 247.7 246.2 41.7 3 2 1.2 425. 0 2.147 24.63 246.3 42.1 3 1.2 425. 0 2.137 24.47 244.7 42.4 1Allergen concentrations lower than the limit of q uantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 101

101 Table 4 40. AD wash removal results for milk allergens on a braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for ea ch sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final c onc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 389.6 2.985 57.16 571.6 571.3 46.7 + 2 1.1 389.6 2.980 57.05 570.5 46. 4 3 1.1 389.6 2.985 57.16 571.6 46.7 1 0.0 0 .0 0.443 <2.5 0.0 0.00 2 0.0 0 .0 0.439 <2.5 0.0 3 0.0 0 .0 0.438 <2.5 0.0 1 1.0 354.2 0.765 3.031 30.31 25.94 91.4 1 2 1.0 354.2 0.737 2.594 25.94 92.7 3 1.0 354.2 0.709 < 2.5 21.56 93.9 1 0.9 318.6 0.652 <2.5 12.66 12.66 96.0 2 2 0.9 318. 6 0.651 <2.5 12.50 96.1 3 0.9 318.6 0.653 <2.5 12.81 96.0 1 1.0 354.2 0.642 <2.5 11.09 12.19 96.9 3 2 1.0 354.2 0.647 <2.5 11.88 96.7 3 1.0 354.2 0.658 <2.5 13.59 96.2 1Alle rgen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

PAGE 102

102 Table 4 41. AD w ash removal results for egg allergens on una braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.3 1321. 5 2.9 47 28.86 288.6 287.0 78.2 + 2 1.3 1321.5 2.937 28.72 287.3 78.2 3 1.3 1321.5 2.921 28.51 285.1 78.4 1 0.0 0 .0 0.318 <2.5 0.0 0.00 2 0.0 0 .0 0.318 <2.5 0.0 3 0.0 0 .0 0.318 <2.5 0.0 1 0.9 914. 9 1.374 7.284 72.84 72.76 92.0 1 2 0.9 914. 9 1.376 7.309 73.09 92.0 3 0.9 914.9 1.370 7.235 72.35 92.1 1 1.0 1016.5 1.305 6.432 64.32 64.77 93.7 2 2 1.0 1016.5 1.312 6.519 65.19 93.6 3 1.0 1016.5 1.309 6.481 64.81 93.6 1 0.9 914. 9 1.382 7.383 73.83 74.28 91.9 3 2 0.9 914.9 1.391 7.494 74.94 91.8 3 0.9 914.9 1.384 7.407 74.07 91.9 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

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103 Table 4 42. AD wash removal results for egg allergens on a braded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dil ution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 914.9 3.000 29.59 295.9 295.9 67.7 + 2 0.9 914.9 3.000 29.59 295.9 67.7 3 0.9 914.9 3.000 29.59 295.9 67.7 1 0.0 0 .0 0.321 <2.5 0.0 0.00 2 0.0 0 .0 0.314 <2.5 0.0 3 0.0 0 .0 0.316 <2.5 0.0 1 1 .0 1016.5 1.524 9.136 91.4 83.00 91.0 1 2 1.0 1016.5 1.418 7.827 78.3 92. 3 3 1.0 1016.5 1.427 7.938 79.4 92.2 1 1.0 1016.5 1.432 8.000 80.0 77.82 92.1 2 2 1.0 1016.5 1.407 7.691 76.9 92.4 3 1.0 1016.5 1.404 7.654 76.5 92.5 1 1.2 1219.8 1.657 10.7 8 108 83.05 91.2 3 2 1.2 1219.8 1.356 7.061 70.6 94.2 3 1.2 1219.8 1.357 7.074 70.7 94.2 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm 2 An absorbance reading equal to 3.000 indi cates the highest value read by the machine.

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104 Table 4 43. Water wash removal results for peanut allergens on unabraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample wa s read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initi al conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.5 3389.7 2.086 16.68 333.6 335.0 90.2 + 2 1.5 3389.7 2.095 16.78 335.6 90.1 3 1.5 3389.7 2.096 16.79 335.9 90.1 1 0.0 0.0 0.540 <2.5 0.0 0.00 2 0.0 0.0 0.531 <2.5 0.0 3 0.0 0.0 0.521 <2.5 0.0 1 1.1 2485.8 1.791 13.32 266.5 267.4 89.3 1 2 1.1 2485.8 1.803 13.46 269.2 89.2 3 1.1 2485.8 1.791 13.32 266. 5 89.3 1 1.0 2259.8 1.449 9.432 188.6 187.9 91.7 2 2 1.0 2259.8 1.448 9.421 188.4 91.7 3 1.0 2259.8 1.440 9.330 186.6 91.7 1 1.1 2485.8 0.672 <2.5 11.85 11.17 99.5 3 2 1.1 2485.8 0.675 <2.5 12.54 99.5 3 1.1 2485.8 0.660 <2.5 9.124 99.6 1All ergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

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105 Table 4 44. Water wash removal results for peanut allergens on abraded surfaces. Initial allergen concentration was calculated from the amount o f protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.5 3389.7 2.429 52.31 1046.3 1047.8 69.1 + 2 1.5 3389.7 2.420 52.09 1041.8 69.3 3 1.5 3389.7 2.447 52.77 1055.3 68.9 1 0.0 0.0 0.432 <2.5 6.957 6.96 2 0.0 0.0 0.435 <2.5 8.261 3 0.0 0.0 0.429 <2.5 5.652 1 0.9 2033.8 1.132 15.57 311.3 307.8 84. 7 1 2 0.9 2033.8 1.121 15.33 306.5 84.9 3 0.9 2033.8 1.119 15.28 305.7 85.0 1 0.9 2033.8 1.150 15.96 319.1 307.1 84.3 2 2 0.9 2033.8 1.120 15.30 306.1 85.0 3 0.9 2033.8 1.097 14.80 296.1 85.4 1 0.9 2033.8 0.818 8.739 174.8 173.3 91.4 3 2 0.9 2033.8 0.815 8.674 173.5 91.5 3 0.9 2033.8 0.811 8.587 171.7 91.6 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

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106 Table 4 45. Water w ash removal results for milk allergens on unabrad ed surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equa tion and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 2033.8 2.945 33.30 666.0 665.3 67.3 + 2 0.9 2033.8 2.959 33.48 669.6 67.1 3 0.9 2033.8 2.923 33.01 660.3 67.5 1 0.0 0.0 0.431 <2.5 6.926 7.099 2 0.0 0.0 0.437 <2.5 7.704 3 0.0 0.0 0.429 <2.5 6.667 1 1.1 389.6 0.540 <2.5 21.06 19.72 94.6 1 2 1.1 389.6 0.522 <2.5 18.73 95.2 3 1.1 389.6 0.527 <2.5 19.38 95.0 1 1.1 389.6 0.546 <2.5 21.84 18.12 94.4 2 2 1.1 389.6 0.535 <2.5 20.42 94.8 3 1.1 389.6 0.471 <2.5 12. 11 96.9 1 1.1 389.6 0.549 <2.5 22.23 14.75 94.3 3 2 1.1 389.6 0.477 <2.5 12.89 96.7 3 1.1 389.6 0.448 <2.5 9.131 97.7 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

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107 Table 4 46. W ater wash removal results for milk allergens on abraded surfaces. Initial allergen concentration was calculated from the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution w as calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) A bsorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 0.9 2033.8 3.000 34.01 340.1 336.5 83.3 + 2 0.9 2033.8 2.993 33.92 339.2 83.3 3 0.9 2033.8 2.923 33.01 330.1 83.8 1 0.0 0.0 0.393 <2.5 1.997 2.387 2 0.0 0.0 0.415 <2.5 4.851 3 0.0 0.0 0.380 <2.5 0.311 1 1.1 389.6 0.428 <2.5 6.537 4.375 98.3 1 2 1.1 389.6 0.421 <2.5 5.629 98.6 3 1.1 389.6 0.385 <2.5 0.960 99.8 1 1.2 425.0 0.348 <2.5 0.0 0.00 100 2 2 1.2 425.0 0. 345 <2.5 0.0 100 3 1.2 425.0 0.343 <2.5 0.0 100 1 1.0 354.2 0.354 <2.5 0.0 0.00 100 3 2 1.0 354.2 0.353 <2.5 0.0 100 3 1.0 354.2 0.346 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expr essed as <2.5 ppm 2 An absorbance reading equal to 3.000 indicates the highest value read by the machine.

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108 Table 4 47. Water wash removal results for egg allergens on una braded surfaces. Initial allergen concentration was calculated from the amount of prot ein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reduction 1 1.1 2485.8 2.775 27.70 277.0 273.95 88.9 + 2 1.1 2485.8 2.747 27.26 272.6 89.0 3 1.1 2485.8 2.744 27.22 272.2 89.1 1 0.0 0.0 0.621 <2.5 0.0 0.00 2 0.0 0.0 0.625 <2.5 0.0 3 0.0 0.0 0.625 <2.5 0.0 1 0.9 914.9 0.913 <2.5 0.0 0.00 100 1 2 0.9 914.9 0.92 0 <2.5 0.0 100 3 0.9 914.9 0.948 <2.5 0.0 100 1 1.0 1016.5 0.674 <2.5 0.0 0.00 100 2 2 1.0 1016.5 0.671 <2.5 0.0 100 3 1.0 1016.5 0.675 <2.5 0.0 100 1 1.1 1118.2 0.677 <2.5 0.0 0.00 100 3 2 1.1 1118.2 0.678 <2.5 0.0 100 3 1.1 1118.2 0.676 <2.5 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

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109 Table 4 48. Water wash removal results for egg allergens on a braded surfaces. Initial allergen concentration was calculated f rom the amount of protein indicated on the label, absorbance for each sample was read at 650 nm, final allergen concentration before dilution was calculated from the standard curve regression equation and multiplied by dilution factor, final concentrations for triplicates were averaged, and % reduction [(initial final conc.)/ initial conc.] Sample Trial # Initial w eight (g) Initial conc (ppm) Absorbance (650 nm) Final c oncentration1 (ppm) Conc after dilution (ppm) Avg c onc after dilution (ppm) %Reductio n 1 1.1 2485.8 2.696 26.46 264.6 254.72 89.4 + 2 1.1 2485.8 2.585 24.72 247.2 90.1 3 1.1 2485.8 2.618 25.24 252.4 89.8 1 0.0 0.0 0.730 4.450 0.0 0.00 2 0.0 0.0 0.711 4.748 0.0 3 0.0 0.0 0.700 4.921 0.0 1 0.9 914.9 0.844 2.657 0. 0 0.00 100 1 2 0.9 914.9 0.798 3.381 0.0 100 3 0.9 914.9 0.791 3.491 0.0 100 1 0.9 914.9 0.606 6.399 0.0 0.00 100 2 2 0.9 914.9 0.599 6.509 0.0 100 3 0.9 914.9 0.598 6.525 0.0 100 1 0.9 914.9 0.610 6.336 0.0 0.00 100 3 2 0.9 914.9 0.59 8 6.525 0.0 100 3 0.9 914.9 0.593 6.604 0.0 100 1Allergen concentrations lower than the limit of quantitation of the test kit (2.5 ppm) are expressed as <2.5 ppm

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110 Figure 410. Peanut allergen reduction across methods in study 1. Each set of bars corresponds to unabraded surface ( U ) and abraded surface ( A ) From left to right, the order of treatments was JPA Type 4, CAD, AD, and Water. Each bar contains 9 observations. The variability is shown with standard error bars. F igure 411. Peanut allergen reduction across methods in study 2. Each set of bars corresponds to unabraded surface (U) and abraded surface (A). From left to right, the order of treatments was JPA Type 4, CAD, AD, and Water. Each bar contains 9 observations. The variability is shown with standard error bars.

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111 Figure 412. Milk allergen reduction across methods in study 1. Each set of bars corresponds to unabraded surface (U) and abraded surface (A). From left to right, the order of treatments was JPA Type 4, CAD, AD, and Water. Each bar contains 9 observations. The variability is shown with standard error bars. Figure 413. Milk allergen reduction across methods in study 2. Each set of bars corresponds to unabraded surface (U) and abraded surface (A). From left to right, the order of treatments was JPA Type 4, CAD, AD, and Water. Each bar contains 9 observations. The variability is shown with standard error bars.

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112 Figure 414. Egg allergen reduction across methods in study 1. Each set of bars corresponds to unabraded surface (U) and abraded surface (A). From left to right, the order of treatments was JPA Type 4, CAD, AD, and Water. Each bar contains 9 observations. The variability is shown with standard error bars. Figure 415. Egg allergen reduction across methods in study 2. Each set of bars corresponds to unabraded surface (U) and abraded surface (A). From left to right, the order of treatments was JPA Type 4, CAD, AD, and Water. Each bar contains 9 observations. The variability is shown with standard error bars.

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113 CHAPTER 5 CONCL USION Food allergies are a major concern for a segment of consumers and have been on the rise in recent years. The Food Allergen Labeling and Consumer Protection Act, FALCPA, is a law that is currently in place to protect people that suffer from allergies, but their inadvertent or accidental presence in foods still remains a big concern for those severely affected. The only proven method towards preventing reactions lies in the avoidance of food allergens. Therefore, complete allergen removal from food proc essing plants that share equipment for allergen and nonallergen containing products is key. However, present techniques are not 100% effective in preventing aller gen cross contamination, and a consensus on the validation of cleaning protocols has not been reached within the food processing industry. The present study compared four types of washes and two types of surfaces in the removal of peanut, milk, and egg allergens. In general, the JPA Type 4 wash and the CAD wash were the most effective methods for a ll allergens and surfaces. The acid d etergent wash was not effective at all for removing milk allergens (88.0%), and had a fair performance for peanut and egg allergens. In addition, the water only wash performed better than the AD protocol, resulting in 100% reduction for egg allergens and almost 97% removal of milk allergens. The latter results were unexpected, especially since water removed more milk residues than the acid, which is a type of detergent commonly used in the dairy industry for cleaning. O verall percent reductions were calculated for each allergen, where both types of surfaces and the two studies were taken into account. For peanut allergens, both JPA Type 4 and CAD washes had 99.8% reduction, followed by AD and water with 93.6 and 92.6% re duction, respectively. For milk allergens, CAD achieved 99.6% reduction, followed by JPA (98.6%),

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114 water (96.9%), and finally AD (88.0%). Lastly, there was 100% reduction for egg allergens with the JPA, CAD, and water washes. The AD obtained a combined reduction of 93.4%. Based on these results, it is possible to say that both the JPA and the CAD washes were the most effective methods across the board. Water was also very successful at removing egg and milk allergens to a certain extent. Conversely, the AD w as unexpectedly ineffective, especially for milk residues. It is important to keep in mind that efficacy is based on 100% reduction which is needed for complete avoidance. T he majority of the results have an average of 88% reduction and above, but the higher the achieved reduction the lower the chances of someone getting sick from the presence of food allergens. A second objective of this research was to validate the cleaning protocol that proved more successful at removing allergens. From the results obta ined, we could say that for egg allergens all except for the acid detergent worked and could be validated. Even though high levels of reduction were seen for milk and peanut allergens with the CAD and modified JPA protocols, more studies need to be done to ensure reproducibility especially since there was variability in some of the samples for the JPA unabraded in study 1. For the peanut allergens, both the modified JPA and CAD methods seemed to work without so much variability and they could be validated i f further studies are conducted proving these results. The benefits of an effective allergen removal protocol are countless, including prevention of cross contamination, which leads to safer products on the shelves, and ultimately the well being of the end user by avoiding dangerous allergic reactions. The food industry will benefit from these techniques by ensuring allergic consumers that their products do not accidentally contain allergens. Interestingly, this study shows that three common cleaning protoc ols regularly used in the food industry did not yield the same results.

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115 APPENDIX STATISTICAL ANALYSIS First Study Table A 1. Statistical analysis of peanut allergen reduction across methods. A) ANOVA. B) Means by surface. C) Means by treatment. A) The ANOVA Procedure Dependent Variable: peanutconc Sum of Source DF Squares Mean Square F Value Pr > F Model 7 1314953.241 187850.463 40.73 <.0001 Error 64 295150.616 4611.728 Corrected Total 71 1610103.857 R Square Coeff Var Root MSE pe anutconc Mean 0.816688 56.46638 67.90971 120.2657 Source DF Anova SS Mean Square F Value Pr > F surface 1 8688.630 8688.630 1.88 0.1747 treatment 3 1193271.742 397757.247 86.25 <.0001 surface*treatment 3 112992.869 37664.290 8.17 0.0001 B) Duncan's Multiple Range Test for peanutconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 4611.728 Number of Means 2 Critical Range 31.98 Duncan Grouping Mean N surface A 131.25 36 AB A A 109.28 36 UN

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116 C) Duncan's Multiple Range Test for peanutconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 4611.728 Number of Means 2 3 4 Critical Range 45.22 47.57 49.13 D uncan Grouping Mean N treatment A 328.16 18 AD B 126.62 18 W C 19.46 18 JPA C C 6.83 18 CAD Table A 2. Statistical analysis of milk allergen reduction across methods. A) ANOVA. B) Means by surface. C) Means by treatment. A) The ANOVA Procedure Dependent Variable: milkconc Sum of Source DF Squares Mean Square F Value Pr > F Model 7 6942.94871 991.84982 3.74 0.0019 Error 64 16970.98449 265.17163 Corrected Total 71 23913.93320 R Square Coeff Var Root MSE milkconc Mean 0.290331 122.905 1 16.28409 13.24932 Source DF Anova SS Mean Square F Value Pr > F surface 1 89.229214 89.229214 0.34 0.5639 treatment 3 4728.944782 1576.314927 5.94 0.0012 surface*treatment 3 2124.774710 708.258237 2.67 0.0549

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117 B) Duncan's Multiple Range Test for milkconc A lpha 0.05 Error Degrees of Freedom 64 Error Mean Square 265.1716 Number of Means 2 Critical Range 7.668 Duncan Grouping Mean N surface A 14.363 36 UN A A 12.136 36 AB C) Duncan's Multiple Range Test for milkconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 265.1716 Number of Means 2 3 4 Critical Range 10.84 11.41 11.78 Duncan Grouping Mean N treatment A 25.603 18 AD B 13.433 18 W B B 10.949 18 JPA B B 3.012 18 CAD

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118 Table A 3. Statistical analysis of egg allergen reduction across methods. A) ANOVA. B) Means by surface. C) Means by treatment. A) The ANOVA Procedure Dependent Variable: eggconc Sum of Source DF Squares Mean Square F Value Pr > F Model 7 114866.8748 16409.5535 523.59 <.0001 Error 64 2005.7778 31.3403 Corrected Total 71 116872.6526 R Square Coeff Var Root MSE eggconc Mean 0.982838 33.32808 5.598239 16.79736 Source DF Anova SS Mean Square F Value Pr > F surface 1 13480.54633 13480.54633 430.13 <.0001 treatment 3 60944.68950 20314.89650 648.20 <.000 1 surface*treatment 3 40441.63900 13480.54633 430.13 <.0001 B) Duncan's Multiple Range Test for eggconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 31.34028 Number of Means 2 Critical Range 2.636 Duncan Grouping Mean N surface A 30.481 36 AB B 3.114 36 UN

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119 C) Duncan's Multiple Range Test for e ggconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 31.34028 Number of Means 2 3 4 Critical Range 3.728 3.922 4.050 Duncan Grouping Mean N treatment A 67.189 18 AD B 0.000 18 CAD B B 0.000 18 JPA B B 0.000 18 W S econd Study Table A 4. Statisti cal analysis of peanut allergen reduction across methods. ANOVA. B) Means by surface. C) Means by treatment. A) The ANOVA Procedure Sum of Source DF Squares Mean Square F Value Pr > F Model 7 614106.5228 87729.5033 37.51 <.0001 Error 64 149704.6851 2339.1357 Corrected Total 71 763811.2079 R Square Coeff Var Root MSE peanutconc Mean 0.804003 85.51009 48.36461 56.56013 Source DF Anova SS Mean Square F Value Pr > F surface 1 9001.2641 9001.2641 3.85 0.0542 treatment 3 561254.6781 187084.8927 79.98 <.0001 surface*treatment 3 43850.5807 14616.8602 6.25 0.0009

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120 B) Duncan's Multiple Range Test for peanutconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 2339.136 Number of Means 2 Critical Range 22.77 Duncan Grouping Mean N surface A 67.74 36 AB A A 45.38 36 UN C) Duncan's Multiple Range Test for peanutconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 2339.136 Number of Means 2 3 4 Critical Range 32.21 33.88 34.99 Duncan Grouping Mean N treatment A 209.11 18 W B 15.75 18 AD B B 1.38 18 JPA B B 0.00 18 CAD

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121 Table A 5. Statistical analysis of milk allergen r eduction across methods. A) ANOVA. B) Means by surface. C) Means by treatment. A) The ANOVA Procedure Dependent Variable: milkconc Sum of Source DF Squares Mean Square F Value Pr > F Model 7 5060593.339 722941.906 238.69 <.0001 Error 64 193838.520 3028.727 Corrected Total 71 5254431.859 R Square Coeff Var Root MSE milkconc Mean 0.963110 45.94537 55.03387 119.7811 Source DF Anova SS Mean Square F Value Pr > F surface 1 505676.018 505676.018 166.96 <.0001 treatment 3 2938571.223 979523.741 323.41 <.0001 surface*treatment 3 1616346.098 538782.033 177.89 <.0001 B) Duncan's Multiple Range Test for milkconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 3028.727 Number of Means 2 Critical Range 25.91 Duncan Grouping Mean N surface A 203.59 36 AB B 35.98 36 UN

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122 C) Duncan's Multiple Range Test for milkconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 3028.727 Number of Means 2 3 4 Critical Range 36.65 38.55 39.81 Duncan Grouping Mean N treatment A 469.63 18 AD B 9.49 18 W B B 0.00 18 JPA B B 0.00 18 CAD Table A 6. Statistical analysis of egg allergen reduction across methods. A) ANOVA. B) Means by surface. C) Means by treatment. A) The ANOVA Procedure Dependent Variable: eggconc Sum of Source DF Squares Mean Square F Value Pr > F Model 7 78382.79862 11197.54266 576.39 <.0001 Error 64 1243.33591 19.42712 Corrected Total 71 79626.13453 R Square Coeff Var Root MSE eggconc Mean 0.984385 23.21396 4.407621 18.98694 Source DF Anova SS Mean Square F Value Pr > F surface 1 128.48045 128.48045 6.61 0.0125 treatment 3 77868.87682 25956.29227 1336.09 <.0001 surface*treatment 3 385.44135 128.48045 6.61 0.0006

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123 B) Duncan's Multiple Range Test for eggconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 19.42712 Number of Means 2 Critical Range 2.075 Duncan Grouping Mean N surface A 20.323 36 AB B 17.651 36 UN C) Duncan's Multi ple Range Test for eggconc Alpha 0.05 Error Degrees of Freedom 64 Error Mean Square 19.42712 Nu mber of Means 2 3 4 Critical Range 2.935 3.088 3.189 Duncan Grouping Mean N treatment A 75.948 18 AD B 0.000 18 CAD B B 0.000 18 JPA B B 0.000 18 W

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125 Hugget A C and Hischenhuber C. 1998. Food manufacturing initiatives to protect the allergic consumer. Allergy 53 (46): 8992. Immer R. 2006. Factors affecting the effectiveness of allergen detection. Detecting allergens in food: The nature of food allergy pp. 330344. Woodhead Publishing: Cambridge, England. Jackson L S Schlesser J E, Beachman Bowden T, Fu T J, Gendel S M, Moorman M A 2004. Effect of cleaning on removal of peanut allergens from foodcontact surfaces. Book of abstracts. Institute of Food T echnologists Annual Meeting Session 54I 1. Jackson L S, Schlesser J E, Al Taher F, Fu T J, Gendel S M, Moorman M A 2005. Effect of cleaning on removal of milk protein from a stainless steel surface. Book of abstracts. Institute of Food Technologists Annua l Meeting Session 49I. Jackson L S, Al Taher F M, Moorman M, DeVries J W, Tippett R, Swanson K M J, Fu T, Salter R, Dunaif G, Estes S, Albillos S, Gendel S M 2008. Cleaning and other control and validation strategies to prevent allergen cross contact in foodprocessing operations Journal of Food Protection 71 (2): 445458. Jensen J M. 1970. Cleanability of milkfilmed stainless steel by chlorinated detergent solution. J ournal of Dairy Science 53 (2): 248 251. Jones GM. 2001. Cleaning and Sanitizing Milkin g Equipment. Publication 404400. Blacksburg,VA. Virginia Cooperative Extension Virginia Tech. Accessed on October 9th, 2009. www.ext.vt.edu/pubs/dairy/404400/404400/html Juice Products Association. 2008. Model Tanker Wash Guidelines For the Fruit J uice Industry. Washington, D.C. Juice Products Association. June 2008. Accessed on August 20th 2008. www.juiceproducts.org Katsuyama A M, Jantschke M, Gombas D E. 1999. Chemical Hazards and Controls. In: Stevens on KE and Bernard DT, HACCP A Systematic Approach to Food Safety 3rd Ed. Pp.5362. The Food Processors Institute: Washington, DC. Katsuyama A M. 1993. Principles of Food Processing Sanitation 2nd E d. Pp. 540. The Food Processors Institute : Washington, D C. Laoprasert N, Wallen N D, Jones R T, Hefle S L, Taylor S L, Yunginger J W 1998. Anaphylaxis in a milk allergic child following ingestion of lemon sorbet containing trace quantities of milk Journal of Food Protection 61 (11): 15221524. Marriott N G and Gravani R B. 2006. Principles of Food Sanitation 5th Ed. Pp. 413. Springer Science and Media, Inc. : New York City. Matsuda T and Nakamura R. 1993. Molecular structure and immunological properties of food allergens. Trends in Food Science and Technology 4: 289293.

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126 Milledge J J and Jowitt R. 1980. The cleanability of stainless steel used as a food contact surface. Proceedings Institute of Food Science and Technology (U.K.) 13 (1): 5762. Morisset M, Moneret Vautrin D A, Kanny G, Gunard L, Beaudouin E, Flabbe J, Hatahet R 2003. Thresholds of clinical reactivity to milk, egg, peanut, and sesame in immunoglobin E dependent allergies: evaluation by double blind or single blind placebo controlled oral challenges. Clin ical and Exp erimental Allergy 33: 1046.1051. National Institute of Allergy and Infectious Diseases (NIAID). 2008. Food Allergy. Accessed on August 26th 2008. http://www3.niaid.nih.gov/topics/foodAllergy/ understanding/quickFacts.htm Newcomer C. 1999. Controlling allergens in the food manufacturing environment Dairy, Food and Environmental Sanitation: 227, 236. Poms R E, Klein C L, Anklam E. 2004. Methods for allergen analysis in food: a review. Food Addi tives and Contaminants 21 (1): 1 31. Reinemann D J, Wolters G, Rasmussen M D 2000. Review of practices for cleaning and sanitation of milking machines. University of Wisconsin, Madison, WI. Presented at the Pacific Dairy Congress in Nagano, Japan. Sampso n H A. 2004. Update on food allergy. J ournal of Allergy and Clin ical Immunol ogy 113 (5): 805 819. Schlegel V Yong A, Yee Foo S 2007. Development of a direct sampling method for verifying the cleanliness of equipment shared with peanut products. Food Cont rol 18: 14941500. Schmidt R H. 2003. Basic Elements of Equipment Cleaning and Sanitizing in Food Processing and Handling Operations. eIFAS Extension Publication. UF IFAS Extension ePublishing Volume FS14. Accessed on August 26th, 2008. http://edis.ifas.ufl.edu/BODY_FS077 Taylor S. 2006. The nature of food allergy. Detecting allergens in food: The nature of food allergy pp. 3 20. Woodhead Publishing: Cambridge, England. Urisu A, Ando H, Morita Y, Wada E, Y asaki T, Yamada K, Komada K, Torii S, Goto M, Wakamatsu T 1997. Allergenic activity of heated and ovomucoiddepleted egg white. Journal of allergy and clinical immunology 100 (2): 171176. U.S. FDA CFSAN. 2006. Approaches to establish thresholds for major food allergens and for gluten in food: Food Allergy. Accessed on August 20th 2008. http://www.cfsan.fda.gov/~dms/alrgn.html U.S. FDA CFSAN. 2004. 21CFR 110 Current good manufacturing practices in ma nufacture, packaging, or holding human food. Accessed on September 3rd, 2009. http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/CurrentGoodManuf acturingPracticesCGMPs/ucm110907.htm

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127 U.S. FDA CFSAN. 2004. Food Allergen Labeling and Consumer Protection Act of 2004 Public Law 108282. Accessed on October 10th, 2009. www.cfsan.fda.gov/~dms/alrgact.html U.S. FDA CFSA N. 2006. Guidance for industry: Questions and answers regarding food allergens, including the Food Allergen Labeling and Consumer Protection Act of 2004 (Edition 4); Final guidance. Accessed on October 10th, 2009. http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocume nts/FoodLabelingNutrition/ucm059116.htm van Hengel A J. 2007. Food allergen detection methods and the challenge to protect foodallergic consumers. Anal ytical and Bioanal ytical Chem istry 389: 111 118. Wal J M. 1998. Allergy Review Series II. An update on allergens: Cows milk allergens. Allergy 53 (11) : 10131022. Wal J M. 2001. Structure and function of milk allergens. Allergy 56 (67): 3538. Watrous GH. 1975. Food soils, water hardness, and alkaline cleaner formulations. Journal of Milk Food Technology 38(3):163 165. Zayas J F. 1997. Functionality of proteins in food. Pp 373. Springer V erlag: Berlin, Germany.

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128 BIOGRAPHICAL SKETCH Yael Spektor was born in 1985 in Montevideo, Uruguay. After moving to the U.S. in 2003, she attended the Univer sity of Florida from 2004 2007 and graduated Summa cum Laude with her B.S. in Food Science. Following her graduation, Yael was offered an assistantship at the University of Florida to pursue her m aster s degree in Food Science, under the supervision of Dr. Rene Goodrich. In the summer of 2009, Yael obtained an internship with Coca Cola North America at their Juice R&D Department in Apopka, FL, where she gained invaluable experience about the business. In December 2009, she graduated with her M.S. in Food Science and she also received a minor in Packaging Sciences. During the course of her graduate degree Yael was the President of the Florida Association for Food Protection (FAFP) Gator Chapter. She was also an avid member of the Food Science and Human Nutrition club, where she served as Treasurer and FL Section IFT Ambassador. Yael has been an active leader and member of the Product Development Competition, and has participated in the Annual Collegiate Dairy Products Evaluation Contest and College Bowl teams. She has also been an active member in organizations in her field, such as the Institute of Food Technologists ( IFT) since 2006, and the International A ssociation for F ood P rotection (IAFP), since 2008.