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1 EFFECT OF PULSED U LTRA V IOLET LIGHT O N THE ANTIGENICITY OF WHOLE SHR IMP HALF CUT SHRIMP AND SHRIMP PROTEIN EXTRACT By SYED ALI SHAMIKH ABBAS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 S yed Ali Shamikh Abbas
3 To those that suffer from shrimp allergy
4 ACKNOWLEDGMENTS Several people deserve recognition in helping me throughout graduate school and offering suggestion and input along the way. First, I would like to thank my major advis never ending support. He introduced me to this field and provided valuable input from the beginning. I would also like to thank my committee members, Dr. Melanie Correll and Dr. Renee Goodrich Schneider, for all of their helpful input and their time. Second, I would like to ackno her dedication and commitment and many hours of sacrifice. I would also like to acknowledge my lab mates for helping me from the beginning and always going over and beyond what one would expect. I d like to give my thanks t o : Akshay Anugu, Tara Faidhalla, Senem Guner, and Cheryl Rock Last, but not least, I would like to acknowledge my family consisting of Mu zaffar Abba s, my dad, and Tahseen Muz a ffar, my mom, and my siblings Al eena Muzaffar, Areesha Muzaffar, and Mohammad Ali Abbas, for all of their generous help and guidance. All of these individuals are fantastic individuals who made everything possible and provided m any memorable moments in graduate school.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 1 1 CHAPTERS 1 INTRODUCTION ................................ ................................ ................................ .... 14 2 OBJECTIVES ................................ ................................ ................................ ......... 19 3 JUSTIFICATION ................................ ................................ ................................ ..... 20 4 LITERATURE REVIEW ................................ ................................ .......................... 21 Protein Biology and Chemistry ................................ ................................ ................ 22 Food Allergens ................................ ................................ ................................ ........ 23 Food Allergy ................................ ................................ ................................ ............ 24 Methods Used For Detecting Food Allergy ................................ ............................. 26 In Vivo Methods ................................ ................................ ................................ 26 Skin Prick Test (SPT) ................................ ................................ ................. 27 Oral F ood Challenge (OFC) ................................ ................................ ....... 27 In Vitro Methods ................................ ................................ ............................... 28 RAST and EAST ................................ ................................ ........................ 28 ImmunoCAP ................................ ................................ ............................... 28 SDS PAGE ................................ ................................ ................................ 28 Immunoblotting ................................ ................................ .......................... 29 ELISA ................................ ................................ ................................ ......... 30 Food Allergen Mitigation ................................ ................................ ......................... 31 Thermal Processing ................................ ................................ ......................... 31 Moist Heat ................................ ................................ ................................ .. 34 Dry Heat ................................ ................................ ................................ ..... 38 Non Thermal Processing ................................ ................................ .................. 40 Pulsed Light ................................ ................................ ............................... 41 Gamma Irradiation ................................ ................................ ..................... 42 High Intensity Ultrasound ................................ ................................ ........... 44 High Pressure Processing ................................ ................................ .......... 45 Genetic Modification ................................ ................................ .................. 46
6 5 MATE RIALS AND METHODS ................................ ................................ ................ 47 Human Plasma Samples ................................ ................................ ........................ 47 Preparation of Protein Extract ................................ ................................ ................. 47 Preparation of Whole Shrimp ................................ ................................ .................. 48 Preparation of Half Cut Shrimp ................................ ................................ ............... 48 Pulsed Ultraviolet Light Treatment ................................ ................................ .......... 49 Temperature Measurement ................................ ................................ .................... 50 Post Processing Preparation ................................ ................................ .................. 50 Protein Concentration Measurements ................................ ................................ ..... 51 SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) .............. 51 Western Blot Analysis ................................ ................................ ............................. 52 Indirect ELISA ................................ ................................ ................................ ......... 54 Statistical Analysis ................................ ................................ ................................ .. 55 6 RESULTS AND DISCUSSION ................................ ................................ ............... 56 Protein Extracts ................................ ................................ ................................ ...... 56 SDS PAGE Analysis of Protein Extracts ................................ .......................... 56 Western Blot for Protein Extracts ................................ ................................ ..... 57 Indirect ELISA for Protein Extracts ................................ ................................ ... 60 Whole Shrimp ................................ ................................ ................................ ......... 62 SDS PAGE of Whole Shrimp ................................ ................................ ........... 62 Western Blot for Whole Shrimp ................................ ................................ ........ 64 Indirect ELISA for Whole Shrimp ................................ ................................ ...... 67 Sensory Remarks for Whole Shrimp ................................ ................................ 68 Half Cut Shrimp ................................ ................................ ................................ ...... 73 SDS PAGE of Half Cut Shrimp ................................ ................................ ......... 73 Western Blot for Half Cut Shrimp ................................ ................................ ..... 75 Sensory Remarks for Half Cut Shrimp ................................ ............................. 76 Other Remarks ................................ ................................ ................................ ....... 80 Optimization of Pulsed Light Illumination ................................ .......................... 80 Temperature and Moisture Change ................................ ................................ .. 80 Antigenicity Variations Caused by Other Processing Methods on Shrimp ....... 82 7 CONCLUSIONS ................................ ................................ ................................ ..... 85 8 FUTURE WORK ................................ ................................ ................................ ..... 87 9 PRELIMINARY RESULTS ................................ ................................ ...................... 88 LIST O F REFERENCES ................................ ................................ ............................... 92 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 102
7 LIST OF TABLES Table page 6 1 Densimetric analysis of Western blot samples for protein extract from ImageJ 1.44 software (NIH). ................................ ................................ ........................... 60 6 2 Densimetric analysis of Western blot samples for whole shrimp from ImageJ 1.44 software (NIH). ................................ ................................ ........................... 67 6 3 Densimetric analysis of Western blot samples for half cut shrimp from ImageJ 1.44 software (NIH). ................................ ................................ ............... 76 6 4 Temperature measurements for whole shrimp samples. Each observation is a mean of three temperatures and the standard deviation. ................................ 81 6 5 Temperature measurements for half cut samples. Each observation is a mean of three temperatures and the standard deviation. ................................ ... 82 6 6 Representative moisture loss measurements for whole shrimp samples. Weight is measured in grams (g). ................................ ................................ ....... 82 6 7 Representative moisture loss measurements for half cut shrimp samples. Weight is measured in grams (g). ................................ ................................ ....... 82
8 LIST OF FIGURES Figure page 5 1 Xenon PL processor (Model LHS40 LMP HSG ). ................................ ................ 50 5 2 Summary of experimental design for protein extract, whole shrimp, and half cut shrimp. ................................ ................................ ................................ .......... 55 6 1 SDS PAGE analysis of protein profile comparing 1) raw shrimp extract sample (R) and 2) pulsed light treated shrimp extract samples, with times of 2 min, 4 min, 5 min, 6 min, and 8 min. 15 uL was added to each well with concentrations of 1:50 for the R and 1:30 for the other samples. Molecular weight marker (M) is shown on the left. Tropomyosin is pointed as 37 kDa. ...... 57 6 2 Western blot analysis of protein extract samples including 1) raw sample (R) 2) boiled sample (B) 3) pulsed light treated sample with time points of 2 min, 4 min, 5 min, 6 min, and 8 min. ................................ ................................ ........... 59 6 3 Indirect ELISA illustrated the changes in IgE binding compared to untreated, boiled, PL treated shrimp protein extracts using pooled h uman plasma containing IgE antibodies against shrimp. The times correspond to the treatment time and a control was used. A = absorbance of the sample; A 0 = absorbance of untreated sample. Data are expressed as mean SEM (n = 5). Results are relative value s, normalized to the untreated sample; untreated is standardized and set to 1. Values that are significantly different are ................................ ................................ ... 61 6 4 SDS PAGE analysis of protein profile of whole shrimp samples comparing 1) raw sample (R) 2) boiled sample, and 3) pulsed light treated whole shrimp samples, with times of 6 min, 8 min, 10 min, 12 min, and 15 min. The se times correspond to pulsed light treatment on one side of whole shrimp, with their being an application of treatment of both sides. 15 uL was added to each well with concentrations of 1:50 for the R and 1:10 for the other samples. Molecular weight marke r (M) is shown on the left. Tropomyosin is pointed as 37 kDa. ................................ ................................ ................................ ............... 64 6 5 Western blot analysis of whole shrimp samples including 1) raw sample (R) 2) boiled sample (B), 3) pulsed light treated sample with time points of 6 min, 8 min, 10 min, 12 min, and 15 min. These times correspond to pulsed light treatment on one side of whole shrimp, with their being an application of treatment of both sides. ................................ ................................ ...................... 66 6 6 Indirect ELISA illustrated the changes in IgE binding compared to untreated, boiled, PL treated whole shrimp extracts using pooled human plasma containing IgE antibodies against shrimp. The times correspond to the treatment time and a control was used. A = absorbance of the sample; A0 = absorbance of untreated sample. Data are expressed as mean SEM (n =
9 5). Results are relative values, normalized to the untreated sample; untreated is standardized and set t o 1. Values that are significantly different are ................................ ................................ ... 68 6 7 Raw shrimp sample was taken as a c ontrol. This raw sample was applied in following experiments in SDS PAGE, Western blot, and indirect ELISA. ........... 71 6 8 Boiled shrimp sample was considered as another control. This sample was prepared with a raw sample that was boiled in distilled water for 10 min. .......... 71 6 9 Whole shrimp sample was treated by pulsed light for 6 min on both sides for a total treatment of 12 min. ................................ ................................ ................. 71 6 10 Whole shrimp sample was treated by pulsed light for 8 min on both sides for a total treatment of 16 min. ................................ ................................ ................. 72 6 11 Whole shrimp sample was treated by pulsed light for 10 min on both sides for a total treatment of 20 min. ................................ ................................ ................. 72 6 12 Whole shrimp sample was treated by pulsed light for 12 min on both sides for a total treatment of 24 min. ................................ ................................ ................. 72 6 13 Whole shrimp sample was treated by pulsed light for 15 min on both sides for a total treatment of 30 min. ................................ ................................ ................. 73 6 14 SDS PAGE analysis of protein profile of half cut shrimp comparing 1) raw shrimp (R), 2) Boiled shrimp (B), and 3) pulsed light treated shrimp samples, with times of 6 min, 8 min, 10 min, 12 min, and 15 min. These times correspond to pulsed light treatment on one side of whole shrimp, with their being an application of treatment of both sides. 15 uL was added to each well with concentrations of 1:50 for the R and 1:10 for the other samples. Molecular weight marker (Std) is shown on the left. Tropomyosin is pointed as 37 kDa. ................................ ................................ ................................ .......... 74 6 15 Western blot analysis of half cut shrimp samples including 1) raw sample (R), 2) boiled sample (B), and 3) pulsed light treated sample with tim e points of 6 min, 8 min, 10 min, 12 min, and 15 min. These times correspond to pulsed light treatment on one side of whole shrimp, with their being an application of treatment of both sides. ................................ ................................ ...................... 75 6 16 Raw shrimp sample for the half cut shrimp sample. This sample was used in SDS PAGE and Western blot. ................................ ................................ ............ 78 6 17 Half cut shrimp was treated by pulsed light for 6 min on both sides for a total treatment of 12 min. ................................ ................................ ............................ 78 6 18 Half cut shrimp was treated by pulsed light for 8 min on both sides for a total treatment of 16 min. ................................ ................................ ............................ 78
10 6 19 Half cut shrimp sample was treated by pulsed light for 10 min on both sides for a total treatment of 20 min. ................................ ................................ ............ 79 6 20 Half cut shrimp sample was treated by pulsed light for 12 min on both sides for a total treatment of 24 min. ................................ ................................ ............ 79 6 21 Half cut shrimp sample was treated by pulsed light for 15 min on both sides for a total treatment of 30 min. ................................ ................................ ............ 79 9 1 SDS PAGE of analysis of protein profile of ground shrimp samples comparing 1) raw sample (R), and 2) pulsed light treated ground shrimp samples, with time s of 8 min, 10 min, 12 min, and 15 min. 15 uL was added to each well with concentrations of 1:50 for the R and 1:10 for the other samples. Molecular weight marker (M) is shown on the left. Tropomyosin is pointed as 37 kDa. ................................ ................................ .............................. 89 9 2 SDS PAGE analysis of protein profile analysis of ground shrimp samples comparing 1) raw sample (R) and 2) pulsed light treated ground shrimp samples, with t imes of 8 min and 15 min. 15 uL was added to each well with concentrations of 1:50 for the R and 1:10 for the other samples. Molecular weight marker (M) is shown on the left. Tropomyosin is pointed as 37 kDa. ...... 90
11 LIST OF ABBREVIATIONS BSA Bovine serum albumin EIA Enzyme immunoassay ELISA Enzyme linked immunosorbent assay IgE Immunoglobulin E IgG Immunoglobulin G OPD O phenylenediamine dihydrochloride P L Pulsed ultraviolet light PVDF P olyvinylidene fluoride SDS PAGE Sodium dodecyl sulfate polyacrylamide electrophoresis
12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Ma ster of Science EFFECT OF PULSED U LTRAVIOLET LIGHT ON THE ANTIGENICITY OF WHOLE SHRIMP, HALF CUT SHRIMP, AND SHRIMP PROTEIN EXTRACT By S yed Ali Shamikh Abbas August 2012 Chair: Major: Food Science and Human Nutrition Tropomyosin is responsible for triggering immune responses in individuals allergic to shrimp. Although allergens can manifest themselves at an early age and continue to adulthood, with sensitivity decreasing as one ages, there is no current cure for indivi duals affected with shrimp allergy except total avoidance of the food. Current developments in non thermal processing, such as pulsed ultraviolet light (P L ), show promise as they have been found to reduce the tropomyosin antigenicity following treatment. However, more research needs to be conducted to evaluate the potential P L effect on shrimp The main objective of this study is to examine the effect of PL on tropomyosin antigenicity on shrimp native protein extract, whole shrimp and half cut shrimp. The samples were treated at a distance of 10 centimeters ( cm ) from the quartz window of the Xenon PL processor (Model LHS40 LMP HSG ). 10 milliliters ( mL ) of protein extract was treated for 2, 4, 5, 6, and 8 minutes ( min ) and both w hole shrimp and half cut shri mp were treated for 6, 8, 10, 12, and 15 min on both sides. SDS PAGE ( sodium dodecyl sulfate polyacrylamide electrophoresis ) was performed to illustrate the electrophoretic profile of tropomyosin, which showed reduction in band intensity of tropomyosin in the protein extract. However, there was no noticeable effect on
13 tropomyosin in whole shrimp and half cut shrimp under the PL conditions treated in this study Th e antigenicity response is the same in Western blot and indirect ELISA ( enzyme linked immunoso rbent assay) Th e results demonstrate both the applica bility and limitation of PL in treating allergenic shrimp samples Future work would be needed to evaluate the commercial potentiality of this technology in developing hypoallergenic shrimp products.
14 CHAPTER 1 INTRODUCTION Allergies t o food are a problem that affects millions of people worldwide when certain foods containing allergens produce an adverse immune response once consumed. Although the allergenic proteins are non toxic, individuals that are allergic produce high levels of antibodies, or immunoglob ulins, that recognize the sites called epitopes on these proteins and elicit an immunological reaction. Starting at birth, this type of hypersensitivity can progress until adulthood, although sensitization can decrease as the age increases ( Ayuso and others 2010 ) The symptoms due to allergen exposure can be minor and may include itching or coughing and possibly fatality, if serious reactions like anaphylactic shock occur. For this reason, persons with food allergies only have one method for dealing with this problem, and that is compl ete avoidance of the allergenic food. The fundamental mechanism behind the allergenic responses is that the body contains immunoglobulins that function to bind and remove foreign matter. nse when a specific protein sequence is present in the body. T he se protein sequences constitute part of the protein in food and trigger antibodies in the body to recognize these sequences as foreign and mediate an immune response, even though no harmful ag ent enters the body Several types of antibodies exist. These include Immunoglobulin E (IgE), IgG, IgM, and IgA, of which all are present in both allergic and non allergic individuals ( Daul and others 1990 ) However, the levels of IgE are much higher in individuals exhibiting allergenic reactions, suggesting that IgE plays an important role with respect to food hypersensitivity ( Daul and others 1987 )
15 B iological techniques as well as novel technologies are being developed which include genetic modification of plant fo ods and the use of emerging technologies such as irradiation and pulsed light (PL) that may be potentially used to eliminate or reduce the target allergen in the food. One particular technology and the focus of this research, PL, refer s to the light spect ra ranging from 1 70 to 26 00 nanometers (nm) (Fellows 2009). It comprises approximately 54% UV light, 26% visible light, and 20% infrared when emitted ( Oms Oliu and others 2010 ) Within the UV light fraction of the PL, there are further subdivisions of several spectra including UV A, UV B, and UV C (200 280 nm) UV C is the largest spectrum and constitutes a peak power of 254 nm. Several studies have shown that PL can be used for different treatments, such as the decontamination of foods ( Gmez Lpez and others 2007 ) and also inactivation of allergens in shrimp extract ( Shriver and others 2011 ) egg proteins ( Anugu and others 20 10 ) and wheat gluten ( Nooji and others 2010 ) There are three main mechanisms proposed for PL interactions with foods: p hoto thermal, photo chemical, and photo physical effects ( Gmez Lpez et al. 2007 ; Shriver and Yang 2011 ; Shriver et al. 2011 ) In the photo thermal effect, heat is generated from both the infrared and the visible parts of the PL spectrum, which causes the top layer of the food to heat if enough energy is emitted (Fellows 2009). However, this phenomenon is not as effective as the photo chemical or photo physical ef fect when the treatment time is relatively s hort (i.e. seconds) since it has been shown that inactivation of microorganisms can occur without an increase in the temperature of the food. In the photo physical effect, microbial inactivation results from the rupture of the cell and release of the cellular contents Lastly the photo chemical effect has been reported to
16 initiate several modifications such as changing the structure of the allergenic proteins, or creating pyrimidine dimers in DNA, which inhibits DNA replication, resulting in the inactivation of microorganisms ( Sizer and Balasubramaniam 1999 ) In conventional equipment of generating continuous UV light, the light is generated with a low pressure mercury vapor lamp (Fellows 2009), whereas for PL, electricity is stored in capacitors and then released in a shorter duration at thousandths of a second into a lamp containing Xenon or Krypton inert ga s ( Gmez Lpez et al. 2007 ; Shriver et al. 2011 ) T his allows the power of the light to intensify and so when the light is emitted from the lamp, it is one of high energy and intensity. As an allergen contains epitope sites for immunoglobulin binding, the PL can alter those sites through mechanisms such as protein denaturation, modification of the protein structure, and protein hydrolysis to change the IgE binding sites. Currently, the F ood a nd Drug Administration lists eight major allergenic foods, which are soy, tree nut, peanut, milk, eggs, wheat, fish, and shellfish Of these, shellf ish are the most common and also accoun t for the most emergency visits ( Clark and others 2004 ) Shellfish include arthropods like lobsters, shrimp, and crab, all of which have a chitinous exoskeleton and dwell in aquatic environments ( Nagpal and others 1989 ) In shrimp, the major compound responsible for causing immunoreactivity in shrimp sensitive individuals is a protein called tropomyosin ( Shanti and others 1993 ) It can also referred to as Pen a 1 Pen m 1, and Lit v 1 depending on the shrimp species ( Ayuso and others 2008 ) Other proteins are arginine kinase, myosin light chain, and sarcoplasmic calcium binding protein, which have been associa ted as shrimp allergens ( Ayuso et al. 2010 ) However, tropomyosin is regarded as the major protein responsible
17 for the majority of allergenic reactions ( Ayuso et al. 2008 ) The weight of the protein is 34 to 38 kiloDaltons (kDa). It is also present in other species too Therefore, cross reactivity can occur. For example, there is cross reactivity between the shrimp tropomy osin and the mite tropomyosin as the amino acid sequence between the two proteins shares an 81% homologous sequence ( Boquete and others 2011 ) This is a problem as individuals that suffer from shrimp allergens also can exhibit symptoms due to mites. Other species that contain tropomyosin are cockroaches and parasites ( Yang and others 2010a ) As a result, several stu dies have focused on changing the protein composition through recombinant technology or altering the protein's structure using other food processing methods, such as PL. Based on a previous study ( Shriver et al. 2011 ) PL light is shown to be effective in reducing the binding of the IgE antibody in shrimp extracts. However, there is no reported resear ch of PL light on whole shrimp. Due to the proposed photo thermal, photo chemical, and photo physical effects of PL, if the whole shrimp, rather than shrimp extracts, could be cooked with the allergen minimized during the PL treatment, it would have a sign ificant impact on public health as well as the technological development for PL The PL technology for simultaneous cooking and allergen reduction is desirable in the food industry as shrimp is consumed mostly as whole. Furthermore, further evaluation of the native tropomyosin protein has not been conducted and is also important, as ongoing research demonstrates that PL is possibly able to alter protein structure. And last, within the limitation of PL to only penetrate the surface of foods, experiments were conducted to in cut shrimp. All of these experiments will illustrate the interaction of PL technology on the
18 tropomyosin protein as it is available either as a protein extract or in a natural state in the different forms of the shrimp sample.
19 CHAPTER 2 OBJECTIVES The overall objective of this research was to examine allergen reduction on the native tropomyosin protein in shrimp treated with PL. A s part of this objective, the effects of PL to on allergen reduction for the whole shrimp and half cut shrimp samples were compared This objective constitutes the foundation of this research and is extended from previous studies on shrimp extracts ( Shriver et al. 2011 ) Th is earlier study had demonstrated that IgE binding to epitope sites decr eased after PL treatment on the shrimp protein. Other studies have also shown allergen reduction in other foods such as milk ( Anugu and others 2009 ) wheat gluten ( Nooji et al. 2010 ) soybeans ( Yang and others 2010b ) and peanuts ( Shriver and Yang 2011 ) Therefore, this research aims to conduct the treatment to the whole and half cut shrimp along with further assessment of the native tropomyosin protein extracts to examine the antigenicity of the shrimp sampl es of different forms after PL illumination The shrimp species used is the Litopenaeus setiferus also known as the Atlantic white shrimp Antigenicity was measured through SDS PAGE, Western blotting, and indirect ELISA, wh ere each analysis serv ed the purpose of indicating protein separation, detecting antibody reactivity with the major shrimp allergen, tropomyosin, and antibody reactivity with all proteins, respectively. Different durations of PL were used to provide various exposure times to PL t
20 CHAPTER 3 JUSTIFICATION According to the National Oceanic and Atmospheric Administration (NOAA), shrimp is the most commonly consumed seafood. It is estimated that 4.1 pounds ( lbs ) of shrimp are consumed per c apita in the United States (NOAA 2010). Several varieties are available and there is a large market for shrimp Therefore, having a hypoallergenic shrimp product would be very attractive to the seafood industry and could expand the consumer options in food choices. Additionally, it would allow for novel food products to be developed, since shrimp could be used as an ingredient in other food products. Furthermore, 1 in 50 Americans are estimated to have shellfish allergy, most of which are caused by prawns a nd shrimp ( Ayuso et al. 2008 ) Although no treatment is currently available for allergies, emerging technologies like PL have shown positive outcomes for allergenic foods such as peanut and milk. Moreover, in a p revious study ( Shriver et al. 2011 ) conducted in the Food Processing and Engineering L aboratory at the University of Florida, the effects of PL on shrimp extracts gave positive results in that pulsed light treatment could reduce the antigenicity of tropomyosin extract. However, more research is required to illustrate and determine the applicabi lity of PL to creating a hypoallergenic shrimp product This could translate into a multitude of beneficial implications, economically and public health wise. With successful development, this technology potentially could be used in commercial processing to cr eate hypoallergenic cooked shrimp.
21 CHAPTER 4 LITERATURE REVIEW In order to better understand allergens and allergie s, a literature review is provided that describes the basics of allergens and allergic responses and the current methods used to diagnose allergies and treat ment of allergenic foods so that they trigger a reduced allergen reactivity This review is broken down into four parts. First, a description of the biology and chemistry of the proteins is given to help clarify the protein nature, with an emphasis on allergenic proteins. Second, the differences between the terms allergen and allergy are defined. Third, the methods used for detecting food allergens are mentioned. These methods include both in vivo and in vitro testing. In vivo testing adverts to testing on a living organism while in vitro testing concerns itself with testing outside a living organism, s uch as in an environment defined by the researcher. The environment is designed so that it simulates the actual component of the living organism. Last, a selective summary is provided that shows the current research regarding the treatment of allergenic fo ods using both thermal and non thermal treatments. The purpose is to highlight the different treatments that exist and also the variability of results from these different treatments The results can demonstrate either an increase or decrease in allergen r eactivity or give mixed results due to the complex food matrix and protein changes. Under the broader scope of allergenic foods, different foods are mentioned, but emphasis is given to current shrimp allergen treatments. In summary, this review will establ ish a background that will help with understanding and analyzing food allergen research, especially for shrimp allergens.
22 Protein Biology and Chemistry Antigenic compounds are simply proteins. Proteins are made up of monomers of compounds called amino aci ds. These monomers contain an amino group, an alpha carbon that is bonded to a hydrogen atom and an R group, and a carboxyl group. The R group can be any other compound that helps designate the amino acid, serving to define its structure and function. Link ed together through a condensation reaction that removes a water molecule, these amino acids make up the polymeric protein. Such a bond is called a peptide bond ( Berg and others 2007 ) As these amino acids combine together, the structure of the protein begins to form. Four levels of structure exist. The prima ry structure refers to the order, or sequence, of amino acids linked together. The secondary structure is the shape of the sequence, which can either be an alpha helix or the beta pleated sheet where the sequence of amino acids folds on itself. Hydrogen bo nds help to retain this structure. Next, the tertiary structure is the 3D shape that the protein takes on. Many kinds of interactions are present here, including hydrogen bonds, ionic bonds, van der Waals interactions, and disulfide bridges. Last, if multi ple polypeptides combine together, they can form the quaternary structure to give a unique structure. This last level of structure is not found in all proteins and similar interactions such as those for tertiary structure are present ( Berg et al. 2007 ) Despite the chemical bonds holding a protein structure toget her the protein can be denatured, and go from a hierarchy of higher protein structures, such as quaternary more primitive structures such as short chains of amino acids. High temperatures, such as those in boiling, can break down hydrogen bonds, acid base reactions can disrupt
23 ionic bonds, and proteases, which are also proteins, can break down peptide bonds. When the protein is denatured, this can result in several changes in that as solubility is decreased, allergenic activity may be destroyed, created, or kept the same, thus affecting digestibility ( Berg et al. 2007 ) Food Allergens A food allergen is a protein found in the matrix of foods. Food allergens are also known as antigens and elicit an abnormal autoimmune respons e in the body when individuals that are sensitive to the protein ingest it. If the allergen is not denatured and retains its form through out digestion, antibodies in the body are produced to bind to the allergen and trigger an immune response. Allergens ar e usually found as glycoproteins. Their molecular weights range from 10 70 kDa and they are water soluble ( Ebo and Stevens 2001 ) In the human body there are several antibodies that can be produced, but the antibody typically responsible for the majority of food allergies is an immunoglobu lin antibody called Immunoglobulin E, abbreviated IgE. When the antibody binds to the antigen, it causes the release of an inflammatory response that releases mediators such as histamines and cytokines ( Kabourek a nd Taylor 2003 ) The region where the allergen, or antigen, binds to the antibody is called the epitope. Epitopes are sequences of amino acids that can either exist either in a linear fashion along the protein or be found as conformational epitopes that exist when the protein folds into a 3 dimensional shape ( Tanabe 2007 ) For an antibody to bind to these epitopes, the allergen must maintain the stability of that linear or conformational epitope. However, l inear epitopes can be modified through fragmentation or genetic modification while conformational epitopes can be
24 modified through partial denaturation crosslinking, chemical modification or aggregation The former involves changes in the sequence of the amino acids while the latter involves structural changes to the 3 dimensional protein ( Sathe and others 2005 ) Therefore, several methods have focused on changing the allergenic protein so that if there is an inhibition of IgE binding, then that would lead to a reduced or eliminated allergenic response. Food Allergy As for food allergies, i n order to better understand them it is better to define a few terms. First, a food allergy falls under the broader scope of adverse food reactions. An adverse food reaction is any abnormal response that results from consumption of a food or food additive. The reaction can be toxic, such as poisoning, or non toxic, where the response is due to hypersen sitivities or food intolerance respectively With respect to mediated or non IgE mediated response to the foreign antigen present in the body. On the other hand, an individual with f ood intolerance is not able to properly digest the food because example is the inability to break down the milk sugar lactose since some individuals do not have the necessa ry lactase enzyme to break the sugar down, hence the term lactose intolerant. For the purpose of this review, the focus is on food hypersensitivities that are IgE mediated since these are prevalent food allergies, are better understood, and are relevant to the PL treatment on shrimp tropomyosin allergen. In the human diet, there are several different foods that one can consume. However, despite this, only a select few foods cause the majority of food induced allergies. These select few foods are the eight m ajor allergenic foods: soy, tree nut,
25 peanut, milk, eggs, wheat, fish, and shellfish. For children, the common allergenic foods are milk, eggs, peanuts, soy and wheat and cause 90% of the reactions For adults, the common culprits are peanuts, tree nuts, f ish, and shellfish and cause 85% of the reactions ( Sampson 1999b ; Sampson 1999a ) Starting at birth, infants are more predispo sed to food allergies than adults. Several symptoms can develop upon ingestion of the allergenic protein. These symptoms form as the gastrointestinal tract allow the passage of these proteins, or antigens, into the body, which are then disseminated to different organs ( Walzer 1942 ) T hese symptoms can either occur immediately within minutes or may have a longer onset, such as in hours. Furthermore, the severity of the symptoms can vary among individual to individual. In order to separate the potpourri of symptoms, these symptoms can b e categorized into cutaneous disorders, respiratory disorders, gastrointestinal disorders, and system wide disorders such as anaphylactic shock. In cutaneous disorders, it can lead to urticaria and angioedema; for gastrointestinal disorders, symptoms inclu de diarrhea, nausea, vomiting, abdominal pain and colic; for respiratory disorders, individuals can suffer from asthma, rhinoconjunctivitis, and bronchospasm;
26 and lastly, there is anaphylactic shock ( Sampson 1999a ) The latter is the most lethal and can lead to fatality. Anaphyla ctic shock is when a series of allergenic reactions can occur throughout the body, from itchiness to breathing problems to shock to ultimately death. In fact, more patients are admitted to hospitals for anaphylaxis that stem from food allergies as opposed to other sources of anaphylaxis ( Yocum and Khan 1994 ) To treat these reactions, sensitized in dividuals can use injections of epinephrine, antihistamines, or other treatments that help alleviate these symptoms ( Clark et al. 2004 ) Devices such as EpiPen or Twinject offer portable injectors that can help suppress these reactions. Immunotherapy is also an option. The individual is subjected to increasing doses of the antigen over a period of several years in order to help the s antigen so that the immune response is weaker with time. This however may not be effective for all individuals. Lastly, the best way to avoid having an allergenic response is to completely avoid the food. However, since every individual can respond diffe rent to an allergen, it is important that they recognize the degree of their sensitivity to the allergen. Many foods contain trace amounts of allergens either from their use as an ingredient or residue left behind from lack of equipment sanitation. Therefo re, people that are extremely sensitive to even these minuscule amounts can trigger autoimmune reactions. Methods Used For Detecting Food Allergy In Vivo Methods For in vivo detection, the skin prick test (SPT) and the oral food challenge (OFC) are used.
27 Skin Prick Test (SPT) Regarding the skin prick test, t his test is used to determine whether a patient has or does not have an allergy. First, the evaluator must prepare a pure extract of the a needle or pin. If a small red circle called a wheal forms and its diameter is greater than 3 millimeters ( mm ) as compared to the control, the patient is considered positive for the allergen. However, a rash, urticaria, or worse, anaphylaxis can form Furthermore, patients that have atopic dermatitis can give false positive results and each evaluation is not the same from one evaluator to th e next due to subjective testing ( Poulsen 2001 ) Oral Food Challenge (OFC) A more reliable method for detecting a food allergy is the oral food challenge. The oral food challenge is considered to be the gold standard of food allergy tests. Although not 100% reliable due to the occurrence of false positives and false negatives that may exist, the test is much more reliable than other diagnostic tests. The patient is given a capsule of foods that cause allergy and placebos that several hours or even days, and the patient is observed of any reactions during this time. Patients that would s uffer from anaphylaxis are excluded from these studies since there is direct exposure to the allergen. However, at times, some patients may exert serious reactions. That was tested by ( Perry and others 2004 ) who found that in this type of testing, 28% of those tested did give off serious reactions. Furthermore, increasing dosages can be given to see if a reaction occurs after a certain threshold. However, all this can be time consuming, expensive, and require different types of foods so they can be difficult to administer A variation of the test is double blind
2 8 placebo controlled food challenge (DBPCFC) where both th e researcher and the patient do not know what is given. In Vitro Methods For the in vitro detection, RAST (radioallergosorbent test) and EAST (enzyme allergosorbent test) are used along with ImmunoCAP testing. Furthermore, there is also SDS PAGE, immunoblotting, and ELISA. The last three were used in this experiment RAST and EAST RAST and EAST stand for radio allergosorbent and enzyme allergosorbent t ests, respectively. RAST in vitro incubated with the allergen bound to a solid phase ( Besler 2001 ) A secondary antibody is added to bind with the primary antibody. The secondary antibody is also bound to a rad ioisotope like I 125 and this helps to quantify the allergen by measuring radioactivity through a standard curve ( Falagiani and others 1994 ) With EAST, enzymes (i.e. alkaline phosphatase) instead of radioisotopes are used and there is measurement of enzyme activity instead. ImmunoCAP The ImmunoCAP test use s the same concept of RAST and EAST tests with a few differences. First, a 3D solid phase is used to help prevent binding of non specific non IgE antibodies. And second, these tests also include more conformational epitopes ( Hamilton and Williams 2010 ) These tests do not take much time and can be done in 20 minutes and they report their units in kilo units per liter (kU / L). SDS PAGE SDS PAGE stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis. The test detects which proteins are present in a sample by separating
29 them into a gel ( Shapiro and others 1967 ) The limitations of SDS PAGE are that large proteins will not pass through the gel and small proteins, or small fragments of proteins after treatment, will pass through t he gel into the buffer without being detected. Smeared bands can also form if protein cross linking occurs, in which the proteins does not pass through the gel in a linear matter because of the different proteins are attached together ( Taheri Kafrani and others 2009 ) Although this test can separate the proteins in a sample, it will not show if the allergens are reactive to the antibodies. All it can show if the allergen of interest is present or not. To show if the allergens are reactive to the antibodies immunoblotting and ELISA methods ar e required. Immunoblotting Immunoblotting consists of the Wes tern blotting and dot blotting ( Towbin and others 1979 ) Western blotting The first step to any immunoblotting is the gel electrophoresis. It is the same gel electrophoresis used for SDS PAGE. However, instead of staining it to see the visible bands, the gel is taken instead and then the proteins are transferred to membrane made of either polyvinylide fluoride (PVDF) or nitrocellulose. The protein on this membrane is then incubated with the primary antibodies followed by the secondary antibodies. There are several washes in between the loading of the primary and secondary antibod ies. After the incubation is over, the membrane is then put under X ray analysis to show the reaction of the antibody with the antigen. If the antigen was present on the membrane, results can be seen. However, if the antigen is not present as a result of t he same limitations found in SDS PAGE, such as having large proteins, small proteins, or protein cross linking, it will not show results. Another problem is that only linear epitopes are
30 usually tested as a result of heating the protein to denature it befo re gel electrophoresis, so all the conformational epitopes may not be detected ( Aalberse 2000 ) Dot blotting Dot blotting is similar to Western blotting in that it analyzes allergen reactivity by binding the antibody to the antigen and then detecting it through the secondary antibody ( Besler 2001 ) However, some differences do exist. First, no denaturing is done and this helps preserve the conformational epitopes. Second, there is no preliminary gel electrophoresis step. The sample is spotted to a membra ne that contains the antibodies ( Besler 2001 ) This means that total protein allergenicity is measured as opposed to single protein allergenicity since there was no protein separation step, unless a single isolated protein is analyzed. The material for the membrane can be made of nitrocellulose or polyester cloth ( Besler 2001 ; Singh 1985 ) ELISA The term ELISA stands for enzyme linked immunosorbent assay. This assay is similar to the other tests that involve a series of attachments between the surface, the antigen, the primary antibody, and the secondary antibody. In indirect ELISA, the antigen is first adsorbed to a soli d phase such as a 96 well plate ( Wachholz and others 2005 ; Kemeny and Chanter 1988 ) Then the primary antibody is added to bind to protein followed by a secondary antibody that is marked by an en zyme. The better that the binding occurs, the stronger the color reaction will be afterwards ( Besler 2001 ) Several types of ELISA tests exist and kits are available to detect whole protein extracts or specific proteins ( Taylor and Nordlee 1996 ) The types of ELISA tests include indirect ELISA, competitive inhibition ELISA (ciELISA), and sandwich ELISA. However, some downfalls of th is method is that the protein may not fully adsorb to the plate due to
31 interactions that interfere with conformational epitopes and also linear epitopes will not be detected if they are not present on the surface. It is also the case that protein denaturation can occur if there is too strong of a binding between the s urface material and the protein ( Butler and others 1997 ) Food Allerg en Mitigation Thermal Processing Thermal treatments can be separated into moist heat and dry heat treatments. With moist heat, there is some level of moisture, as is implied in the name for the treatment. The source of the moisture can be water or steam or any cooking oil, among others. Boiling, frying, heat sterilization and extrusion are examples of moist heat treatments. Heat transfer by moist heat happens either by conduction, where heat moves through the molecules in the food, or by convection, where heat is transferred by a movement of the molecules. The latter is seen more in gases and liquids as opposed to solids where molecular movement is more flexible and the movement of the molecules causes warmer and cooler regions to be cooler together as a r esult of density changes. Furthermore, depending on the source used, heating is done either directly or indirectly. With direct heating, the source comes into direct contact with the food. With indirect heating, a heat exchanger is commonly used that separ ates the source of heat and the food product such as steaming over the oven ( Fellows 2009 ) On the opposite end, there is dry heat and it includes microwaving, baking, and roasting. The principle of dry heat is that moisture is r emoved from the product while at the same time heat is applied. In microwaving, electromagnetic waves pass through the food and indirectly heat the food by converting the electromagnetic energy into heat energy. This form of heat transfer is through radiat ion. For baking and roasting, these
32 are the same operation and involve the passage of heated air that goes through the food by conduction. The two names just refer to the different target foods for each treatment, such as baking for bread and roasting for meats ( Fellows 2009 ) Regardless of how the heat is applied, such changes can have numerable effects on food. Microorganisms can be destroyed. There can be sensory changes from food interactions happening at th e molecular level. And most important, there can be nutritional changes, from either protein modification to protein inactivation that affects how the protein allergen of interest can be made available to the body. For this reason, it was believed that th ermal processing could be used to destroy allergenic proteins in the food. If the temperature is high enough, it can destroy covalent or non covalent bonding. However, due to the complex chemical and physical changes that can take place, allergenicity of t he food may incre ase, decrease, or stay the same. Some conformational changes may occur which can destroy epitopes, but other changes can follow which can create new allergenic sites. These new sites are called neoantigens ( Davis and others 2001 ) To better understand how these changes occur on the allergen and only the allergen as opposed to changes in digestibility that can occur in the body, it is important to mention the Maillard reaction. The Maillard reaction, also known as the browning reaction since it is responsible for giving many foods their brown color, is an important and common reaction that occurs during heating. Sinc e the food can contain protein, reducing carbohydrates, or sugars, and water, this reaction is made possible by the addition of heat. The Maillard browning reaction is a non enzymatic reaction that starts by the reaction of the carbonyl carbon on the reduc ing sugar to the nitrogen located on
33 the amino acid and the removal of water to give a compound called a glycosamine. This intermediate compound then goes what is called an Amadori rearrangement to give another intermediate compound called a ketoamin e als o known as an Amadori compound ( Bucala 1996 ) The Amadori compound can then enter multiple pathways to give end products such as melanoidin pigments, 5 hydroxymethy furfural (HMF), or reductones. Collectively, all these different end pr oducts that from their various pathways are called advanced glycation end products ( Davis et al. 2001 ) Other changes can occur too. Reactions with oxidized lipids are possible and there can be direct oxidation when oxygen intermediates form ( Doke and others 1989 ; Kalluri and others 2000 ) Since amino acids are used in this reaction, it may either lead to a loss of amino acids or new neoantigens may form. What makes this critical is whether the B cell epitopes that result can be detected by the immune system, or in other words, whether the a llergen is intact and can be absorbed to stimulate IgE antibodies ( Davis et al. 2001 ) It is possible that while the patient may not r eact to the uncooked product, an IgE mediated response can occur once the product has been heated or cooked. This will be demonstrated below along other select studies that survey thermal processing on allergenic foods. Nonetheless, the final result for t he Maillard browning reaction is that there can be a change in protein quality along with changes to the appearance and taste of the food. While thermal processing can affect the allergenicity potency of the food, it is important to consider that the ideal situation is to have higher temperatures that are used for shorter times so that the food is edible ( Fellows 2009 )
34 Moist Heat Foods that show allergen reduction when treated with moist heat are peanuts depending on how they a re prepared What makes peanuts interesting is the manner in which they are treated. It is estimated that in the U S about 0.6% of the population suffers from peanut allergy and it is on an upward trend ( Sicherer and others 1999 ) In boiling and frying peanuts, which is the Chinese method of cooking, allergenicity is found to be less than the conventi onal method of roasting peanuts in the U .S. ( Beyer and others 2001 ) Furthermore, half of the peanut consumed in the United States is consumed as peanut butter ( Sanford 1998 ) The common varieties of peanut include Runn er and Valencia, both of which are consum ed mostly in a roasted form ( Beyer et al. 2001 ) In treating the two major peanut allergens A r a h1 and Ara h2 by roasting peanuts researchers found that on SDS PAGE there were bands that corresponded to Ara h1 (65kDa) and Ara h2 (isoforms of Ara h2 at 16kDa and 18kDa) and also a band for Ara h1 ( 148 kDA, which represents the trimeric form of Ara h1 ) ( M ondoulet and others 2005 ) However, these bands were not found in boiled and fried peanut preparations ( Beyer et al. 2001 ) These trimers that form are stable and might explain why binding increased for the roasted peanuts Furthermore, i t was also shown that purified Ara h1 and Ara h2 were bound by IgE antibodies the most when they came from roasted peanut. On the other hand, other studies show that Ara h1 decre ases in fried peanuts ( Beyer et al. 2001 ). In fish, the major allergens are parvalbumins (PV) which are pr oteins that are involved in the skeletal muscle of fish and help it function, along with binding free calcium ions. M ultiple forms of PV can exist in fish, ra ng ing from two to eight forms, but
35 usually three to five forms are found. Furthermore, p arvalbumins are also pres ent in frog and for that reason patients can cross react with frog PV because of similar IgE binding epitop es and the same isoform ( Gillis 1985 ; Chikou and others 1997 ; Huriaux and others 2002 ; Focant and others 2003 ; Arif and others 2007 ; Arif 2009 ) However, PV does maintain stability when heat treated. In a study ( Arif 2010 ) by boiling three PV isoforms of Channa marulius for 90 o C for 3 hours, researchers found that PVs were able to resi st the denaturation as these ba n d s were still visible on SDS PAGE at 10 kDa In egg, the major allergens are ovalbumin (45 kDa) and ovamucoid (28 kDa). These make up either 54% or 11% of the protein in egg white, respectively. Egg, along with some other allergenic foods, such a s cow milk, soy, and wheat, is one of the frequent foods implicated in food allergies in children ( Worm and others 2009 ) Fortunately, these allergies only persist in childh ood and become transient as one ages ( Tryphonas and others 2003 ) In testing both ovalbumin and ovamucoid ( Bernhisel Broadbent and others 1994 ) researchers found that most of the antibody response was to ovamucoid as it demonstrated a greater IgE response when testing on humans and mouse models. It was suggested that because ovamucoid has hardy physical characteristics, it retains its potency and resides in the body for several years, helping memory B cells recognize the antigen when it is ingested. These memory B cells are responsible for helping trigger immune responses and the reason that these memory B cells exist is because the antigen is present in the body for several yea rs ( Tew and others 1990 ) Other egg allergens are lysozyme and ovamucin, but these are not as prevalent.
36 Next, f or soybean ( Glycine max ), the major allergen is ca lled P34, or Gly m Bd 30K, or Gl y m 1, even though there are a total of at least 21 identified allergens ( Babiker and others 1998 ) P34 is similar to one of allergen Ara h1 main allergen, 2 S1 casein, because it shares a 70% homology and a 50 70% homology respectively. So ybean allergies affect 1 to 6% of the infant population ( Tryphonas et al. 2003 ) but like other common infant allergies, it can be outgrown when the infant reaches 3 years of age ( Kabourek and Taylor 20 03 ) In soybean, the major proteins are the 11S globulin unit, which contains glycinin, and the 7S globulin unit, which contains conglycinin. Both glycinin and conglycinin have subunits that each participate in some level of antibody response. However, the majority of the response comes from the P34 glycoprotein. It consists of 257 amino acids that are attached to the 7S globulin protein by disulfide bonds ( Wilson and others 2005 ) It is coded by a single gene that comprises 2% 3% of all protein and it is proposed that by removing that gene, the nutritional value loss would be minimal ( Babiker and others 2000 ; Helm and others 1998 ; Ji and others 1998 ) 12 linear epitope regions have been considered to be involved in IgE binding and it is proposed that there is a diversity of amino acids present However different experiments have demonstrated different results. In one study, it was shown that by inserting an alanine amino acid at an individual site by genetic modification, it is possible that allergenicity ( Helm and others 2000 ) With respect to heat processing, the use o f steam in autoclave treatment can cause P34 allergenicity to increase ( Yamanishi and others 1995 ) Yet, when the soy is prepared as
37 a textur ed soy protein, there were reduced levels of allergenicity with extrusion at high temperatures ( Franck and others 2002 ) Lastly, for milk, it is an allergenic food that is more common among infants than adults, since this allergy can disappear for most children at around 4 years of age. Since milk contains all the nutrients for a newborn and is the most appropriate food for infants, it can be a problem when cow milk is substituted for human milk, which can lead to nutritional and immunological problems ( El Agamy 2007 ) There can be an immediate reaction within hours or a delayed reaction (can go from hours to days). As a result, CMA is more serious in early infancy ( Hill and Hosking 1996 ; Jrvinen and others 2002 ) About 20 allergens have been found to cause allergi es, but the main proteins are casein and whey ( Gjesing and others 1986 ; Cavagni 1994 ; Docena and others 1996 ) F ro m the casein side, there can be and k casein while on the whey side, the important allergen is lac t ogl obulin ( lg) Other proteins are lactalbumin, bovine serum albumin (BSA) and immunoglobulin (IGs). As a result of all these proteins, different results can occur after heat treatment depending on what the milk protein is. These proteins reflect different stabilities. BSA is the most heat labile milk protein, lg is comparatively heat stable ( Bahna and Gandhi 1983 ) And despite casein being the most stable, heating milk at 120 o C for 15 min did not affect it ( Hanson and M Nsson 1961 ) However, the BSA and I Gs milk proteins did lose their antigenicity at 70 o C 80 o C or at 100 o C ( Fiocchi and others 1998 ; Hanson and MNsson 1961 ) However, it is important to consider the implications of these heat treatments on milk, which can cause nut ritional loss and an inferior product.
38 Dry Heat Another common allergen which has been treated with moist thermal processing is wheat. It is used in several ways, from making bread to pasta to dough, which can give other kinds of products ( Simonato and others 2001 ) In wheat, the proteins responsible for the majority of allergenic responses are gluten proteins. These pr oteins are made up of monomeric gliadins and polymeric glutenins. In one study ( Pasini and others 2001 ) the researchers evaluated the antigenicity of the wheat flour prote ins. The sources for the proteins were unheated wheat dough, bread crumb, and bread crust. For both the crumb and the crust, two different heat treatments were applied, one at 100 o C and one at 180 o C. The researchers discovered that after this heat treatme nt, these samples were more easily digested using an in vitro system as compared to the unheated dough However, they also discovered in a previous study that antigenicity did increase as a result of protein modification and browning reactions ( Hansen 1975 ; Hansen 1979 ; Pasini et al. 2001 ) Subsequently, lower weight proteins, such as those allergens at 16 kDa, were found to be digestable. These allergens play a role in happens after an individual breathes in flour particles ( Gmez and others 1990 ) But, by baking the bread, that 16 k D a all ergen did disappear in crumb and crust samples. Regarding tree nuts allergies, hazelnut allergy affects individuals who may als o be bir c h pollen allergic ( Worm et al. 2009 ) C or a 1.04 is the major pollen in hazelnut and because it is part of the pathogenesis related protein family 10, it shares a high sequence with the allergen behind birch pollen, Bet v 1 ( Kleine Tebbe and others 2002 ; Lttkopf and others 2002 ; Worm et al. 2009 ) However, unlike the Bet v 1 birch pollen allergen, which is labile to heat treatment, the food hazelnut allergen shows a spectrum of heat tolerance ( Vieths and others 1999 ) It may be that these allergens in hazelnut
39 belong to a more heat resistant protein family or because of the different heat tolerant allergenicity decrease s ( Hansen and others 2003 ; Worm et al. 2009 ) Furthermore, i n another study ( Worm et al. 2009 ) researchers also discovered that oral allergy syndrome can occur f rom hazelnut digestion or a systematic reaction, rather than just by contact. B y giving the patients capsules filled the antigen, it was postulated that organ specific reactions could occur. Other allergens also present in hazelnut include Cor a 2, Cor a 8, Cor a 9, Cor a 11, 2S albumin and a thaumatin like protein ( Ballmer Weber 2002 ; Beyer and others 2002 ; Pastorello and others 2002 ; Schocker and others 2004 ) Finally, a nother tree nut, almond, is also a common allergenic food. The major antigen is amandin, or almond major protein (AMP). This tree nut, like some other allergens, also gives different results. When roasted or blanched (moist heat), there is no elimination of the antigenicity ( Roux and others 2001 ) However, when analysis is extended to evaluate all the total allergens and more extreme treatments are used, the results can be the opposite. Researchers tested different commercial processing methods on the almond proteins ( Prunus dulcis L.) ( Venkatachalam and others 2002 ) They found that microwaving at extreme conditions (3 min) caused a reduction in antigenicity; roasting also decreased it (320 o C for 20 and 30 min; and that blanching decreased antigenicit y too (2 10 min). The reason that these extreme conditions were more effective is due to the protein structure. While heat can affect protein conformation, reduction in antigenicity in these samples would result from changes in
40 linear epitopes. For stand ard commercial processing, the proteins from 39 kDa to 66 kDa, which are IgE binding epitopes, were stable ( Venkatachalam et al. 2002 ) Non Thermal Processing Non thermal processing methods include those that minimize the amoun t of heat that is applied to the food. As mentioned earlier, heat can be a cooking method and is necessary to prepare some food such as wheat and heat. It also can destroy microorganisms and give food their final properties. Too much heat, however, can be detrimental. It can lead to nutritional and sensory loss in the food. This kind of produ c t is not favored by consumers and is considered an inferior product. To circumvent such potential issues, a trend towards non thermal processing methods is being inve stigated. Non thermal processing refers to minimal application of heat in order to give a food that is of high quality and also safe. Methods of non thermal processing include pulsed ultraviolet light (pulsed light), gamma irradiation, ultrasound, high pre ssure processing, genetic modification, pulsed electric field, and oscillating magnetic fields ( Fellows 2009 ) Some of these technologies have also been discovered to have an effect on proteins, including allergenic proteins, and show an inherent capacity to mitigate food allergens in some samples. Antigenicity can increase or decrease depending on the sample and treatment. Notwithstanding the results, these technologies are still in development and while results do not always give good prospects always, there is a possibility that in the future these technologies may play a more dominant role in allergen reduction. The technologies of pulsed light, gamma irradiation, ultrasound, high pressure processing, and genetic modificatio n are highlighted below to give an overview of the use of these technologies on food allergens.
41 Pulsed Light As described earlier, pulsed light is the application of the light spectrum from 170 2600 nm through a lamp filled with an inert gas, like Xenon, in short duration pulses. It is also possible for ultraviolet light to be emitted as a continuous phase, but these traditional systems lack the intensity of the pulsed light, which can be many thousand times stronger ( Dunn and others 1995 ) PL can cause photo chemical, photo thermal, and photo physical changes in the food because of its high energy and the a pplication of other waves such as visible waves and infrared waves. Regarding PL treatment on food allergens, a previous study on shrimp extract ( Shriver et al. 2011 ) show ed that protein extract after PL treatment of 4 6 minute showed reduction of the major shrimp tropomyosin protein. In the SDS PAGE, the tropomyosin protein was not visible, which was also confirmed by Western blot analysis. Shriver et al. then went to further test the 4 minute sample under different conditions This time point was chosen because it presented the ideal shrimp since the 5 minute and 6 minute samples showed significan t mois ture loss. Having shrimp with high moisture loss can make it unsuitable for consum ption if the treatment ever was translated into more commercial processing methods. Thus, by further testing only the 4 minute sample again with boiling, it showed that compared to the raw sample, the pulsed and pulsed + b oiling sample showed reduction while the boiled sample alone showed no reduction While tropomyosin is considered a highly resistant protein to thermal treatment, it was suggested by Shriver and others that the high peak energy of PL may have caused intense localized heating that could h a ve denatured the protein. Furthermore, other changes from protein cross linking or protein fragmentation due to the Maillard reaction or photo chemical effects could al so have caused these changes.
42 This is possible because other studies have shown that the Maillard reaction can reduce tropomyosin ac tivity in squid tropomyosin ( Nakamura and others 2006 ) but another study showed that it does not in sca llop tropomyosin ( Nakamura and others 2005 ) To demonstrate another research utilizing pulsed light on food allergen ( Chung and others 2008 ) treated peanut extracts and liquid peanut butter compared to boile d samples. They found that the major allergens, Ara h1 (63 kD a) and Ara h3 (~50 kD a) were not found in the PL treated sam ples while bands for 18 to 20 kD a which represented Ara h2, were visible in the peanut extracts These proteins might either be insolu ble aggregates or precipitates. However, the boiled treated extract showed no missing bands and w a s similar to the control It showed the presence of the 50 kDa and 63 kDa proteins, indicating that the proteins after boiling were soluble non aggregates. In other words, reduction in peanut allergy could be attributed to the formation of aggregates. As for the liquid peanut butter sample, it should no presence of the 50 kDa, 63 kDa, and 18 20 kDa proteins. For these samples, the same reasons were proposed, in that the cause for this reduction in peanut allergens in liquid peanut butter was attributed to insoluble protein aggregation. Gamma Irradiation Gamma irradiation refers to ionizing radiation from the gamma portion of the electromagnetic spectrum. It ca n disrupt the chemical bonds in a food through free radicals. There are different uses for gamma irradiation in food applications, including food decontamination, optimization of yield, and other quality functions. With regard to allergens, it has been fou nd that irradiation can have varying results in shrimp ( Zhenxing and others 2007b ) When working with the protein extract, the researchers demonstrated gamma irradiation could decrease antigenicity. However, when scaled up
43 to the shr imp muscle, antigenicity i ncreased up to a dosage of 5 kiloGrays ( kGy ) after which it decreased when dosage levels exceeded 10 kGy. This however is the high limit for safe human consumption. The researchers explained that the reason for shrimp muscle bein g less susceptible to gamma irradiation could have been due to the protection of tropomyosin from free radicals by other compounds such as lipids and that as the radiation dose increased, reduction could be seen because of stronger radical forces that were able to break the chemical linkages in proteins. Several reactions may have taken place such as deamination, decarboxylation, and epitope breakage. Furthermore, they also discovered that on the SDS PAGE, a band of 45 kDA w as noticed at the 7 kGy and 10 kGy mark for the shrimp muscle. This new band may have been the outcome of protein cross linking or protein aggregation and shows the disruptive effect of gamma irradiation. In another study ( Zhenxing and others 2007a ) the researchers expanded the treatment to include gamma irradiation and heat on peeled shrimp. Dosages from 1 15 kGy were applied and the heat source included blanchin g in boiling water for times of 5, 10, and 15 minutes. After extracting the protein, this combined treatment showed better reduc tion than irradiated shrimp alone. There was a 5 to 30 fold decrease compared with the untreated shrimp for the IC 50 (half maxim al inhibitory concentration) value, which measures how effective a compound is at inhibition. The basis for an increased reduction can be that radiation first destroyed or exposed the allergens through its free radicals and then when heat treatment followe d, this facilitated destruction of those exposed epitope sites.
44 High Intensity Ultrasound High intensity ultrasound, which is also known as power ultrasound, is a technology that uses waves of ultrasound frequency from 20 100 (kilohertz) kHz with high in tensities of 10 1000 watt per square meter ( W cm 2 ) The use of these high intensity waves causes the waves to impact the food matrix and create cavitation bubbles in the food. The cavitation bubbles are the result of opposite compression and shearing wa ves. As these bubbles form and then eventually implode, it creates areas of high localized pressure and heat that go up to 1000 atmospheric pressure ( atm ) and 5000 Kelvins ( K ) along with the formation of free radicals in the food which subsequently cause physical and chemical disruptions in the food structure. Researchers ( Zhen xing and others 2006 ) used ultrasound 30 hertz (H z ) 800 watts ( W ) for 30 180 minutes on shrimp muscle and protein extract. They discovered that ultrasound showed more reduction in the pure all ergen as opposed to the shrimp muscle, although the shrimp muscle itself did show allergen reduction. The explanation for this difference was attributed to the other food components present in the shrimp muscle, which would have created a layering effect. The researchers also went to discover a proportional relationship between the treatment time and the amount of reduction. One interesting observation was that immunoblotting results showed greater reduction than ELISA results at the same treatment time. Tw o possible explanations were given. First, there is the fact that immunological reactions can be different from one patient to another and also the reactive antibodies differed too. Second, ultrasound treatment of the samples causes some epitopes to be sep arated, which could only be detected in ELISA and not immunoblotting.
45 High Pressure Processing High pressure processing is the use of high pressures, from 100 megapascals ( MPa ) to 1000 MPa. It has been found that by applying such high pressures on the food for a time duration that can be from seconds to minutes, proteins can be inactivated. Changes in the food may or may not occur depending in the amount of pressure applied. Weak bonds in the protein can be disrupted while covalent bonds are not disrupted ( Fellows 2009 ) This technology has been extended to food allergens too as i t can affect the tertiary and quaternary structures ( Silva and others 2001 ) In one study ( Peas and others 2011 ) researchers treated soybean seeds tofu, and sprouts at 300 MPa for 15 m in at 40 o C. They discovered that the t reatment did increase allergens in the soybean seeds and sprout s while nothing happened in the tofu sample, as compared to each respective raw sample. In another study ( Troszynska and others 2007 ) the results were were the oppos ite in which it was seen that soybean allergenic ity decreased when soybeans were germinated. This may be due to the soybean species and also the conditions for germination. However, w hen those same sprouts w er e t r eated with HHP, they did show reduction. In another study, researchers looked at the application of high pressure on milk proteins. It was show n that lactoglobulin and lactalbumin can be denatured if the pressure ranges from 100 300 MPa, but the denaturation is reversible ( Suzuki and Taniguchi 1972 ) However, when 400 MPa is r eac hed, the denaturation is irreversible. A n other study ( Kleber and others 2007 ) sho w ed that antigenicity of lactoglobulin increased with increasing pressure and holding ti me, form 200 600 MPa. This was exp l ai ned by the weak ening of the non covalent bonds and this allowed better accessibility of the antibo die s.
46 Genetic Modification Genetic modification refers to changes that occur at the genetic level in order to elicit some desirable property in the food. While genetic engineering can be controversial and consumers are unsure of its long term safety it does however demonstrate potential. Gly m Bd 30 K, also known as P34, is the major immunodominant allergen in soybean t hat affects over 65% of patients ( Helm et al. 1998 ; Helm et al. 2000 ; Ogawa and others 1991 ; Ogawa and others 1993 ) By using molecular techniques the gene that codes for Gly m Bd 30 K was silenced through the insertion of P34 complementary DNA (cDNA) that did not code for the P34 gene itself. Genetic modification was necessary because no n atural species of soybean plants was found that lacked this gene ( Yaklich and others 1999 ) No observable phenotypic changes were present and no new pr otein bands were found in the gel electrophoresis system. Only the P34 band was absent. This showed that removal of allergenic proteins is possible. However, the researchers did not evaluate whether the food itself would be deemed hypoallergenic to sensiti ve individuals.
47 CHAPTER 5 MATERIALS AND METHOD S Human Plasma Samples Human plasma samples were provided by PlasmaLabs International (Everett, WA, U S A). The samples were obtained as pooled plasma of three individuals having specific IgE antibodies for major shrimp allergens, since every individual reacts differently to an allergen. In addition, samples from three individuals that have no history of shrimp hypersensitivity served as a control in distinguishing any false positive or negative results. Ther efore, a total of six individuals were included: half with and half without a history of shrimp allergies, respectively. The concentration of the plasma was measured at 76 kilounits per liter ( kU / L ) based on measurements from ImmunoCAP testing (Phadia A B, Uppsala, Sweden). The plasma is used for immunob lotting in Western blotting and testing with indirect ELISA. Preparation of Protein Extract Frozen whole shrimp (Atlantic white Shrimp) was purchased from Publix Supermarkets (Gainesville FL, U.S.A ). Prio r to experimentation, t he samples were purchased de headed and de veined, thawed in a plastic zip bag in water at room temperature for 1 hour ( h ) and de shelled afterwards. In order to extract the protein, shrimp were weighed out to 25 grams ( g ) and ground in a food processor at high speed for 5 minutes ( min ) in a buffer of 0.6M KCL in 0.01M NaH 2 PO 4 (pH of 7). Subsequently, the ground shrimp mixture was homogenized using a BioSpec BioHomogenizer (Bartlesville, OK, U.S.A.) at high speed for 5 min and aliquots were placed in 15 mL Screw Cap Conical Bottom centrifuge tubes (Corning, Corning, NY, U.S.A.) for centrifugation ( Beckman Allegra X 15R, Beckman Coulter, Brea, CA, U.S.A.) at 5000
48 times g ravity (x g ) for 120 min. The supernatant obtained serve d as the reservoir for the protein extract samples. 10 milliliters ( m L) was added to aluminum weighing dishes ( Fisher Scientific, Pittsburg, PA, U.S.A.) and placed on ice in a Styrofoam cooler and covered before treatment. The time points for the protein e xtract were raw (R) boiled (B) 2, 4, 5, 6, and 8 min. For the boiled shrimp, 25 g of shrimp were boiled at 100 o C for 10 min before the subsequent steps of grinding, centrifugation, and homogenization. Preparation of Whole Shrimp Whole shrimp samples were prepared exactly the same as the protein extrac t samples, with few significant and noticeable differences. The shrimp was purchased from Publix Supermarkets (Gainesville FL, U.S.A). Next the shrimps were thawed by transferring them into plastic plast ic zip bags and placing them in water for 1 h before use. The whole shrimp were de headed, de veined, and de shelled. Afterwards, the shrimp s were weigh ed and placed on aluminum dishes and placed on ice in a Styrofoam cooler and covered prior to treatment. A set of three shrimp were used for each time point, which included raw, boiled, 6, 8, 10, 12, and 15 min for treatment on each side. Therefore, the total exposure time to PL for the whole shrimp treated on both sides totaled 12, 16, 20, 24, and 30 min, r espectively. The boiled samples were boiled at 100 o C for 10 min. Preparation of Half Cut Shrimp In the third experimental group, the half cut shrimp were prepared similar to the whole shrimp. After purchase of de headed, de veined, and de shelled shrimp, plastic bags were used to thaw the shrimp for 1 h by placing the bag in water. The shrimp were removed from the bag and cut along the longitudinal axis from the head to the tail by making the incision on the dorsal side, or back side, of the shrimp A stan dard kitchen
49 knife was used and the cut went up to the surface of the ventral side, or belly of the shrimp. Two symmetrical halves were formed and full separation did not occur since the shrimp was still intact at the ventral end. In other words, the shrim p had been unfolded from the back to reveal the interior muscle. Considered as one unit, three shrimps were weighed together on aluminum dishes and stored and covered in a Styrofoam cooler containing ice. Three shrimps were then treated for the same time d uration used for the whole shrimp experimental group. Treatment points were comprised of the points raw, boiled, 6, 8, 10, 12, and 15 min Also coinciding with the whole shrimp samples, both sides were treated to make the total exposure times for half cut shrimp amount to 12, 16, 20, 24, and 30 min, respectively. The boiled samples were prepared exactly the same as other boiled shrimp samples at 100 o C for 10 min. Pul sed Ultraviolet Light Treatment All the shrimp samples were treated with PL at a distance of 10 cm from the light source on the aluminum dishes. Using the Xenon PL processor (Model LHS40 LMP HSG ) (Xenon Corp., Wilmington, MA, U.S.A) ( Figure 5 1 ), treatment was carried out at 3 pulses / s with a width of 360 us in batch mode. For the protein extract, a single tray was placed underneath the lamp for each time point For the whole and half cut shrimp, t hree individual shrimp or shrimp units were placed together in three separate dishes for each time point and flipped halfway to treat the other side. All the shrimp samples for each group were treated together. In other words, whole shri mp samples for all the time points were treated one after another and half cut shrimp samples for all of the treatment times were treated one after another. This is the same for the protein extract samples.
50 Figure 5 1 Xenon PL processor (Model LHS40 LMP HSG ) Temperature Measurement A non contact infrared thermometer (Omega OS423 LS, Omega Engineering, Inc., Stamford, CT U.S.A. ) was used to measure temperature of the PL treated shrimp before and immediately after treatment. Fo r the whole shrimp and half cut shrimp this was also done prior to flipping the shrimp. We were not able to record the highest temperature since that would have been during PL illumination. Instead, after a 5 10 s delay in removing the samples from the PL processor, we recorded the highest temperature reading available Post Processing Preparation In order to work with the samples after treatment, some post pro cessing preparation was needed for the protein extract, whole shrimp, and half cut shrimp. For the protein extract, the only post processing step involved homogenization for 5 min following treatment. For the whole shrimp and half cut shrimp, each group se t of shrimp for each time point were ground together in a food blender in a buffer of 0.6 KCL in
51 .01M NaH 2 PO 4 (pH of 7) and then homogenized for 5 min at high speed The mixture for each group for each time point was used in subsequen t testing. Protein Concentration Measurements After PL treatment, the samples were analyzed for protein concentration using the Coomassie Plus (Bradford) protein assay kit (Pierce, Rockford, IL, U.S.A), using bovine serum albumin protein (Pierce) as a standard (0 0.05 mL). The supernatant (protein extract), whole shrimp and half cut shrimp mixture obtained from the post processing steps was appropriately diluted using PBS 1X buffer ( Fisher Scientific Phosphate Buffered Saline 10X Solution, distilled water ). Then, 10 of the diluted samples were added to a 96 clear well plate (Fisher Scientific) in duplicates. The dilutions used were : 1:50 for all raw samples in each experimental group; 1:30 for the protein extract samples, including its boiled sample, and 1:10 for the whole shrimp and half cut shrimp samples, including each of their boiled samples. This was followed then b y the addition of 300 uL of Coomassie Blue ( Thermo Scientific, Rockford, IL, U.S.A. ) that had been previously warmed to room temperature. Protein concentration values were then determined using a SpectraMax 340PC384 Absorbance Microplate Reader at 595 nm (Molecular Devices, Sunnyvale, CA, U.S.A.) and then diluted to 5 ug/uL using PBS 1X for gel electrophoresis and 0. 0 2 ug/ 100 u L for ELISA t esting. SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) For the SDS PAGE, all the concentrated shrimp samples after the protein concentration measurements were prepared into a 1:1 ratio with 2X Laemmli sample buffer (62.5 mM Tris HCL, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, and mercaptoethanol, pH 6.8) (BioRad Laboratories, Hercules, CA, U.S.A). The samples were boiled for 10 min before being transferred to a pre cast tris glycine
52 minigel (4 20%) (Bio Rad) The only excep tion to this was the treated protein extracts, which were not boiled since the native tropomyosin prote i n was of interest. A Protein Plus All Protein All Blue protein stain ( Bio Rad ) was added and used as the standard. A ll the samples were added in duplicates except for the marker, which had one well Next, the gels were placed in a Mini P ROTEAN Tetra Cell tank apparatus ( Bio Rad ) to begin gel electrophoresis. The settings used for protein separation were 150V for 1.5 hr at room temperature. Upon com pletion, the gel was removed from its casing and washed with distilled water for 5 min on a rocker and then stained with GelCode Blue reagent ( Pie r ce). The gel was left overnight to facilitate adequate staining. Distilled water was used again to remove any excess GelCode Blue reagent and the gels were then scanned with a Canon Pixma MP160 scanner. Western Blot Analysis The Western blot procedure also follows the same procedure as SDS PAGE except that after gel electrophoresis, the gels are not stained to id entify the protein bands. Instead, the gels are similarly washed with distilled water for 5 min and then transferred on to an Immobilon P Transfer Membrane (Millipore Corporation, Bedford, MA, U.S.A ) made of polyvinylidene fluoride (PVDF). The transfer pr ocedure involves cutting two Protean Extra Thick Blot Papers ( Bio Rad ) and the transfer membrane to the size of the gel The size of the papers and the membrane must match or be similar to the gel so that all the proteins can be transferred to it. Collecti vely, these materials are then soaked in Transfer Buffer (Bio Rad 10X T G distilled water, ethanol ) for 1 min. Next the transfer membrane is removed and covered with methanol on both sides. Everything is then placed in a Trans Blot SD Semi Dry Transfer Ce ll (Bio Rad) For the
53 placement order, starting from the bottom, it is : the blot paper (Protean Extra Thick Blot Paper ), the transfer membrane, the gel, and finally the second blot paper. All bubbles were removed and a setting of 15V and 30 minutes was used. Next, the blotting membrane which is the transfer membrane that has had the proteins transferred to it from the gel, was put in a small plastic container and washe d with distilled water for 5 min on the rocker. The distilled water was removed and Ponceau S Solution ( Sigma Aldrich, St. Louis, MO, U.S.A. ) was added for 5 min to give a red indication to show if the proteins transferred properly. After this, t he blottin g membrane was washed with TBS 1X ( Fisher Scientific Tris Buffered Saline 10X Solution, distilled water ) for 10 min and then covered with StartingBlock T 20 Coating Blocking Buffer (Pierce) for 1 hr. This would permit antibody specificity to the protein of interest. Following this, the membrane was washed with TBST ( T BS 1X, Fisher Scientific Tween 20 ) for two times for 5 min each. Incubation with the anti shrimp IgE (primary antibody) ( Phadia AB ) follow ed at a dilution of 1:20, using PBS 1X as the diluent. 10mL of the primary antibody was used and incubation last ed for 2 hrs. Afterwards, subsequent washing continue d with four washes of 5 min each using TBST. Finally, the transfer membrane was prepped for X ray analysis. A 1:1 solution of SuperSignal West Pico Chemiluminescent substrate ( Thermo Fisher Scientific, Rockford, IL, U.S.A. ) was prepared as the enhanced chemiluminescence (ECL)
54 substrate. It was poured over the transfer membrane and al lowed to stand for 5 min. The mixture was then poured off and the tr ansfer membrane placed inside a n X ray cassette ( Soyee Products, Thompson, CT, U.S.A.) and a ll the bubbles were removed. Lastly, u sing Classic Blue Autoradiography Film ( MIDSCI, St. Louis, MO, U.S.A. ) the film was developed using an X ray machine ( ImageWorks, Elmsford, NY, U.S.A.) Exposure times are optimized to give the sharpest distinction among the bands and the least visible background. Indirect ELISA For the ELISA, all the concentra ted shrimp samples were placed into Costar EIA polystyrene 96 well plates (Corning ) at a dilution of 0.2 ug/ 100mL using PBS 1X to dilute the mixture. 100 uL of each sample was added to each well and repeated three times to give triplicates. Furthermore, in addition to the sample tests, a control sample was used from the human plasma that comes from individuals who do not have any shrimp allergies. The samples are then incubated at 37 o C for 1 hr. Next, 200 uL of StartingBlock T 20 Blocking Buffer ( Pierce ) was added and allowed to incubate the samples for 1 hr. Following this, the wells were washed by flipping over the samples into a sink and quickly adding PBST ( PBS 1X, Tween 20 ) to each well. The PBST is also poured out immediately and the 96 well plate is patted on brown napkins to dry. This is repeated two more times with PBST. The primary antibody (Phadia AB ) is added next. The dilution used is 1:10 with PBS 1X serving as the diluent. Incubation occurs for 1 hr followed by subsequent washing with PBST for three times. A secondary antibody ( Invitrogen ) diluted to 1:3000 with PBS 1X is then added and incubation continues for another hour. After this, the plate is washed again using the same methods from above and an OPD Solution
55 ( Thermo Fisher Scientific ) is added to each well. 100 uL is added and immediately afterwards, the absorbance values are read at 490 nm using a Spectramax 340 PC 384 spectrophotometer (Mo lecular Devices ). Statistical Analysis Densitometric analysis was performed on the samples in the Western blot to quantify the binding and describe the changes using ImageJ 1.44 software (National Institute of Health) In addition, o ne way analysis of variance ( ANOVA ) was also performed using SAS 9.2 software (Cary, N.C.) with significant differences b etween Figure 5 2 Summary of e xperimental design for protein extract, whole shrimp, and half cut shrimp Treatment Groups Half Cut Shrimp Raw (Control), Boiled, 6, 8, 10, 12, 15 min SDS PAGE, Western blot Whole Shrimp Raw (Control), boiled, 6, 8, 10, 12, 15 min SDS PAGE, Western blot, and ELISA Native Protein Extract Raw (Control), Boiled, 2, 4, 5, 6, 8 min SDS PAGE, Western blot, and ELISA
56 CHAPTER 6 RESULTS AND DISCUSSI ON Even though shrimp samples are rich in different proteins, the protein of interest that causes the majority of allergenic reactions is tropomyosin. It is responsible for more than half of the auto immune response in individuals. This heat stable protein is resistant to denaturation and can tolerant even extreme changes and therefore the effects of pulsed light treatment have given mixed results depending on the sample. Protein Extracts SDS PAGE Analysis of Protein Extracts The SDS PAGE for the native protein extracts ( Figure 6 1 ) shows a total of 13 bands. The bands of interests are the tropomyosin bands as marked with a pointer a t 37 kDa. From left to right, the bands are the standard marker, the raw (R) samples, 2 min, 4 min, 5 min, 6 min, and 8 min PL treated samples. All the samples were added in duplicates except for the standard marker. The boiled sample data are not shown. SDS PAGE is a technique for detecting protein profiles by separating those protein samples into different bands. The protein bands can be differentiated by a protein marker that has a known protein molecular weight. By being able to see and match the prote ins of interest to the standard marker, we can determine what proteins are present in the sample, or those that exist in the gel electrophoresis system, and also which bands are not present in the treated samples. As Figure 5 1 illustrate s the raw shrimp samples have several different protein bands These correspond to the proteins that would be present in the raw sample. Unlike the pulsed light treated samples, which show a reduction of the whole protein profile, more bands are present in the raw samples and with greater protein presence.
57 One protein band, at the 37 kDa mark, however appears to be the most resistant to degradation from PL This band represents tropomy osin, which is a protein characterized as being resistant to thermal denaturation or degradation. While it does show resistance at shorter treatment times, it does diminish as treatment time increases. This may be due to the fact that p ulsed light treatmen t can cause intense localized heating, which may result in the reduction of tropomyosin ( Shanti et al. 1993 ) As discuss ed below the surface temperature of treated shrimp did show a significant increase. Figure 6 1 SDS PAGE analysis of protein profile comparing 1) raw shrimp extract sample (R) and 2) pulsed light treated shrimp extract samples with times of 2 min, 4 min, 5 min, 6 min, and 8 min 15 uL was added to each well with concentrations of 1:50 for the R and 1:30 for the other samples Molecular weight marker (M) is shown on the left. Tropomyosin is pointed as 37 kDa. Western B lot for Protein Extracts In the Western Blot analysis ( Figure 6 2 ), it can be seen that there is a notable decrease in IgE binding to tropomyosin following pulsed light treatment, which is indicated by the reduced black intensity of the bands. As expected, the boiling heat 37kD
58 treatment did not appear to affect tropomyosin IgE immunoreactivity. For the boiled sample, IgE binding was similar to the raw sample (control). However, IgE binding to tropomyosin was reduced in pulsed light treated sample s as compared to the control and boiled sample. The densimetric analysis for the protein extract samples is provided in Error! Reference source not found. Table 6 1. ANOVA was applied for stastical analysis. Results showe d the significant reduction at 2min treatmen t compared to the control. Furthermore, the reduction of the bands density and IgE binding seen in the pulsed light treated sample s were not simply due to hydrothermal effects. Other changes such as photo chemical and photo physical changes may also have c ause d protein propert y variation. Pulsed light energy can cause protein modifications including protein fragmentation, denaturation, or intra or inter molecular protein cross linkage. Fragmented protein sections that are smaller than the limit of the SDS PAGE gel may travel through the acrylamide pores more quickly than other protein molecules. On the other end, those cross link ed proteins that are much larger than the native proteins can be h eld on the top the gel which means that they cannot run down th e gel during gel electrophoresis. This part of the protein cannot be detected in both the SDS PAGE and the W estern blot. In addition, although this is not the case here, covalent proteins formed by intramolecular crosslinking can migrate through polyacryla mide gels with difficulty P rotein bands corresponding to modified proteins can cause a smear on the W estern blot analysis ( Taheri Kafrani et al. 2009 ) This indicates that the smearing of allergens is formed because of the Maillard reac t ion and other non enzymatic browning
59 reactions. However, these reactions can also play a role in reducing IgE binding reactivity. Such reactions like the Maillard reaction, which is a complex reaction between carbohydrate s and protein s, can cause the modification of these proteins so that upon digestio n, the IgE binding epitopes are not accessible or available to the antibodies. The effects of the Maillard reaction appear to be dependent on the sample and the amount and type of reducing sugars present. Furthermore, i n shrimp, t he concentration of reduci ng sugars and amino acids are different among the various species and can change in response to climate For instance, t he concentration of reducing sugars and amino acids can increase during cold seasons ( Karanova and Andreev 2010 ) Subsequently, o ther n on enzymatic browning reaction s can also be accelerated by the increases in temperature with an extended pulsed light treatment. Thus m odification of proteins via non enzymatic browning could also explain the effect of pulsed light on shrimp allergen IgE binding activity. In one study ( Taheri Kafrani et al. 2009 ) it was demonstrated that this phenomenon can cause smearing of modifie d milk allergen reactivity in W estern blot. Moreov er, other previous studies also revealed that the M aillard reaction in roasted peanuts and pulsed light treated peanuts could increase the IgE immunoreactivity ( Chung et al. 2008 ; Maleki and Hortense 2002 ) Figure 6 2 Western blot analysis of protein extract samples including 1) raw sample (R) 2) boiled sample (B) 3) pulsed light treated sample with time points of 2 min, 4 min, 5 min, 6 min, and 8 min.
60 Table 6 1. Densimetric analysis of Western blot samples for protein extract from ImageJ 1.44 software (NIH). Sample Mean Statistical Comparison R 11141.9 A B 7519.518 B 2 min 4225.18 C 4 min 3406.558 CD 5 min 2541.376 CDE 6 min 1691.42 DE 8 min 1167.305 E Samples include 1) raw samples (R) 2) boiled samples (B), and 3) pulsed light treated sample with time points of 2 min, 4 min, 5 min, 6 min, and 8 min. Samples Indirect ELISA for Protein Extracts ELISA is one type of immunoassay that can be used to test single proteins or total proteins in a sample, depending on the antibodies employed. To describe the technique b riefly proteins are adsorbed on a surface of polystyrene 96 well plate and detect ed with the appropriate antibodies (Wachholz et al. 2005). The limitation is that i n some cases, hydr ophobic interactions which are the main force that cause protein adhesion to the plate, can interfere with or mask conformational epitopes of allergen proteins. Nonetheless, these in vitro tests are more reliable than other tests and are commonly used. In this experiment, indirect ELISA was applied to determine the reactivity of the protein extracts. The results from the ELISA ( Figure 6 3 ) supported the Western blot analysis, illustrating a decrease in IgE binding to pulsed light treated samples as compare d to the boiled and raw samples. Total reductions in IgE binding were likely explained by changes in tropomyosin as seen in the W estern blot. The b oiled sample showed a
61 higher IgE reactivity than the raw sample and the rest of the pulsed light treated samp les. Statistical analysis was done to compare the reactivity between each sample P ulsed light treated shrimp sample at 6 min and 8 min have a significant reduction compared to both the min, most allergen reactivity c ould not be detected. Boiled shrimp sample also increase d allergenicity due to conformation changes of epitopes of trypomyosin as indicated and expected by Shanti et a l. (1993 ) The r eduction in IgE binding of pulsed light treated sample s can be explained by the changes in the amount of detectable tropomyosin as described above for SDS PAGE ( Figure 6 1 ) and W estern blot analysis ( Figure 6 2 ). Due to the energy absorbed from pulsed light, the proteins in shrimp have been changed via photo thermal, photo chemical, and photo physical effects. Overall, pulsed light processing showed positive results in reducing the IgE immunoreactivity of solubl e allergen levels in shrimp extracts. Figure 6 3 Indirect ELISA illustrated the changes in IgE binding compared to untreated, boiled, P L treated shrimp protein extracts using pooled human plasma containing IgE antibodies against shrimp. The times correspond to the treatment time and a control was used. A = absorbance of the sample; A 0 = 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Raw Boiled 2min 4min 5min 6min 8min control IgE Binding (A/A0)
62 absorbance of untreated samp le. Data are expressed as mean SEM (n = 5). Results are relative values, normalized to the untreated sample; untreated is st andardized and set to 1. Values that are significantly different are analyze Whole Shrimp Despite the fact that tropomyosin properties can be changed in shrimp extracts after pulsed light treatment further evidence was needed to evaluate pulsed light effects on whole shrimp. T he results from treatment of the whole shrimp are shown below. This includes the SDS PAGE, Western blot, and ELISA, each of which is shown in Figures 6 1 to 6 3 respectively. SDS PAGE of Whole Sh rimp For the SDS PAGE (Figure 6 4 ), there are a total of 15 bands. From left to right, these are the standard marker, the raw samples, the boiled samples, and the treated samples that range from 6 min, 8 min, 10 min, 12 min, and 15 min. More specifically, the treated samples time points correspond to the treatment of one side of the whole shrimp, so in effect, the total time point for each sample is double. For example, the 6 min sample was treated for a total of 12 min and the 15 min whole shrimp sample wa s treated for a total of 30 min. The bands for tropomyosin are marked and reflect protein bands found in the 34 3 8 kDA range. The intensity of the band visibility coincides with the effectiveness of pulsed light to alter the structural properties of tro pomyosin. Darker bands indicate higher concentration levels of tropomyosin found in shrimp whereas the lighter bands suggest that the levels have decreased. As shown in the SDS PAGE, the raw and boiled bands give the reference point for which to compare t he pulsed light treated samples. It is seen between the raw and
63 boiled samples that the bands for the boiled sample are much more prevalent. The reason for the darker band intensity may be due to the conformation al changes of tropomyosin after boiling ( Shanti et al. 1993 ) The raw sample may contain tropomyosin in a native state and after boiling, the protein and its epitopes are more accessible. In effect, the protein would form the nutritional and struct ural value of consumed whole shrimp. Shr imp is high in protein content and s ince most of the consumed shrimp is muscle, it would be expected to contain a high level of tropomyosin in the meat. With respect to the treated samples, it can be seen that the tropomyosin band is present up until the 15 min mark. Absolutely no effect can be observed for the 6 min to 12 min samples. The tropomyosin band for these treated samples resembles that of the boiled sample, which as stated earlier, h as a higher intensity than the raw sample. Pulsed light technology, which contains a photo thermal spectral component, may be responsible for the heating that may have occurred during treatment from 6 min to 12 min. For the same reasons as stated earlier, this could have caused conformational changes in the protein. As for the 15 min sample, it does show a reduced intensity indicating that the allergenic protein concentration is lower. It would seem promising, but the reality is that the shrimp sample itse lf is burnt and shows discoloration due to the formation of white spots. Rather, this reduced intensity can be attributed to browning reactions such as the Maillard reaction. As described in the shrimp extract results, the Maillard reaction and other non e nzymatic reactions can cause browning. This is evident in the 15 min treatment sample and can be seen in Figure 6 13 The sensory remarks for each of these shrimp samples are described in the sensory remarks section
64 Figure 6 4 SDS PAGE analysis of protein profile of whole shrimp samples comparing 1) raw sample (R) 2) boiled sample and 3) pulsed light treated whole shrimp samples with times of 6 min, 8 m in, 10 min, 12 min, and 15 min. These times correspond to pulsed light treatment on one side of whole shrimp, with their being an application of treatment of both sides 15 uL was added to each well with concentrations of 1:50 for the R and 1:10 for the ot her samples Molecular weight marker (M) is shown on the left. Tropomyosin is pointed as 37 kDa Western Blot for Whole Shrimp T he results for the Western blot ( Figure 6 5 ) for whole shrimp samples are shown above. From left to right the data points are in duplicates. These are raw, boiled, 6 min, 8 min, 10 min, 12 min, and 15 min samples. Once again, these points reflect pulsed light treatment to a single side of the whole shrimp. The total time for each shrimp sample is double the time shown in F igure 6 5 Furthermore, as mentioned previously, the visibility of the bands represents the effectiveness of pulsed light to reduce the level of tropomyosin. Only tropomyosin bands will be shown in the Western blot since the purpose of Weste rn blot is to separate the proteins in a sample during gel electrophoresis and then isolate the protein of interest using antibodies. Std Raw Boiled 6 min 8min 10min 12min 15min 37kD
65 In the Western blot, it can be seen that throughout the entire data set, no significant reduction is present for any of the time points with the exception of the 15 min ute set. The bands are solid for the most part and show no significant reduced visibility that suggests tropomyosin in shrimp is lower. However, it is worth pointing out that for the 10 min mark and 15 min ma rk, the bands are broken and lighter, respectively. For the 10 min mark, this broken band may be the result of loading, failure of proper washing during the Western blot procedures, or possible human error that could have resulted in this. It does not sign ify reduction at the 10 min mark. As for the 15 min mark, it is seen on both the SDS PAGE and this Western blot that there is reduction. While this may be the case, and is reinforced by the ELISA (shown below), the 15 min shrimp sample itself is not edible The shrimp is over treated and the final form of the shrimp can be described as charred and crispy. The smell following treated resembled that of a burnt nature and no moisture within the shrimp remained. The crispiness of the 15 min shrimp occurs due to the fact that there is a significant moisture loss. To obtain further data of the percentage of signal binding reduction, Image J was applied to analyze d ensimetric value for each band as shown in Table 2. ANOVA was applied to analyze different treated g roups o f shrimps. Results showed that r aw sample, boiled sample, 6min, 10min, and 12min have similar immunoreactivity with human IgE, however, 8min treatment is significantly high signal than other treatment and 15min has a significant low IgE immunoreacti vity compared to other groups. This can be explained as the proteins in shrimp at 8min were partially unfolded and more epitopes were exposed, but less than 8min treatment, even boiled shrimp samples do not have much
66 changes on the epitopes compared to raw sample. In addition, the last treatment (burned shrimp) has the lowest signal due to protein cross linkage, protein aggregation and protein fragmentation that m ay related to non enzymatic browning reactions during pulsed light illumination. From the pict ure for PL treated shrimp, it was obvious that the maximum treate d shrimp become burned that can not be edible. These results also showed that penetration is a major limitation factor to affect protein changes. For sterilization purpose s pulsed light provide s a high degree of penetration in most food samples. H owever, a previous study also showed that pulsed light still had a limited penetration into honey. Their results indicated that the heat generated within the pulsed light system did not appear to have a synergistic effect on the inactivation of C. sporogenes in honey (Demirci and Panico 2008). Our results also demonstrated that the penetration of pulsed light is one of the major limitations t hat can not produce obvious photo thermal, photo physical, and photo chemical effects on whole shrimp samples. Further research about reducing the thickness of shrimp samples were needed to demonstrate the effects of pulsed light. Figure 6 5 Western blot analysis of whole shrimp samples inclu ding 1) raw sample (R) 2) boiled sample (B) 3) pulsed light treated sample with time points of 6 min, 8 min, 10 min, 12 min, and 15 min. These times correspond to pulsed light treatment on one side of whole shrimp, with their being an application of treat ment of both sides
67 Table 6 2 Densimetric analysis of Western blot samples for whole shrimp from ImageJ 1.44 software (NIH). Sample Mean Statistical Comparison R 4376.494 A B 4303.342 A 6 min 6384.853 A 8 min 9708.198 B 10 min 4733.676 A 12 min 5127.004 A 15 min 1259.795 C Samples include 1) raw samples (R) 2) boiled samples (B), and 3) pulsed light treated sample with time points of 6 min, 8 min, 10 min, 12 min, and 15 min. Samples 0.05). Indirect ELISA for Whole Shrimp F rom the ELISA ( Figure 6 6 ) we observed that the results do show reduction after the 6 min mark. Allergenic protein levels increase from the raw to the boiled to the 6 min shrimp sample and then decreases afterwards for the other time points. These results are different from those of the SDS PAGE and Western blot, which showed no significant reduction. Therefore, even though r ed uction in allergenicity is indicated for the 8, 10, 12, and 1 5 min mark, this is due to the fact that the pulsed light treatment affected the conformational structure of the allergen prote i n. The fragmentation effect is not as prevalent. Instead, it is possible that the photo thermal effect of pulsed light inf l uen c e d non covalent bon d s such as hydrogen bonding and van der W aals forces so that it made less of the epitopes available on the surface adsorb to the ELISA plate. In other words, epitopes that are on the interior are unavailable to bind to the plate and those epitopes would not be detected. Only a fraction of the allergenic sites would be detected, which would portray reduced allergenicity even though not all the epitope regions are analyzed. The linear epitopes and any intramolecular conformational
68 epitopes a re masked by the surface conformational shape and the antibodies will only bind to the accessible surface antigens. Figure 6 6 Indirect ELISA illustrated the changes in IgE binding compared to untreated, boiled, P L treated whole shrimp extracts using p ooled human plasma containing IgE antibodies against shrimp. The times correspond to the treatment time and a control was used. A = absorbance of the sample; A0 = absorbance of untreated sample Data are expressed as mean SEM (n = 5). Results are relativ e values, normalized to the untreated sample; untreated is standardized and set to 1. Values that are significantly different are analyze Sensory Remarks for Whole Shrimp A normal shrimp has moisture content of 81.1%, a protein content of 17.4%, a fat content of 0.4% and a NaCL content of 0.7% ( Zeng and others 2005 ) As another previous illustrated thermal effects co uld affect the color and texture changes on shrimp. Yellow and red color ha d significant increase s However, changes in texture did not show clear tendencies. F or example, texture did not become harder or softer with increasing temperature. But when a fin al temperature went over than 85C, the tend erness has a significant change, meaning that it become harder ( Schubring 2009 ) It was assumed that changes were mainly due to heat denaturation of intracellular 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Raw Boiled 6min 8min 10min 12min 15min control IgE Binding (A/A0)
69 proteins, which developed gradually from 30C to higher temperature. Above 70C the denaturation occurred so intensely that the muscle meat shows a heavy shrinkage ( Mizuta and others 1999 ) Traditional roasted shrimps are specially used as an ingredient for tempura, sushi and rice snacks. Roasted shr imp emanates an aroma which is strong and pleasant to the Japanese consumer Pulsed light as a novel processing technology has a strong thermal effects as treatment time increases and thus this technology can be considered as a parallel to traditional roas t ing technology. As both of these two technologies produce the similar sensory properties on shrimp, special aromas are produced due to the M aillard reaction during the process ( Tachihara and others 2004 ) The color changes on the s urface are always caused by a non enzymatic browning reaction or M aillard reaction The acceptability of pulsed treated shrimp at each ti me point needs to be optimized. The observations from our experiments are as follows. After the first treatment, there is a good color development for the 6 min shrimp. The shrimp looks good, but has a repulsive smell. The top is dry, but moist on the bottom and center. Follow ing the second treatment, the underside shows some burnt orange skin. The smell is still the same However, t he feel is soft and squishy and some darker color has developed. The first treatment for the 8 min shrimp gives off a smell that is repulsive, but less than the first treatment for the 6 min shrimp. There is a squishy feel and the shrimp seems edible. The underside is more burnt than the top. The surface is not as moist. Next, the second treatment gives a shrimp where there is a reduction in size a foul
70 smell, and hardness of the shrimp with some moisture in the middle. The tail appears crunchy and the body is hard on the surface with some softness inside. For the 10 min shrimp sample, the top is white and pink on the bottom. There is a faint smell that is not foul, but shrimp like. The shrimp also looks more treated and seems edible Coincidently, the shrimp treated time for the pulsed light treatment i s the same as that for a 10 min boiling time for shrimp. It feels soft and squishy. These observa tions were for the first treatment. For the second treatment giving a total time of 20 min changes in shrimp were that the underside is more burnt than before and the top is pink like with a white surface. The smell also is more foul. The tail is crunchy and appears that it will break under pressure and there is some moisture in the middle. Moving on to the next time point, the 12 min shrimp after the first treatment has a white color on the top and the underside is burnt orange. The smell is foul, but n ot so strong. Also, the shrimp feels less soft than the shorter time treated shrimps for the first treatment. After the second treatment, there are some white spots, size reduction is present, and the center is moist. Burnt color is visible on both sides, the smell is faint, and the shrimp feels crunchy on the surface. For the last sample for 15 min shrimp, the first treatment gives an underside that is burnt orange. The smell also is the most foul here. The skin is flaky and there is a softness feel in th e center and also moisture in the middle. On the top, the color is white with some flour spots. The shrimp is not edible at all and has been destroyed by the pulsed light treatment.
71 Figure 6 7 Raw shrimp sample was taken as a control. This raw sample was applied in following experiments in SDS PAGE, Western blot, and i ndirect ELISA. Figure 6 8 Boiled shrimp sample was considered as another control. This sample was prepared with a raw sample that was boiled in distill ed water for 10 min. Figure 6 9 Whole shrimp sample was treated by pulsed light for 6 min on both sides for a total treatment of 12 min.
72 Figure 6 10 Whole shrimp sample was treated by pulsed light for 8 min on both sides for a total treatment of 16 min Figure 6 11 Whole sh rimp sample was treated by pulsed light for 10 min on both sides for a total treatment of 20 min Figure 6 12 Whole shrimp sample was treated by pulsed light for 12 min on both sides for a total treatment of 24 min
73 Figure 6 13 Whole shrimp sample was treated by pulsed light for 15 min on both sides for a total treatment of 30 min Half C ut S hrimp SDS PAGE of Half Cut Shrimp For the half cut shrimp SDS PAGE ( Figure 6 14 ) the bands are loaded, in order from left to right, with the standard marker first, then the raw and boiled shrimp samples second, and finally followed by 6, 8, 10, 12, and 15 min shrimp samples. With the exception of the standard marker, all other samples were loaded in duplicates. The times are also parallel to that for whole shr imp samples. Maximum treatment time for the treated samples is 12, 16, 20, 24, and 30 min, respectively. As can be seen by the area marked as 34 3 8 kDA, to represent the tropomyosin protein again, it can be noticed that the visibility of the bands conti nues to remain the same. The boiled protein shows greater intensity as a result of cooking shrimp. However, the other shrimp samples also show an increased intensity up until the 10 min sample, after which a significant reduction can be observed at the 15 min mark. All this suggests that the concentration of tropomyosin at that time point decreases. This is exactly the same trend for the whole shrimp sample. At longer duration times, the band for the 15 min tropomyosin decreases. However, like the whole shr imp sample, the
74 wholesomeness of the shrimp is, without doubt, inedible. The entire sample is charred and burnt. It cannot be consumed and is covered in flour spots. Therefore, the possible explanation for the reduction of the tropomyosin protein is that t he high treatment L heating, but also the limitation that exists of PL penetrat ion not being deep enough to destroy the protein without destroying the sample first. And despite slicing the shrimp in half, the sample itself is still too thick because it has similar results with the whole shrimp sample. The same reasons for whole shrimp samples are cited to explain the same outcome pattern in half shrimp, in that PL penetration ma y not be deep enough and the role of the Maillard reaction and non enzymatic reactions in causing shrimp browning. Figure 6 14 SDS PAGE analysis of protein profile of half cut shrimp comparing 1) r aw shrimp (R) 2) Boiled shrimp (B) and 3) p ulsed light treated shrimp samples with times of 6 min, 8 min, 10 min, 12 min, and 15 min These times correspond to pulsed light treatment on one side of whole shrimp, with their being an application of treatment of both si des 15 uL was added to each well with concentrations of 1:50 for the R and 1:10 for the other samples Molecular weight marker (Std ) is shown on the left. Tropomyosin is pointed as 37 kDa 37kD
75 Western Blot for Half Cut Shrimp The results for the Western blot analysis ( Figure 6 14 ) coincide with the SDS PAGE ( Figure 6 13 ) The bands are loaded, from left to right: standard marker, raw (R) boiled (B) 6, 8, 10, 12, and 15 min. While the SDS PAGE showed reduction at the 15 min point, this is not visible in the Western blot. Densimetric analysis is shown in Error! Reference source n ot found. ANOVA was applied for stastical analysis. For all the data points, no reduction is present Since we did not see any significant reduction in whole shrimp, this half shrimp study was based on previous whole shrimp to inv estigate the effects of pulsed l ight. Results showed that, the penetration was still a limitation factor for shrimp sample. N o indirect ELI SA was done for this experiment as the purpose of the half cut shrimp was to assess further treatment of pulsed light on a thinner sample. More optim izations of distance and time were recommended for further study, such as choosing lower distance s. For example, distances of 7 cm or 5 cm can be used. This would bring the shrimp samples closer to the pulsed light source. In addition, times could also hav e to be evaluated relative to these distance changes. These time changes can be similar to the ones used or different times. Figure 6 15 Western blot analysis of half cut shrimp samples including 1) raw sample (R) 2) boiled sample (B) an d 3) pulsed light treated sample with time points of 6 min, 8 min, 10 min, 12 min, and 15 min. These times correspond to pulsed light treatment on one side of whole shrimp, with their being an application of treatment of both sides
76 Table 6 3 Densimetric analysis of Western blot samples for half cut shrimp from ImageJ 1.44 software (NIH). Sample Mean Statistical Comparison R 8331.258 A B 9312.785 A 6 min 9713.371 A 8 min 6046.371 A 10 min 9530.451 A 12 min 10123.87 A 15 min 9535.167 A Samples include 1) raw samples (R) 2) boiled samples (B), and 3) pulsed light treated sample with time points of 6 min, 8 min, 10 min, 12 min, and 15 min Samples Sensory R emarks for Half Cut Shrimp Sensory observations were also done for the half cut shrimp. For the 6 min shrimp, treatment of the first half showed that the shrimp is dry on the top and the underside is still moist. The shrimp is kind of soft and has a foul like sm ell. After the second treatment, it is dry on both sides, still soft like, and the foul smell of the shrimp has increased. The top is burnt and some of the skin sticks to the plate. The color is faded white with orange burnt spots and orange skin lines. T he 8 min shrimp shows burnt sides on both sides of the shrimp, it is squishy, and the smell is not strong. At the end of the second half of the treatment, the shrimp is still soft and squishy, burnt on both sides, and there is an internal pink color visibl e on some areas while the top of the shrimp is faded white. A huge size reduction is also visible and the smell is not as strong. The first treatment for the 10 min shrimp gives off a foul shrimp smell and size reduction is present. The shrimp feels somew here between soft and hard and the bottom shows some moisture. The second treatment gives more size reduction and the loss of some parts which were hard. The shrimp smell is also not as strong so it is
77 tolerable. There is some pink on the inside at the bot tom and the color is faded white. There are also some flour spots that are visible and the shrimp is hard with crunchy spots. Next, the 12 min shrimp sample was burnt on the bottom following the first treatment. The surface is dry and the feel is soft with a faint shrimp smell. A lthough the faint smell is not a repulsive smell, in this instance th e smell resembles more of raw shrimp rather than spoiled shrimp. Following the second treatment, s everal parts have been burnt and are charred. The shrimp has become stiffer Parts of the shrimp have folded and some parts stick to the tray. The smell is the same as before although it lingers more as an after smell. Lastly, the 15 min shrimp after the first treatment shows many changes. There was size reduction folding of the shrimp, and the shrimp was burnt on some parts. The smell was very, very foul. As for the feel, it was soft, but the texture of the surface was very dry and rough like. This would be the expected results from the longer treatment of the pu lsed light. Specifically, the photothermal effect of pulsed light would be responsible for the dryness and moisture loss that is present. The color was faded with some visible pink on the bottom underside. The second treatment continued this trend. The end shrimp is charred on some parts and other parts show an orange crispiness. The smell resembles a burnt sample. A huge size reduction can be seen along with the addition of shrimp folding inward White flour spo t s have also formed. In addition, some parts can also be seen to have fallen off on the tray. These can be due either during the flipping of the shrimp sample or from the fragileness of the burnt and crispy sample.
78 Figure 6 16 Raw shrimp sample for the half cut shrimp sample. This sample was use d in SDS PAGE and Western blot. Figure 6 17 Half cut shrimp was treated by pulsed light for 6 min on both sides for a total treatment of 12 min Figure 6 18 Half cut shrimp was treated by pulsed light for 8 min on both sides for a total treatment of 16 min
79 Figure 6 19 Half cut shrimp sample was treated by pulsed light for 10 min on both sides for a total treatment of 20 min Figure 6 20 Half cut shrimp sample was treated by pulsed light for 12 min on both sides for a total treatment of 24 min Figure 6 21 Half cut shrimp sample was treated by pulsed light for 15 min on both sides for a total treatment of 30 min
80 Other Remarks Optimization of Pulsed Light Illumination Based on a previous study of the effect of pulsed light illumination on shrimp allergen protein (36kD) ( Shriver et al. 2011 ) this study attempted to establish a series of proper durations and a proper distance for shrimp extracts, whole shrimp samples and half cut samples. In the previous study, researchers investigated durations at 0 (control), 1, 2 3, 4, 5, and 6 min. It was found that the reduction of IgE immunoreactivity of tropomyosin can be clearly found at 4 to 6 min. This study continue d to investigate extended durations on shrimp extract, but by using a crude protein extract in its native fo rm. The parameters selected were 3 pulses / s for the PL treatment and a distance of 10 cm from the light source. The maximum treatment time was selected at 8 min. Changes in the reactivity of tropomyosin at each time point were detected via Western blot analysis. Figure 6 3 indicates that IgE binding to tropomyosin was reduced from 4 min to 8 min. Moreover, at the 8 min mark, the signal of IgE reactivity was hardly detected. Temperature and Moisture C hange As a previous study reported ( Keklik and others 2009 ) the temperature can increase significantly a s treatment time increases. The surface temperatures of the shrimp extracts can a ttain measurements up to 8 0C. It is worth mention ing that instanta neous temperatures may be higher during the treatment, but we did not detect this kind of temperature. Temperature mea surements are given in Table 6 3 and Table 6 4 below. Furthermore, based on the previous report, it was shown that color changes of shrimp could be detected as high as 120 o C, indicating that temperature changes
81 were present. From this, we can conclude that the instantaneous temperatures of the treated shrimp could be higher than 120 o C ( Keklik et al. 2009 ) Moisture loss was higher in the pulsed light treated samples because the samples were not enclosed during the pulsed light treatment and heat evaporation was also present ( Chung et al. 2008 ) also indicated that around 40% volume reductions can be found after pulsed light treatment s In our experiments, we can see moisture losses that exceed 40% and go up to roughly 76%. More moisture loss can be see in the thinner samples and these can be due to the pulsed light being able to have more of a surface area to treat and penetrating more on the surface. A closed system may be benefici al for preventing moisture loss. H owever, the pulsed light penetration into the sample might be compromised. Another solution would be to fit the pulsed light equipm ent with a cooling s ystem to absorb the heat produced during the whole treatment. This phenomenon was also found in extended treatment of shrimp samples. Moisture loss can be achieved to 50% of total sample for the longer treated samples. The moisture loss is given in Table 6 5 and Table 6 6 Table 6 4 Temperature m easurements for whole shrimp samples. Each observation is a mean of three temp e ratures and the standard deviation Sample Initial Temperature Mean SD First Treatment Mean SD Second Treatment Mean SD Raw 20.57 6 Min 20.67 0.60 56.63 7.07 57.13 5.52 8 Min 19.13 1.30 44.27 0.85 62.17 3.36 10 Min 19.63 0.55 71.50 1.57 46.83 5.09 12 Min 20.20 0.78 60.70 0.56 35.53 0.95 15 Min 19.83 0.64 54.03 4.09 44.37 14.91 The samples include 1) raw samples (R), and 2) pulsed light treated samples with time points of 6 min, 8 min, 10 min, 12 min, and 15 min.
82 Table 6 5 Temperature m easurements for half cut samples. Each observation is a mean of three temp e ratures and the standard deviation Sample Initial Temperature Mean SD First Treatment Mean SD Second Treatment Mean SD Raw 20.13 0.21 6 Min 20.17 0.12 45.67 9.25 60.90 2.55 8 Min 20.10 0.44 44.33 4.74 51.50 5.29 10 Min 19.97 1.17 54.63 10.00 48.10 0.76 12 Min 20.83 0.51 83.13 4.37 57.30 2.61 15 Min 20.60 0.70 71.20 1.18 82.20 0.17 The samples include 1) raw samples (R), 2) pulsed light treated sample with time points of 6 min, 8 min, 10 min, 12 min, and 15 min. Table 6 6 Representative moisture loss m easurements for whole shrimp samples. Weight is measured in grams (g). Treatment Initial Weight (g) Final Weight (g) Percent Loss (%) R 18.89 0 B 21.68 16.48 23.99 6 min 20.49 14.00 31.67 8 min 19.71 11.74 40.46 10 m in 18.96 10.61 44.04 12 min 20.68 9.28 55.13 15 min 20.51 7.44 63.71 The samples include 1) raw samples (R) 2) boiled samples (B), and 3) pulsed light treated sample with time points of 6 min, 8 min, 10 min, 12 min, and 15 min. Table 6 7 Representative moisture loss m easurements for half cut shrimp samples. Weight is measured in grams (g). Treatment Initial Weight (g) Final Weight (g) Percent Loss (%) R 18.41 0 B 22.95 16.96 26.11 6 min 20.62 12.43 39.75 8 min 21.32 10.99 48.45 10 min 20.34 8.48 58.34 12 min 20.74 7.82 62.30 15 min 18.87 4.52 76.03 The samples include 1) raw samples (R) 2) boiled samples (B), and 3) pulsed light treated sample with time points of 6 min, 8 min, 10 min, 12 min, and 15 min.
83 A nti genicity V ariations C aused by O ther P rocessing M ethods o n S hrimp Tropomyosin is considered a thermal stable protein in shrimp. However, p revious studies reported that shrimp allergen was liable to enzyme hydrolysis ( Shi makura and others 2005 ) High intensity ultrasound is also an effective processing method that could significantly reduce the allergen reactivity Immunoblot and ELISA results showed decreased allergenicity in ultrasound treated shrimp samples ( Zhen xing et al. 2006 ) Furthermore, the same group also demonstrated the allergenic potency was greatly lower ed after a combined irradiation and heat treatment as compared to the raw sample ( Zhen xing et al. 2007a ) In contrast it i s interesting to note that allergenicity of irradiated shrimp was increased up to 10 kGy and then decreased after 15 kGy ( Zhenxing et al. 2007b ) Combine d with our research, pulsed light could sign ificantly reduce the allergenicity of tropomyosin in shrimp extracts after a 4 min treatment under a 10 cm distance from the lamp However, it did not show the obvious reduction in whole shrimp and half cut shrimp Penetration of pulsed light on the surfa ce of shrimp samples was considered as one major reason to prevent further protein inactivation. It is obvious from Figure 6 13 (15 min whole shrimp) and Figure 6 21 (15 min half cut shrimp) that shrimps were burned after extended treatment especially on the surface which ha s a roasted orange red color. The smell of th e pulsed light treated shrimps w as so strong that it had similar smells with that of traditional roasted shrimps. The major allergen of shellfish, tropomyosin, has a molecular weight estima ted to be from 34 38 kDa. A number of IgE binding epitopes have been identified in this protein molecul e though they may vary from one shrimp species to another. P rocessing by cooking may destroy existing epitopes on a protein or may generate new epitop es as
84 a result of change s in the protein conformation ( Maleki 2003 ) In a study that looked at the effect of boiling on these epitopes, the b oiling shrimp extracts showed lower IgE binding than the raw shrimp samples. However, the IgE bind ing activity of tropomyosin increase d in response to longer boiling times. It was proposed that the b oiled tropomyosin may form protein protein interaction s during boiling, which may cause the increase in allergenicity ( Liu and others 2010 ) Lastly another reason that could explain pulsed light effects on the whole shrimp and half cut shrimp and why the a nti genicity did not change as much for SDS PAGE and Western blotting results, it may be due to the presence of other compounds. S ince shrimp is seafood high in water content, that may have caused a lower tempe rature increase and therefore the structure of tropomyosin did not change enough to destroy those conformational epitopes. The linear epitopes on the protein chain could also have not been destroyed to achieve a reduction in allergenicity With regard to the study on gamma irradiation treatment on shrimp ( Zhenxing et al. 2007b ) the resistance of proteins at low dosage levels to denature was suggested to be due to layers of lipids that protect the protein from free radical damage.
85 CHAPTER 7 CONCLUSION S In conclusion we discovered that the effect of PL treatment on native shrimp protein extract, whole shrimp samples, and half cut shrimp samples c ould give different results due to the physical and chemical changes that occur red at the molecular level of proteins. While the native shrimp protein extract elicit ed reduced tropomyosin a nti genicity, as indicated by SDS PAGE, Western blotting, and ELISA, the whole shrimp and half cut shrimp d id not give significant results elsewise under the current conditions tested Protein modification involving protein aggregation and the Maillard reaction, along with other non enzymatic browning reactions are suggested t o play an important role in the availability of epitope regions to the IgE antibodies. In treat ing whole shrimp and half cut shrimp samples, it is suggested that linear epitopes and conformational epitopes could be made more accessible and that the brownin g discoloration that occurred in the longer treated samples could be attributed to the non enzymatic type reactions. PL penetration would also be a factor, as PL technology is regarded as a surface treatment and would not be able to affect the tropomyosin protein that comprises a good portion of the shrimp muscle. On the other hand, for the protein extract, these same changes would give the opposite effect. Not only does the native shrimp protein extract remove the layering effect, in which other compounds that might protect tropomyosin from PL, it also can be subjected to the photo thermal and photo chemical effects of PL to a greater extent. Although we did not elucidate the molecular mechanisms, it is suggested that protein modifications could have resul ted in modifying the allergenic sites such that protein
86 denaturation, fragmentation, and cross linking could have resulted in reducing binding of antibodies to the antigens.
87 CHAPTER 8 FUTURE WORK Regarding future work, more research would need to be don e to support the pulsed light effects on the allergen levels of the whole shrimp with respect to optimization of distance of light from the sample and the time of treatment. A decrease in distance is suggested to determine the effects of pulsed light on allergen changes when the lamp source is closer. PL penetration is only on the surface and is a limitation of the technology. It would also indicate that thinner samples, with a thickness in probably millimeters, might give different results as a thinner sample would be used. The half cut shrimp results were similar to the whole shrimp samples. A sample thinner than half cut shrimp would push the boundary for new product development as shrim p would be prepared in a newer form. A preliminary study was done to investigate the effect of pulsed light on ground shrimp at different volumes. The ground shrimp samples were thinner than the half cut shrimp samples and are discussed below. Furthermore, other future work would involve in vitro digestion research which could also be used to detect tropomyosin allergenicity after digestion If a hypoallergenic food is possible, it could be transitioned over to in vivo testing in animals and eventually hum ans. Sensory testing would also have to be done to evaluate the organoleptic properties and also whether such foods would be considered acceptable for consumers. Lastly, f or human subject studies in sensitized individuals other early studies could focus o n skin prick tests with an extract and later studies could focus on food consumption by such individuals
88 CHAPTER 9 PRELIMINARY RESULTS Following are some preliminary results that were done to simply evaluate the application of pulsed light technology o n ground shrimp samples. Ground shrimp samples are shrimp that are prepared exactly the same way as protein extract samples with the exclusion of the centrifuge step. The shrimp is purchased de headed and de veined. After thawing in a plastic zip bag under water for 1 h, the shrimp is de shelled and then ground in a food processor and homogenized afterwards. The shrimp is ground for 10 min at high speed using 0.6M KCL in 0.01M NaH 2 PO 4 (pH of 7) buffer and homogenized for 10 minutes with a BioSpec BioHomogen izer (Bartlesville, OK, U.S.A.). This ground mixture is then added in different volumes to aluminum plates, 20 mL and 10 mL. The purpose of using ground shrimp was to evaluate the effects of pulsed light on thinner samples. Whole shrimp and half cut shrimp did not show any noticeable reduction, which is due to penetration limitation of pulsed light and therefore thinner samples in the form of ground shrimp were used. In another study that evaluated shrimp allergy ( Zhenxing et al. 2007b ) the researchers found that irradiated shrimp showed an increase in antigenicity at low dosages, but when dosages reached to 10 kGy, antigenicity decreased. This was done on shrimp muscle that had been sliced down to 2 millimeters (mm). Our whole shrimp a nd half cut shrimp sample however was much thicker than 2 mm. Therefore, ground shrimp was prepared and added in small volumes to the aluminum plates and then placed underneath the pulsed light for treatment. 20 mL and 10 mL volumes corresponded to a thick ness of 2 mm and 1 mm in the aluminum plate. The samples were treated for 8, 10, 12, and 15 min for the 20 mL sample and 8 and 15 minute for the 10 mL sample. This refers to a single
89 treatment and not treatment on both sides, as was the case for whole shri mp and half cut shrimp. After treatment, the treated samples were analyzed by SDS PAGE. The SDS PAGE technique is exactly the same as the SDS PAGE for the protein extract, whole shrimp, and half cut shrimp samples. Samples were prepared to a 1:1 ratio usin g 2X Laemmli sample buffer boiled for 10 minutes, and then loaded into the 4 20% Tris glycine minigels. The standard marker was used was Protein Plus All Protein All Blue protein stain. This would help identify the protein of interest. For the loading, 15 uL of each sample and the standard was added to each well. The concentration that was used for all of the proteins was 1:50 for the R and 1:10 for all of the PL treated samples and prepared using PBS 1X buffer. Figure 9 1 SDS PAGE of analysis of protein profile of ground shrimp sam ples comparing 1) raw sample (R) and 2 ) pulsed light treated ground sh rimp samples with times of 8 min, 10 min, 12 min, and 15 min. 15 uL was added to each well with concentrations of 1:50 for the R and 1:10 for the other samples Molecular weight marker (M) is shown on the left. Tropomyosin is pointed as 37 kDa M R R 8 8 10 10 12 12 15 15 37kD
90 Figure 9 2 SDS PAGE analysis of protein profile analysis of ground shrimp samples comparing 1) raw sample (R) and 2 ) pulsed light treated ground shrimp samples with times of 8 min and 15 min. 15 uL was added to each well with concentrations of 1:50 for the R and 1:10 for the other samples Molecular weight marker (M) is shown on the left. Tropomyosin is pointed as 37 kDa Based on the SDS PAGE, no noticeable reduction can be seen on the tropomyosin protein for the 20 mL s ample for all the treatment times On the other hand, based on the SDS PAGE, the 10 mL treated sample does suggest noticeable reduction of the tropomyosin protein at the 15 min mark The 10 mL is thinner than the 20 mL sample and measures at 1 mm, a full m illimeter below the 2 mm sample that was used for the gamma irradiated shrimp. While at 2 mm, gamma irradiation was able to give reduction of shrimp allergen, reduction was not present for pulsed light until a thickness of 1 mm was used. This may be due to the fact that pulsed light measures at a lower frequency than gamma irradiation, where the latter can modify compounds at a more sub cellular level ( Fellows 2009 ) The 20 mL sample may be too thick to allow enough pulsed penetrat ion and other compounds such as lipids or stronger protein linkages may prevent tropomyosin from being modified ( Zhenxing et al. 2007b ) While these M M R R 8 8 15 15 37kD
91 results are preliminary, these conditions do suggest that future work would have to be d one to evaluate the potential of pulsed light technology on ground shrimp.
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102 BIOGRAPHICAL SKETCH Syed Ali Shamikh Abbas was born in Karachi, Pakistan in 1986. He graduated from the University of Florida in 2008 with a b b iology. Afterwards, he took a year off, working and gaining real world experience, before discovering the field of food science. After r ealizing his passion for the field, he entered the academi c arena again and chose to pursue a m f ood s cience. He was accepted into the University of Florida Food Science and Human Nutrition Department, after which he did research on shrimp allerg ens, particularly the major shrimp allergen, tropomyosin. He was an active member in the FSHN Club and also the FSHN Graduate Student Association. He received an assistantship from his major professor, volunteered with the Institute of Food Technologists (IFT) and helped to present at the IFT Annual Meeting and Food Expo 2011 and 2012 He graduated in August 2012 and is now pursing law school at Stetson University to combine his knowledge of f ood science with a legal career.