Pulsed Ultraviolet Light Reduces Immunoglobulin E Binding to Atlantic White Shrimp (Litopenaeus setiferus) Extract

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Pulsed Ultraviolet Light Reduces Immunoglobulin E Binding to Atlantic White Shrimp (Litopenaeus setiferus) Extract
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International Journal of Environmental Research and Public Health
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Shriver, Sandra
Yang, Wade
Chung, Si-Yin
Percival, Susan
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allergen
allergy
shrimp
pulsed ultraviolet light
PUV
tropomyosin
IgE antibodies
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Pulsed ultraviolet light (PUV), a novel food processing and preservation technology, has been shown to reduce allergen levels in peanut and soybean samples. In this study, the efficacy of using PUV to reduce the reactivity of the major shrimp allergen, tropomyosin (36-kDa), and to attenuate immunoglobulin E (IgE) binding to shrimp extract was examined. Atlantic white shrimp (Litopenaeus setiferus) extract was treated with PUV (3 pulses/s, 10 cm from light source) for 4 min. Tropomyosin was compared in the untreated, boiled, PUV-treated and boiled+PUV-treated samples, and changes in the tropomyosin levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IgE binding of the treated extract was analyzed via immunoblot and enzyme-linked immunosorbent assay (ELISA) using pooled human plasma containing IgE antibodies against shrimp allergens. Results showed that levels of tropomyosin and IgE binding were reduced following PUV treatment. However, boiling increased IgE binding, while PUV treatment could offset the increased allergen reactivity caused by boiling. In conclusion, PUV treatment reduced the reactivity of the major shrimp allergen, tropomyosin, and decreased the IgE binding capacity of the shrimp extract.
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Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund.

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Int. J. Environ. Res. Public Health 2011 8 2569 2583 ; doi:10.3390/ijerph80 7 2569 International Journal of Environmental Research and Public Health ISSN 1660 4601 www.mdpi.com/journal/ijerph Article Pulsed Ultraviolet Lig ht Reduces Immunoglobulin E Binding to Atlantic White Shrimp ( Litopenaeus setiferus ) Extract Sandra Shriver 1 Wade Yang 1 *, Si Yin Chung 2 and Susan Percival 1 1 Department of Food Science & Human N u trition, University of Florida, P.O. Box 110370, 359 FSHN Bldg. Newell Drive, Gainesville, FL 32611, USA ; E Mails: sandrakshriver@ufl.edu (S.S.); percival@ufl.edu (S.P.) 2 Southern R egional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Blvd., Bldg 001 SRRC, Ne w Orleans, LA 70124, USA ; E Mail: siyin.chung@ars.usda.gov Author to whom correspondence should be addressed; E Mail: wade.yang@ufl.edu; Tel : +1 352 392 1991 ex t 507; F ax : +1 352 392 9467 Received: 18 May 2011 / Accepted: 19 June 2011 / Published: 24 June 2011 Abstract: Pulsed ultraviolet light (PUV), a novel food processing and preservation technology, has been shown to reduce allergen level s in peanut and soybean samples. In this study, the efficacy of using PUV to reduce the reactivity of the majo r shrimp allergen, tropomyosin (36 kDa), and to attenuate immunoglobulin E (IgE) binding to shrimp extract was examined. Atlantic white shrimp ( Litopenaeus setiferus ) extract was treated with PUV (3 pulses/s, 10 cm from light source) for 4 min. Tropomyosin was compared in the untreated, boiled, PUV treated and [boiled+PUV] treated samples, and changes in the tropomyosin level s were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). IgE binding of the treated extract was anal yzed via immunoblot and enzyme linked immunosorbent assay (ELISA) using pooled human plasma containing IgE antibodies against shrimp allergens. Results showed that levels of tropomyosin and IgE binding were reduced following PUV treatment. However, boiling increased IgE binding, while PUV treatment could offset the increased allergen reactivity caused by boiling In conclusion, PUV treatment reduced the reactivity of the major shrimp allergen, tropomyosin and decreased the IgE binding capacity of the shrimp extract. OPEN ACCESS

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Int. J. Environ. Res. Public Health 2011 8 2570 Keywords: allergen; allergy; shrimp ; pulsed ultraviolet light; PUV, tropomyosin ; IgE antibodies 1 Introduction In the United States, approximately 6% of children and 3.7% of adults are aff ected by one or more food allergies. Eight majo soybean and wheat, account for approximately 85% of all food allergies [1] Allergies to shellfish such as shrimp affect 0.1% of American children and 2% of American adults making shellfish allergies the most common type of food allergy in adults [2 ] The major heat stable allergen of shrimp is a 36 kDa protein known as tropomyosin, also referred to as Sa II or Pen a 1 [3 5] Tropomyosin plays a n important role in musc ular contraction, as well as in the regu lation of cell ular structure and motility [6] Although present in both vertebrates and invertebrates, tropomyosin is known to elicit an aller gic reaction only when it is derived from invertebrate sourc e s such as crustaceans, arachnids, insects and mollusks [5] Two additional shrimp allergens have been identified: myosin li ght chain ( 20 kDa) [7] and arginine kin ase ( 40 kDa) [8 10] H owever, studies suggest that the majority of shrimp allergenicity can be attributed to tropomyosin protein alone [3,5, 11] where tropomyosin was recognized by 82% of patients with shrimp allergies a nd was shown to inhibit IgE binding to whole body shrimp extract in 85 95 % of patients. According to Jeong and others [6] t he most frequent symptoms of shrimp induced allergies include itching, hives, swelling of the lips and tongue, pulmonary symptoms, gastrointestinal symptoms, and anaphylactic shock Methods su ch as oral and sublingual i mmunotherapy have been used in attempt to prevent shrimp induced allergies yet the only completely effective approach to date is total avoidance [12] Complete a voidance is often difficult and inconvenient considering the prevalence of food allergens in a multitude of products. Thus, r esearchers are seeking diverse ways such as post harvest treatment methods, to reduce the allergen reactivity of food products before they reach the consumer When an allergen enters the body, i mmunoglobulin E (IgE) antibodies elicit an immune response by binding to s pecific epitopes on an allergen, which can be linear or conformational [13] P ostharvest methods have the potential to alter these epitopes by disrupting or masking amino acid sequences (e.g., protein fragmentation or genetic modification) or by altering the conformat ion of the protein (e.g., protein denaturation, protein crosslinking, or aggregation). Postharvest methods, including power ultrasound [14] gamma irradi ation [15] and high hydrostatic pressure processing [16] have been shown to al ter allergen reactivity by modifyin g allergen structure. M ore r ecently, pulsed ultraviolet light (PUV) an emerging technology, has been employed to reduce the allergen reactivity of peanut products [17 1 8 ] and soy extract s [1 9 ] Pulsed ultraviolet light is considered more effective in food processing (specifically microbial inactivation) than convent ional, or continuous UV light, because of its instantaneous high energy pulses and greater capability to penetrat e In a PUV system, electrical energy is captured and stored in a capacitor and is ultimately released in short pulse s as ultravio let infrared and visible light ( approximately 54%, 2 0 % and 2 6 % respectively) [ 20 ] The resultant burst s can be several thousand

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Int. J. Environ. Res. Public Health 2011 8 2571 times more intense than continuous UV light [ 21 ] Upon coming into contact with a sample the light interacts wi th m olecules, which are excited and upon returning to ground state liberate energy as photons or heat, which can induce chemical changes. T hus t he efficacy of P UV has been attributed to photochemical, photothermal, and photo physical reactions [ 21 ] These effects may contribute to changes in protein structure and reduction in IgE binding to allergens. At a shorter exposure (e.g., seconds), PUV is normally regarded as nonthermal, as the temperature rise of food is insignificant. H owever, at a longer e xposure (e.g., minutes) PUV can generate significant photothermal effect s and incur considerable temperature rise and moisture loss t o the sample [17 19, 22 25 ] It has also been found that prolonged UV light treatment caused formation of insoluble complex es in food, depolymerization of starch, peroxidation of unsaturated fatty acid s carbohydrate crosslinking, protein crosslinking, and protein fragmentation [ 26 29 ] The significant photothermal effect of PUV after an extended exposure enabl es the PUV technology to be applied directly to solid food s, e.g., whole almond [ 2 3 ] fo r allergen mitigation. Li [ 2 3 ] exposed the whole almond k ernels to PUV for 4 7 min It was found the whole almond treated with PUV for 4 min had a desirable and plea surable roasted almond flavor and taste and its IgE biding capacity was pronouncedly reduced, although the exact mechanism of PUV interacton s with the solids is still unknown. Based on the studies of PUV treatment on peanut and soybean allergens [17 19 ] we hypothesized that PUV treatment of Atlantic white shrimp extract would alter the reactivity of the major shrimp allergen, tropomyosin and consequently reduce the overall allergenic pote ntial of the shrimp extract Our objective w as to exa mine the efficacy of PUV exposure on the inactivation of major shrimp allergen by measuring changes in tropomyosin level and IgE binding 2. Experimental Section 2.1. Materials Frozen Atlantic white shrimp ( Litopenaeus s etiferus ) were purchased de headed and shelled from Publix Supermarkets, Inc. (Lakeland, FL). Coomassie Plus (Bradford) Protein assay, bovine serum albumin (BSA), StartingBlock/Tris buffered saline/Tween 20 (StartingBlock) GelCode Blue gel staining reagen t o phenylenediamine dihydrochloride (OPD) and SuperSignal We st Pico Chemiluminescent (ECL) s ubstrate were purchased from Thermo Fisher Scientific Inc. (Rockford, IL). Electrophoresis equipment and reagents including pre cast Tris HCL minigels (4 15%), M ini PROTEAN Tetra cell tanks, Laemmli sample buffer, Tris glycine transfer buffer, Tris glycine SDS running buffer, nitrocellulose membrane and Trans blot SD Semi dry Transfer cell s were purchased from Bio Rad Laboratories, Inc. (Hercules, CA). Pooled human plasma from 3 patients with history of shrimp allergy was obtained from PlasmaLabs International (Everett, WA) for immunoblo t ti ng and enzyme linked immunosorbent assay ( ELISA ) The plasma specific IgE level for pooled human plasma with antibodies specific to shrimp was measured by the ImmunoCAP method performed by PlasmaLabs and was det ermined to be 92 k U L 1 A s econdary detecti on antibody, mouse anti human IgE conjugated to horseradish peroxidase (HRP), was obtained from Invitrogen (Carlsbad, CA). Rat monoclonal anti tropomyosin (IgG isotype) and rabbit polyclonal anti rat IgG H&L conjugated to HRP antibodies were purchased fro m Abcam Inc. (Cambridge, MA).

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Int. J. Environ. Res. Public Health 2011 8 2572 Costar Enzyme Immunoassay (EIA) polystyrene 96 well plates (Corning, NY) and Immobilon P blotting polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Bedford, MA) were used for immunological assay s. 2.2 Preparation of Atlantic White Shrimp Crude Protein Extract Shrimp e xtract was prepared following the methods of Motoyama and others [ 30,31 ] with modification Briefly, s hrimp (25 g) was ground in a food processor at low speed for 10 s A volume of 0.6 M KCl in 0.01 M phosphate buffer ( 200 mL, pH 7) w as added to the processed tissue, and the mixture was homogenized at the high speed setting (10,000 rpm) for 1 min using a BioSpec BioH omogenizer (Bartlesville, OK) The buffer was carefully selected in order to solubilize the major allergen while maintaining its native s tate Protein concentration was measured with Bradfor d assay using BSA protein standards. The extract was diluted to a concentration of 5 mg/mL with 0.6 M KCl in 0.01 M phosphate buffer (pH 7). Samples were treated immediately or stored packaged on ice in a s tyrofoam container placed a t 4 C for no longer th an one week. 2.3 P reparation of Boiled Shrimp Extract A common way of preparing shrimp is by boiling, but l iterature shows the allergen immunoactivity of shrimp is not reduced in general or in some cases is even increased by boiling [3 2 3 4 ] C onsu mption of boiled or steamed shrimp resulted in more severe allergy skin test responses in some patients than raw shrimp [ 3 3 3 4 ] Liu and others [ 3 5 ] reported that a lthough iELISA d emonstrated that the raw shrimp extracts had higher IgE binding than t he boiled shrimp extracts, dot blot results showed higher IgE binding to the purified tropmyosin from boiled shrimp than raw shrimp. In this study, boiled shrimp extract s were also prepared by heating in boiling water for 4 min and then tested f or its IgE binding capacity, which was compared to the PUV treated samples. A volume of 10 mL of crude shrimp protein extract was placed in b oiling water for 4 min in a loosely c apped 15 mL centrifuge tube Following heat treatment s amples were cooled on ice, and pr otein concentra tion was measured 2.4 Treatment of Shrimp Extract with PUV Raw and boiled samples (10 mL 5 mg/mL ) were treated with a Xenon Steripulse XL RS 3000 batch PUV ster ilization unit (Wilmington, MA) as descr ibed by Chung and others [17] to examine the effect of PUV on the immunoreactivity of both raw and boiled shrimp samples The treatme nt of boiling followed by PUV was to exe mine if synergistic or antagonistic eff ect on allergen reactivity exist ed between boiling and PUV treatments. Pulses were emitted at a rate of 3/s with a pulse width of 360 s, and samples were positioned at a distance of 10 cm from the quartz window of the PUV lamp in aluminum dishes with a d iameter of 7.2 cm Under these conditions the maximum energy lev el of the emitted radiation was 0.27 J/cm 2 per pulse as per the factory calibration Temperature of the samples was recorded using an Omega OS423 LS non contact infrared thermometer (Omega Engineering, Inc., Stamford, CT ), and the samples were cooled on ice following treatment. Changes in volume were measured and p ro tein concentration was determined with Bradford assay

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Int. J. Environ. Res. Public Health 2011 8 2573 2.5 Electrophoresis of Treated Shrimp Extract Samples were analyzed under reducing conditions as described by Laemmli [ 36 ] Briefly, a sample containing protein (12 g) was combined with sample buffer (62.5 mM Tris HCL pH 6. 8, 2% SDS, 25% glycerol, 0.01% bromophenol b lue 0.05% mercaptoethanol) in a microcentrifuge tube. The mixture was heated in boiling water for 5 min The sample was then subjected to electrophoresis within a 4 15% Tris glycine gel for 1.5 h at 15 0 V endations Subsequently, t he gel was stained with GelCode Blue reagent for 2 h and destained for 1 h with deionized water. The protein bands were scanned with a Canon Pixma MP160 scanner 2.6 Determinatio n of IgE and IgG Binding to Tropomyosin with Weste rn Blot Following electrophoresis, proteins were transferred onto a PVDF blotting membrane at 15 V for 30 min Nonspecific binding sites were blocked for 1 h at room temperature ( RT ) with StartingB lock blocking buffer. The membrane was incubated overnight at 4 C with pooled human plasma containing anti shrimp IgE antibodies (1:80) diluted in blocking buffer After washing with Tris buffered saline containing 0.1% Tween 20 (TBST), t he blot was then incub ated in mouse anti human IgE HRP diluted 1:1,000 in bl ocking buffer for 1 h at RT The blot was again washed, and incubated in Super Signal West Pico Chemiluminescent substrate for 5 min at RT The protein bands were developed on an X ray film. To visualize changes in tropomyosin band intensity the above pro cedure was replicated replacing the primary antibody with rat monoclonal anti tropomyosin (IgG) (1:1 000) and the secondary antibody with ra bbit polyclonal anti rat IgG HRP (1:40,000) 2.7 Determination of IgE Binding to Treated Shrimp Extract with Dot Blot A n itrocellulose membrane was blotted with 1. 2 5 and 2.5 g of raw, boiled, PUV treat ed, and [ boiled+ PUV ] treated shrimp extract protein and allowed to dry at 4 C. Following blocking with StartingB lock blocking buffer the blot was incubated overnight at 4 C in a pooled human plasma containing anti shrimp IgE antibodies (1:80) diluted in blocking buffer T he blot was washed and then in cubated in mouse anti human IgE HRP diluted 1:1 000 in blocking buffer ( 1 h ; RT) The blot was again washed, and incub ated in Super Signal West Pico Chemiluminescent substrate for 5 min at RT The protein spots were developed on an x ray film. 2.8 Determination of IgE Binding to Treated Shrimp Extracts with Indirect ELISA P olystyrene 96 well plates were coated overnight a t 4 C with untreated boiled, PUV treated and [ boiled+ PUV ] treated shrimp extract diluted in phosphate buffered saline ( PBS ) to a concentration of 20 per well in triplicates ). The plate s were subsequently washed with TBST and blocked with StartingBlock blocking buffer per well) at room temperat ure for 2 3 h P ooled hum an plasma containing IgE antibodies specific for shrimp allerge ns was diluted in PBS (1:1 0) and added in e qual amounts to each well ( 10 ) T he plate was incubated at room temperature with gentle shaking for 1 h After washing with TBST e ach well was then incubated with secondary antibody, monoclonal mouse anti hum an IgE conjugated to HRP ( 1:3, 00 0 ), for 1 h per well) with gentle shaking The wells were again washed, and an OPD substrate (0.5 mg/mL ) dissolved in

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Int. J. Environ. Res. Public Health 2011 8 2574 0.1 M citrate buffer (pH 5.5) and 0. 0 3% hydrogen peroxide was added to each well (1 per well) The reaction was stopped at 15 30 min with 2.5 N sulfur per we ll), and a bsorbance was measured at 490 nm using a Spectramax 340 384 spectrophotometer (Molecular Devices, Inc. Sunnyvale, CA) 2.9 Statistical Analysis Statistical analysi s was conducted using one way analysis of variance (ANOVA) with the SAS 9.2 software package (Cary, N.C.) S ignificant between means of the untreated (control) and treated ( boiled, PUV treated, and [b oiled+PUV] treated ) samples for t otal IgE binding were determined using least significant difference (LSD) test s 3. Results and Discussion 3.1 Optimal PUV Treatment Time for Shrimp Extract To establish an appropriate treatment time, s everal time courses for PUV treatment were tested. SDS PAGE and Western blot analyse s were used to determine the minimum exposure time at which PUV treatment led to a reduction in both tropomyosin level and IgE binding An SDS PAGE analysis (Figure 1) illustrated that tropomyosin bands (36 kDa) remained in the extract following PUV treatment s of 1 2 and 3 min but were reduced in samples treated for 4 6 min Densitometry analysis verified the reduction in tropomyosin band intensity at 4 6 min. It is noted that Figure 1 also show s other two minor allergenic proteins (16.5 kDa and 20 kDa), which have also be en reported in other studies [ 37 ]. Figure 1. An SDS PAGE profile of shrimp extract treated with PUV at 0 1, 2, 3, 4, 5, and 6 min. Molecular weight marker (M) is shown. The band corresponding to tropomyosin (36 kDa) is highlighted with an arrow. Western blot analysis o f the PUV treated extracts at 0 6 min (Figure 2) showed that IgE binding to tropom yosin was markedly reduced at 4 6 min, comp ared to 0 3 min. Because pattern s between

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Int. J. Environ. Res. Public Health 2011 8 2575 4 and 6 min were similar and a higher moisture loss was observed at 6 min, 4 min was chosen as the optimal treatment time for subsequent experimentation. Figure 2. Western blot analysis of shrimp extract samples treated with PUV at 0 (control), 1, 2, 3, 4, 5, and 6 min using pooled human plasma from 3 individuals containing IgE antibodies against shrimp. T ropomyosin bands (36 kDa) are highlighted within a box. 3. 2 Changes in Tropomyosin Band Intensity of Untreated, Boiled, P UV Treated and [Boiled+ PUV ] Treated Shrimp Extracts As illustrated in the SDS PAGE profile ( Figure 3 ) a 36 kDa band representing tropomyosin w as present following treatments with boiling, PUV and boiling+PUV A decrease in tropomyosin was observed in both PUV and [ boiled+PUV ] treated samples (l anes 3 and 4) whereas the boiled extract (lane 2) did not show a change in tropomyosin density compared to the control These results were verifi ed using densitometry analysis. Figure 3. An SDS PAGE profile of untreated (1) boiled (2), PUV treated (3), and [ boiled+PUV ] treated (4) shrimp extract s. Molecular weight marker is shown (M). An arrow highlights the bands corresponding to tropomyosin (36 kDa).

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Int. J. Environ. Res. Public Health 2011 8 2576 Like Figure 1, Figure 3 also shows the behavior of other two allergen ic proteins (16.5 kDa and 20 kDa) under boiling and PUV treatments. The 20 kDa protein followed a similar trend to 16.5 kDa protein, but PUV caused it to be al most undetectable as illustrated on lanes 3 and 4. Resistance to thermal denaturation or degradation is characteristic of the tropomyosin protein, as it is recognized for its heat stability [3] However, instantaneous pulses of energy generated during prolonged PUV treatment may cause more intense localized heating [ 38 39 ] which may contribute to the reduction of tropomyosin. On the othe r hand, UV exposure, which is basically nonthermal, can also cause protein crosslinking or fragmentation [ 27 29 ]. So, we believe both the nonthermal and photothermal effects of PUV played a role in reducing the aller genic reactivity of shrimp proteins in this study. 3.3 IgE and IgG binding to Untreated Boiled PUV treated, and [ Boiled + PUV ] Treated Shrimp Extracts 3.3.1 Western Blot The four treated samples were analyzed with Western blot using IgE antibodies against shrimp, to evaluate allergen r eactivity, and an anti tropomyosin antibody (IgG) to evaluate tropomyosin levels. Figure 4 demonstrates a notable decrease in IgE binding and tropomyosin levels following PUV treatment as evidenced by decreases in band intens ity The marked difference be tween the control and PUV treatment further supports the SDS PAGE data (Figure 1) that tropomyosin levels were indeed reduced at 4 min B oiling however, appeared to increase IgE binding, based on densitometry analysis of the control versus boiled. Figure 4. Western blot s using ( a ) pooled human plasma containing IgE antibodies against shrimp and ( b ) monoclonal anti tropomyosin antibody (IgG) to analyze u ntreated (1), boiled (2), PUV treated (3) and [ boiled+PUV ] treated (4) shrimp extracts Tropomyosin (36 kDa) is highlighted using a box. Presently, it is still unknown exactly how the IgE binding was reduced by PUV radiation, but it is believed to relate to l ocalized phot o chemical photothermal and photo physical effects of PUV which,

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Int. J. Environ. Res. Public Health 2011 8 2577 as mentioned earlie r, can cause protein modif ications including protein fragment ation denaturation, or cross linking and affect IgE binding F ragmented protein sections that are smaller than the resolution limit of the gel may travel through the acrylamide pores more quickl y causing them to ult imately be lost in to the chamber buffer. Conversely, proteins that have been modified by cross linking are often too large to migrate through the gel or migrate with slight difficulty, thereby resulting in a smeared appearance in SDS P AGE and Western blots. Chung and others [ 40 ] have demonstrated the presence of protein smears due to the cross linking of peanut allergens caused by enzymatic reactions. Also, protein band smearing h as been observed in the allergens of peanuts that have been heated or roasted [ 41 ] While band smearing was not detected in the blot with IgE (Figure 4a), they can be seen in blots using an anti tropomyosin antibody. As shown in Figure 4b, tropomyosin bands and others were notably smeared in PUV treated samples, but not in boiled and co ntrol samples. This finding indicates that modification or cross linking of tropomyosin might have occurred during the PUV treatment. Additionally, Taheri Kafrani and others [ 42 ] have illustrated the smearing of m ilk allergens that have been modified via the Maillard reaction, which is a non enzymatic browning or protein carbohydrate reaction. Several studies [ 42 44 ] have linked glycation or Maillard reaction to the mod ification of allergens and their subsequent changes in IgE binding. A study on squid tropomyosin [ 44 ] found that IgE binding was suppressed with the progression of the Maillard reaction. However, in one study u sing scallop tropomyosin [ 43 ] an increase in the allergen potency was d isplayed with the progression of Maillard reaction. Whether Maillard reaction could occur in the shrimp extract during PUV treatment is not known. Considering the high energy produc ed by PUV and the instantaneous heat absorbed by the molecules [ 39 ] ( i.e. proteins/carbohydrate in the shrimp extracts), it is possible that a Maillard reaction may occur, but such a postulation needs to be further investiga ted 3.3.2 Dot Blot Dot blot analysis was performed to determine the allergen reactivity of the shrimp protein extract as a whole that is tropomyosin and other possible allergens that were not dete cted via Western blot analysis IgE binding to whole shri mp extract was greatly reduced following PUV treatment (Figure 5 ). However, there was an increase in IgE binding followin g boiling treatment. Interestingly PUV appeared to attenuate or negate the boiling effect, a s shown in the [ boiled+ PUV ] treated sample ; IgE binding was notably reduced compared to the boiled only sample. Of all the treatments, PUV alone displayed the most reduction in IgE binding. The finding that boiling or heating can lead to an increase in IgE binding (Figure 5) is not unusual, becaus e t he effect s of thermal processing on food allergen s have been studied extensively, and, in different studies, heating has been shown to either decrease or increase allergen potency. For example, one study [ 41 ] describe d a 90 fold increase in IgE binding of roasted peanuts over r aw peanuts, whereas another study reported that roasting actually decrease d the overall allergen reactivity of hazelnuts [ 45 ] It has also been noted that children with milk allergies show a tolerance to extensively heated milk [ 46 ] In this dot blot analysis (Figure 5) boiling caused an increase in IgE binding possibly not just due to tropomyosin itself, but also due to other proteins present in the whole extract. These proteins could include a rginine kinase (40 kDa) [8, 9] and myosin light chain (20 kDa) [7 38 ] which have both been shown to play a minor role in shrimp allergy. Also, the minor 16.5 kDa pr otein was also persistent during the boiling treatment (Fig ure 3). The effects of boiling on shrimp reactivity

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Int. J. Environ. Res. Public Health 2011 8 2578 are consistent with the results of Carnes and others [3 2 ] who also found that boiled shrimp extracts w ere more immunoreactive in both in vivo skin prick trials and in vitro direct ELISA results. Figure 5. Dot blot analysis of untreated, boiled, PUV treated, and [ boiled+PUV ] treated shrimp extract using pooled human plasma containing IgE antibodies against shrimp. 3.3.3 Indirect ELISA To support the dot blot data in Figure 4, IgE binding of the four t reated shrimp extracts was also determined us in the PUV treated extract, compared to the control (Figure 6). An increase in IgE binding was 0.05) in IgE binding of the [boiled+PUV] treated extract, as compared to the control. The finding was in agreement with the data of dot blot (Figure 4). Figure 6. Indirect ELISA illustrating changes in IgE binding compared to untreated, boiled, PUV treate d, and [ boiled+PUV ] treated shrimp extracts using pooled human plasma containing IgE antibodies against shr imp. A = absorbance of the sample; A 0 = absorbance of untreated sample. Data are expressed as mean SEM (n = 5). R esults are relative values normali zed to the untreated sample; untreated is standardized and set to 1. V alues that are significantly diff are annotated as **

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Int. J. Environ. Res. Public Health 2011 8 2579 Reduction in IgE binding of PUV treated extract was likely explained by changes in the amount of detectable tropomyosin described above in SDS PAGE (Figures 1 and 3) and Western blot analysis (Figures 2 and 4). In the [boiled+PUV] treated sample, IgE binding did not appear to change, compared to the control This was because tropomyosin inc reased after boilng but was offset to the control level after the PUV treatment That is to say, t he increase and decrease in IgE binding, respectively, due to boiling and PUV t reatment, may negate each other, thus resulting in a negligible change in IgE b inding of the [boiled+PUV] extract. Such an effect may be deemed as an antagonistic effect. 3.4 Temperature and Volume Changes Following Treatment of Shrimp Extract As mentioned earlier, PUV is considered a nonthermal method when used for brief periods of time (several seconds); however, following a PUV treatment period of 4 min, a sample surface temperature of 68.3 2.5 C was detected using an infrared thermometer immediately after the PUV pulses stopped and the treatment chamber door was opened. It mus t be noted that there was a 5 10 s delay in probing the sample surface temperature, while the instantaneous temperatures of the sample during t he PUV treatment c ould be higher. Previous trials using thermocouples to monitor temperature during PUV treatmen t were unsuccessful, because extended PUV exposure of the metal probe confounded the readings. Fiber optic temperature sensing may be a way to go for recording accurately the sample temperature during PUV treatment, which was not conducted in this study. F or shorter PUV treatments, a type K thermocouple produced by Omega Engineering, Inc. (Stanford, CT), has been utilized [ 47 ] for temperature mea sur ement without confounding a complication in temperature reading. A study that analyzed PUV treatment for decontamination of shell eggs [ 47 ] reported an i ncreased surface temperature of 10.5 1.2 C after 30 s at 9.5 cm distance from the quartz window of the PUV lamp. Following boiling, PUV, and [boiling+PUV] treatments, moisture loss in each sample was measured: boiled only (5.83 2.3%), PUV only (29 3.6%), and boiled with PUV (39.7 2.5%). Moisture loss was higher in the PUV treated samples because the samples were not enclosed during PUV treatment (the purpose was to ensure the maximum ab sorption of the PUV radiation). By contrast, moisture loss wa s minimal in the boiled sample because the samples were loosely capped during boiling. Chung and others [17] also noted volume reductions of approximately 40% following PUV treatment. To correct for moisture loss in the samples, protein measurements were taken after treatments, and these values were used for subsequent experiments. Excessive m oisture loss is indicative of a sample temperature during the PUV tre atment that was well above the boiling point of water at atmosphere, which caused the water to evaporate. The significant temperature increases due to the photothermal effect of PUV and the capability of PUV in mitigating allergens may potentially be used in conjunction with food preparation to simultaneously heat the food and reduce its allergenic potency Li [ 2 3 ] took advantage of this unique feature of PUV radiation to desirably roast the whole almond after 4 7 min exposure and yet considerably reduce its IgE biding capacity This can also be a potential method to cook the peeled whole shrimp and significantly reduce its allergen although this idea was not tested yet in this study.

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Int. J. Environ. Res. Public Health 2011 8 2580 4. Conclusions A marked decrease in IgE binding of the shrimp extract following PUV treatment has been demonstrated. The decrease was likely due to a reduction in the detectable level of tropomyosin in the PUV treated extract as shown in SDS PAGE (Figure 1) and Western blot (Figure 2). Furthermore, the appearance of protein band smearing, as illustrated in Figure 4, is likely due to the m odification, such as crosslinking, of tropomyosin and may contribute to the decrease in IgE binding. Boiling increased IgE binding to whole shrimp sample ; however, the effect of boiling was o ffset when it was combined with PUV treatment. Overall, PUV was found to be capable of reducing the allergenic potency of shrimp extracts. Further optimization is still needed before the PUV technology can be adopted. In vivo studies are also needed to ver ify the reduction in allergenic potency of the PUV treated shrimp extract s Acknowledgment s The authors gratefully acknowledge Melanie Correll, University of Florida, Department of Agricultural and Biological Engineering, for her intellectual input concerni ng this manuscript References 1. Ellman, L. K .; Chatchatee, P.; Sicherer, S.H.; Sampson, H.A. Food hypersensitivity in two groups of children and young adults with atopic dermatitis evaluated a decade apart. Pediatr Allergy Immunol 2002 13 295 298. 2. Sicherer S.H.; Sampson, H.A. Food allergy. J Allergy Clin Immunol 2010 125 S116 125. 3. Shanti, K.N.; Martin, B.M.; Nagpal, S.; Metcalfe, D.D.; Rao, P. V., Identification of tropomyosin as the major shrimp allergen and character ization of its IgE binding epitopes. J Immunol 1993 151 5354 5363. 4. Jeoung, B. J.; R eese, G.; Hauck, P.; Oliver, J.B.; Daul, C.B.; Lehrer, S. B. Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody based sand wich ELISA. J Allergy Clin Immunol 1997 100 229 234. 5. R eese, G.; Ayuso, R.; Lehrer, S.B. Tropomyosin: an invertebrate pan allergen. Int Arch Allergy Immunol 1999 119 247 258. 6. Jeong, K.Y.; Hong, C.S.; Yong, T.S. Allergenic tropomyosins and their cross reactivities. Protein Pept Lett 2006 13 835 845. 7. Ayuso, R.; Grishina, G.; Bardina, L.; Carri llo, T.; Blanco, C.; Ibanez, M.D.; Sampson, H.A.; Beyer, K. Myosin light chain is a novel shrimp allergen, Lit v 3. J Allergy Clin Immunol 20 08 122 795 802. 8. Yu, C. J .; Lin, Y.F.; Chiang, B.L.; Chow, L.P. Proteomics and immunological analysis of a novel shrimp allergen, Pen m 2. J Immunol 2003 170 445 453. 9. Garcia Orozco, K. D.; Aispuro Hernandez, E.; Yepiz Plascenci a, G.; Calderon de l a Barca, A. M.; Sotelo Mu ndo, R.R. Molecular characterization of arginine kinase, an allergen from the shrimp Litopenaeus vannamei. Int Arch Allergy Immunol 2007 144 23 28.

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