Directive EC 2003/89/EC of the European Parliament and of the Council states that certain ingredients and products derived there of known to cause allergen reactions must always be declared. Furthermore labelling is mandatory irrespective of the amount included. The National Food Administration therefore needs methods for monitoring the presence of allergens in food. Methods already exist for most of the allergens on the EU-list, but an operational method for celery (Apium graveolens) is missing. A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be specific for celery, producing a 113 bp fragment with two celery varieties and negative results with other closely selected species commonly present together with celery in food products (12 samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome copies. When evaluated with model samples of celery in meat, a detection limit of less than 0,01 % was determined. When used to analyse food products from the market, six out of seven products declared to contain celery were correctly identified as positive.

A new European labeling directive (2003/89/EC) states that certain foods and products derived thereof must always be declared. Among the tree nuts specified is Macadamia nut (Macadamia spp.). During the last few years, cases of IgE-allergic reactions, even severe anaphylaxes, have been reported. Reliable methods for the detection of this nut are needed. Protein from Macadamia nuts was isolated. Polyacrylamide gel electrophoresis in SDS revealed two main protein bands of about 20 and 50kDa. These protein bands were cut and extracted from the gel and rabbits were immunized with each protein. Immunoblotting showed dominant reactivity with the respective antigens. The antisera were further tested for specificity in immunodiffusion and in rocket immunoelectrophoresis. In addition, a specific DNA-method was developed, based on Real-Time PCR using Macadamia vicilin as target sequence. Two different primer pairs were tested. Specificity was tested against potentially related nuts. Optimisation of primer and probe concentrations was performed. The limit of detection was 2-4 pg DNA, corresponding to a macadamia nut concentration of 50 to 100 μg per g. In a background of soybean DNA, down to 0,01 % macadamia DNA could be detected.

The present study examines three non-marital, romantic relationship dissolution types: (1) normal dyadic dissolution, (2) fatal attraction dissolution, and (3) social allergen dissolution among a sample of 321 emerging adults, who are between ages eighteen and twenty-nine. Results of an online survey revealed that normal dyadic dissolution occurred in 62%, social allergen dissolution occurred in 27%, and fatal attraction dissolution occurred in 11% of the participants’ relational demises. Results also revealed that there is a surprising amount of overlap between the three dissolution types, and that age is not specifically correlated with a particular dissolution type. However, there are two predicators of dissolution type within an emerging adult population: perceived family support and whether or not the couple cohabitate.

Milk is one of the eight major food allergens. Cow's milk allergy is the most common allergy in children under 2 years of age. About 1.6 to 2.8 percent of children under this age are reported to have cow's milk allergy. Casein, β-lactoglobulin and α-lactalbumin are major milk protein allergens. Nonthermal treatments like high intensity ultrasound, ultraviolet (UV) light and nonthermal plasma treatments have been reported in the literature to be effective in reducing the allergenicity of different food proteins. Hence it was expected for these treatments to reduce cow milk allergenicity. The objective of this study was to investigate the effect of high intensity ultrasound, nonthermal atmospheric plasma and UV-C light treatments in reducing the allergenicity of isolated major milk proteins. Sonics Vibracell VC 505 ultrasonic liquid processor was used to perform high intensity ultrasound treatments. UV light treatments were performed using a DDK Scientific Corporation UV tunnel. A nonthermal atmospheric plasma setup assembled in Department of Microbiology lab was used to perform plasma treatments. Samples were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate the change in protein concentration and enzyme linked immuno sorbent assays (ELISA) to observe the change in IgE binding. A one-way analysis of variance was conducted to evaluate the relationship between treatment time and percent IgE binding at 95% confidence level. Further investigation was conducted with nuclear magnetic resonance (NMR) spectroscopy on treated casein to assess any change in the structure of protein. SDS-PAGE results for ultrasound and plasma treatments didn't show any change in gel band intensities for casein, β-lactoglobulin and α-lactalbumin indicating no significant change in protein concentration. Ci-ELISA analysis showed that there was no significant difference (p>0.05) in IgE binding values for control and treated samples in ultrasound and plasma treatment conditions tested in this study. The intensities of all the three protein bands in SDS-PAGE gel were reduced by UV-C light treatment at 15 min treatment time. In Ci-ELISA, there was a significant difference (p< 0.05) in IgE binding values for control and treated samples and a reduction in allergenicity of proteins (25% reduction for casein and 28% reduction for whey protein fractions) was observed. Further investigations using in vivo clinical trials need to be conducted to confirm this result. NMR results didn't show any noticeable changes in the structures of casein with all three different treatments. In conclusion, UV-C light treatment can reduce the allergenicity of isolated major milk proteins to some extent. High intensity ultrasound and nonthermal atmospheric plasma treatments failed to generate effective results for reducing allergenicity at the conditions tested in this study. Higher intensity and longer treatment conditions might yield better results with ultrasound treatment. Different power and gas flow rates used to generate plasma with direct exposure of proteins might yield better results towards reducing the allergenicity of major milk allergens.

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-03Bitstream added on 2014-06-13T20:29:34Z : No. of bitstreams: 1 pinto_jras_me_rcla.pdf: 1057969 bytes, checksum: 2e29026d533c6190983ed253a062779d (MD5) / Os venenos dos insetos da ordem Hymenoptera contêm uma variedade de proteínas alergênicas. As ferroadas desses insetos podem induzir reações alérgicas e ocasionalmente, anafilaxias fatais. Há um crescente interesse nos componentes químicos dos venenos desses insetos, sobretudo no campo da alergia e imunologia clínica. As principais reações desses venenos são as reações inflamatórias e/ou imunológicas em suas vítimas, podendo ocorrer alguns efeitos sistêmicos. Dentre os Hymenoptera sociais, os venenos de abelhas e vespas têm sido extensivamente estudados e muitos de seus componentes moleculares já foram isolados e identificados. Fosfolipase, hialuronidase, antígeno-5 e serinoprotease são proteínas antigênicas de elevada massa molecular que, quando injetadas durante o ato de ferroar, iniciam uma resposta imune peculiar, sensibilizando alguns indivíduos. Porém, poucos estudos têm sido realizados para a identificação e o mapeamento dos epítopos desses alérgenos. O conhecimento sobre a interação molecular dos principais alérgenos destes venenos certamente deverá contribuir para o aperfeiçoamento dos diagnósticos de alergia, bem como para o desenvolvimento de tratamentos mais seletivos de reações alérgicas mediadas por IgE. No presente estudo identificamos e mapeamos os epítopos lineares de células-B do antígeno-5 do veneno da vespa social Polybia paulista. O antígeno-5 é conhecido como um dos principais alérgenos do veneno de Hymenoptera com massa molecular em torno de 23 kDa e função biológica desconhecida, porém muitos estudos demonstram a sua alergenicidade. Tendo conhecimento da sequência primária do antígeno-5, a identificação e o mapeamento dos epítopos foram realizados através da síntese múltipla de peptídeos, utilizando as técnicas do SPOT-Synthesis, imunodetecção e método indireto do ELISA com soro de pacientes sensíveis... / The venom insects of Hymenoptera order contain a variety of allergenic proteins. The stings of these insects can induce allergic reactions and occasionally fatal anaphylaxis. There is a growing interest in the knowledge about the chemical components of the venom of these insects, especially in the field of allergy and clinical immunology. The main reactions of these venoms are inflammation and / or the induction of immune process in the victims; sometimes systemic effects also may be observed. Among the social Hymenoptera, the venoms of bees and wasps have been extensively studied and many of their molecular components have been isolated and identified. Phospholipase, hyaluronidase, antigen-5 and serine protease are antigenic proteins of high molecular weight, which may initiate a peculiar immune response to sensitize some individuals. However, few studies have been performed for the identification and mapping of the epitopes these allergens. The knowledge about the molecular interaction of the major allergens of these venoms with IgG and/or IgE will certainly contribute for improving the diagnosis of allergy, as well as to develop more selective treatments of reactions IgE-mediated allergy. In this study we identified and mapped the linear epitopes of B-cells of the antigen-5 from the venom of the social wasp Polybia paulista. The antigen-5 is known as one of the major allergens from Hymenoptera venoms with molecular weight around 23 kDa and unknown biological function, but many studies show its allergenicity. The aim of this study was to identify and to map the linear epitopes of B-cells of this allergen. Taking into account the knowledge the primary sequence of antigen-5, the identification and mapping of epitopes was performed by using multiple synthesis of peptides with the combination of different protocols: SPOT-Synthesis, immunoblotting and indirect method of ELISA with the sera... (Complete abstract click electronic access below)

Orientador: Flavia Maria Netto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T21:18:27Z (GMT). No. of bitstreams: 1 Pessato_TassiaBatista_M.pdf: 1693373 bytes, checksum: c84093b37be90c1b31ce17c6722db6a1 (MD5) Previous issue date: 2014 / Resumo: O leite bovino é um dos alimentos mais alergênicos na primeira infância, sendo as caseínas e as duas principais proteínas do soro - ?-lactoalbumina (?-La) e ?-lactoglobulina (?-Lg) - os principais alérgenos. A hidrólise enzimática reduz a antigenicidade de proteínas e pode ser realizada com enzima livre ou imobilizada. A enzima imobilizada não requer inativação ao final da hidrólise, sendo retirada por filtração enquanto que a enzima livre precisa ser inativada, geralmente por aquecimento, para interromper a reação. Diferenças na atividade catalítica e de inativação podem acarretar em características diferentes dos hidrolisados formados. O objetivo desse trabalho foi avaliar os efeitos da hidrólise do isolado proteico de soro de leite bovino (IPS) com Alcalase livre (AL) e imobilizada em glioxil-agarose (AIm) nas características dos hidrolisados e na detecção de ?-La e ?-Lg por teste ELISA. As melhores condições de hidrólise do IPS com AL ou AIm foram estabelecidas por delineamento composto central rotacional (DCCR) 22. As variáveis independentes foram pH (7,0 a 9,0) e temperatura (45 a 65°C), a variável dependente foi grau de hidrólise (GH), determinado pelo método de pH-stat. Os hidrolisados foram caracterizados quanto ao perfil de hidrofilicidade por cromatografia líquida de alta eficiência de fase reversa (CLAE-FR) e, as análises posteriores, foram realizadas em hidrolisados tanto com AL (HAL) quanto com AIm (HAIm) obtidos em três condições de hidrólise. O perfil de massa molecular (MM) foi avaliado por cromatografia líquida de alta eficiência de exclusão molecular (CLAE-EM) e eletroforese (SDS-PAGE e SDS-PAGE/Tricina). A agregação dos hidrolisados foi estimada por turbidez e hidrofobicidade superficial (S0) e a detecção dos alérgenos foi realizada pelo método de Enzyme-Linked Immunosorbent Assay (ELISA) utilizando kits comerciais. Sob as mesmas condições de hidrólise, os valores de GH obtidos nos ensaios do DCCR com a AIm (9,5 a 22,2 %) foram, menores do que os obtidos com a AL (18 a 24 %), possivelmente em função de impedimentos estéricos resultantes da imobilização. As melhores condições de hidrólise do IPS com AIm, dentro das faixas de pH e temperatura estudadas, foram obtidas em temperaturas acima de 60°C. O DCCR com a AL não resultou em modelo devido à pequena variação dos resultados entre os ensaios. Os perfis cromatográficos (CLAE-FR) dos HAIm apresentaram mais picos na região de baixa hidrofilicidade do que os HAL. Os HAIm apresentaram peptídeos de MM maiores que os HAL, o que está relacionado aos menores GH produzidos com a AIm. Os HAL apresentaram menores valores de S0 que os HAIm, sugerindo a ocultação de sítios hidrofóbicos no interior dos agregados. As análises de turbidez em diferentes solventes mostraram que interações hidrofóbicas, pontes de hidrogênio e pontes dissulfeto são as principais forças envolvidas na formação dos agregados. A hidrólise com AL e AIm diminuiu significativamente a detecção das proteínas alergênicas. A detecção dessas proteínas foi maior nos HAIm devido, possivelmente, aos menores GH desses hidrolisados. Porém, os resultados sugerem que as características dos hidrolisados resultantes das condições de hidrólise e a formação de agregados estabilizados por diferentes interações também tiveram efeito na detecção dos alérgenos / Abstract: Cow's milk is one of the most allergenic foods in infancy, with the two major caseins and whey proteins - ?-lactalbumin (?-La) and ?-lactoglobulin (?-Lg) - the main allergens. Enzymatic hydrolysis considerably reduces the antigenicity of proteins, and can be performed with free or immobilized enzyme. The immobilized enzyme does not require inactivation at the end of hydrolysis and can be removed by filtration, while the free enzyme must be inactivated, usually by heating, to stop the reaction. Differences in catalytic activity and inactivation may result in hydrolysates with distinct characteristics. The objective of this study was to evaluate the effects of hydrolysis of whey protein isolate (WPI) with free Alcalase (AL) and Alcalase immobilized on glyoxyl agarose (Alm) on the characteristics of hydrolysates and detection of ?-La e ?-Lg by ELISA test. The best conditions for the hydrolysis of WPI using AL or Alm were established by 22 central composite rotational design (CCRD). The independent variables were pH (7.0 and 9.0) and temperature (45 to 65 °C), whereas the dependent variable was the degree of hydrolysis (DH) determined by the pH-stat method. All hydrolysates were characterized for the hydrophilicity profile by reverse phase high performance liquid chromatography (RP-HPLC), and the subsequent analyses were performed in both hydrolysates by AL (HAL) and Alm (HAlm) from three conditions of hydrolysis. The molecular weight profile (MW) was assessed by size exclusion high performance liquid chromatography (SE-HPLC) and electrophoresis (SDS-PAGE and SDS-PAGE/Tricine). The aggregation of hydrolysates was estimated by turbidity and surface hydrophobicity (S0) and the allergens ?-La and ?-Lg in the hydrolysates were detected by the Enzyme-Linked Immunosorbent Assay (ELISA) method using commercial kits. Under the same hydrolysis conditions, the DH values obtained with the Alm as defined by CCRD (9.5 to 22.2%) were lower than the values found for AL (18 to 24%), possibly due to the steric hindrance resulting from immobilization. The best conditions for hydrolysis of WPI by Alm within the pH and temperature ranges of this study were found at temperatures above 60 °C. The CCRD with the AL did not provide a model due to the little variation between trials. The chromatographic profiles (RP-HPLC) of HAlm exhibited more peaks in the hydrophilic low-complexity region than HAL. HAlm showed peptides with higher MW than HAL, which is related to the lower DH produced with Alm. The HAL had lower S0 values than HAlm, suggesting the hiding of hydrophobic sites in the interior part of the aggregates. The turbidity analyses showed that hydrophobic interactions, hydrogen bonds, and disulfide bonds were the main forces involved in the formation of aggregates. The hydrolysis with AL and Alm significantly decreased the detection of the allergenic proteins, which was higher in HAlm probably due to the lower DH of these hydrolysates. However, the results suggested that the characteristics of the hydrolysates resulting from the conditions of hydrolysis, and the formation of aggregates stabilized by different interactions also had an effect on the detection of allergens / Mestrado / Nutrição Experimental e de Alimentos / Mestra em Alimentos e Nutrição

Denna studie undersöker hur märkning på livsmedelsförpackningar kan göras tydligare för personer med nötallergi. Tidigare studier visar att det finns brister i dagens märkningar och att detta i sin tur skapar problem. Därför har denna studie som avsikt att ta reda på hur allergeninformation för nötallergiker kan förbättras och göras tydligare. Detta tillämpas genom att studiens forskare tar fram ett designförslag som ska komplettera obligatorisk märkning på livsmedelsförpackningar. För att ta reda på hur detta designförslag ska utformas inleds designprocessen med en litteraturstudie och där en cross-case-analys genomförs. Genom denna analys hittas trender och mönster i tidigare studier. Därefter tillämpas en design space-analys där trenderna, olika designprinciper samt forskarnas egna kunskaper kombineras för att utforska olika designalternativ. Alternativ som i slutändan skapar en grund för en design. Studiens forskare skapar symboler, närmare bestämt piktogram. Eftersom jordnötsallergi och nötallergi är olika typer av allergier skapas två stycken piktogram. Piktogrammen, som är cirkelformade, får ett varnande budskap som förmedlas med hjälp av färgerna gul och svart. Piktogrammen utvärderas och testas genom att fem personliga intervjuer genomförs. Intervjuerna görs med personer som är allergiska mot jordnötter och/eller nötter. Forskarna testar piktogrammen genom att placera dem på en livsmedelsförpackning och ställer sedan frågor om bl.a. första intryck, färger och placering. Analysen visar att piktogram mycket väl kan användas som kompletterande märkning på livsmedelsförpackningar. Vidare konstateras det att valet av färg inverkar på den association som sändaren vill förmedla. Intervjuerna visar även att piktogrammen fördelaktigt kan placeras på förpackningens framsida. Studiens syfte har uppnåtts till viss mån, då intervjuerna visar att det ena piktogrammet behöver utvecklas för att tydligare kommunikation för nötallergiker ska kunna skapas.

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Le projet de thèse à pour but de produire et caractériser un nouvel allergène de pollen d'ambroisie. Le projet est décliné en cinq axes: production d'une forme recombinante mature et en conformation native, caractérisation structurale, étude de la fonction enzymatique, étude de l'allergenicité et l'immunogénicité et évaluation du potentiel thérapeutique. / The goal of the thesis project is to product and caracterize a novel ragweed pollen allergen.The project have five axes: production of recombinant mature and native form,structurale caracterization, study of enzymatique function, study of allergenicity and immunogenicity and evaluation of therapeutic potential.

<p>The Canadian prevalence for all food allergies is estimated to be 7.5%. The only management strategy for an allergic individual is a strict avoidance diet, which is heavily reliant on the effectiveness of food labels used on commercial food products. This thesis explores the effects of food allergen labels on the well-being of affected Canadians, using a social constructionist framework and a mixed methods approach.</p> <p>The quantitative portion of this study drew upon primary survey data. Respondents were asked to self-report household allergic status, as well as purchasing behavior and attitudes towards precautionary statements (n=1380). Results from the quantitative analysis were used to inform and develop the qualitative interview schedule for the second phase of this thesis. Qualitative interviews in a grocery store setting were conducted with 12 anaphylactic individuals, or parents of anaphylactic children.Respondents were observed during the course of product selection and questioned about their shopping habits, perceptions of and preferences for allergen labels.</p> <p>Results indicate that current Canadian allergen labels are not as effective as expected, since affected consumers reported not heeding precautionary statements. Allergic families were found to be less diligent than indirectly affected families, and also less likely to find precautionary statements helpful. Through qualitative interviews, it was found that prior experience, not allergen information, is the primary factor guiding purchasing decisions. Even though precautionary statements were found to be easy to understand, terminology, font sizes, and contrast issues on labels were reported to be key areas of improvement. Results from the two phases reflect differences in individuals‟ social constructions of risk, which ultimately shape purchasing and consumption behaviors. This research is the first to explore the effects of allergen labels on affected Canadians, using a social constructionist approach. Ultimately, results will help effective policy change in Canada and help affected consumers make safe consumption choices.</p> <p>This research contributes to the number of health studies that utilized a mixed methods approach, which is an emerging paradigm. The use of mixed methods allowed for extension and refinement of results. Future research directions identified in this research include the need for pharmaceutical labeling in Canada, as well as the need to explore purchasing behaviors of indirectly affected families, as they also utilize allergen labels to guide consumption choices when shopping for allergy-controlled environments.</p> / Master of Arts (MA)