Detecção de avelã como potencial alergénio em chocolates por técnicas de biologia molecular

A avelã está incluída nos oito grupos de alimentos responsáveis por cerca de 90% das alergias alimentares mediadas pela IgE. Deste modo, a Comissão Europeia obriga a rotulagem de ingredientes potencialmente alergénicos, através da Directiva 2007/68/CE. De acordo com o exposto, a detecção de vestígios de avelã em produtos alimentares torna-se essencial para salvaguardar a saúde dos consumidores alérgicos. O recurso a técnicas de biologia molecular, como a Reacção em Cadeia da Polimerase (PCR), constitui uma opção bastante confiável para a detecção de quantidades muito baixas de um determinado ingrediente potencialmente alergénico. Com o presente trabalho procedeu-se à optimização da técnica da PCR para a detecção de avelã em chocolates. Deste modo, tornou-se necessária a comparação de diversos métodos para a extracção adequada de ADN de avelã em chocolates modelo. Numa primeira etapa do trabalho, usando primers específicos para a avelã, foi possível atingir um limite de detecção de 0,005% (50 ppm) de avelã numa matriz de massa de trigo, visando a optimização das técnicas moleculares utilizadas. Com o uso de misturas de referência de avelã em chocolate pretendeu-se, numa segunda etapa, a comparação de diferentes métodos de extracção de ADN: CTAB, Wizard® Magnetic DNA Purification System for Food, NucleoSpin® Food e uso parcial do kit Wizard® Plus Minipreps DNA Purification System. O método baseado no uso do kit NucleoSpin® Food foi o que evidenciou melhor eficiência em termos de reprodutibilidade e sensibilidade na amplificação por PCR convencional e PCR em tempo real com sondas TaqManTM, permitindo a detecção de 0,005% (50 ppm) de avelã em chocolate. Relativamente à detecção de avelã em amostras de chocolates comerciais, foi possível detectar, por ambas as técnicas de PCR, quantidades muito baixas de avelã em chocolates que não rotulavam a avelã como ingrediente, mas que continha a menção “pode conter vestígios de…”. Assim, pode afirmar-se que as amostras de chocolate em questão foram eventualmente contaminadas com pequenas quantidades de avelã, durante o processo de produção. Os resultados obtidos por PCR em tempo real confirmaram em geral as rotulagens das amostras de chocolate, mesmo os chocolates que não amplificaram positivamente para a avelã. Os resultados demonstram ainda que um método eficaz de extracção de ADN é a base para a obtenção de níveis de sensibilidade adequados, para detecção e quantificação de ingredientes potencialmente alergénicos, em matrizes alimentares complexas, como o chocolate. Hazelnut is included in one of the eight food groups responsible for approximately 90% of IgE-mediated food allergies. Thus, European Commission obligates the labeling of potential allergenic ingredients in foods, through the Directive 2007/68/CE. Accordingly the detection of hazelnut traces in food products becomes useful to protect allergic consumers’ health. The use of molecular biology techniques, as Polymerase Chain Reaction (PCR), is a rather reliable option to detect very low amounts of a potential allergenic ingredient. With the present work the optimization of PCR technique, aiming the detection of hazelnut in chocolate model, was achieved. Therefore, it was necessary to compare different DNA extraction methods of hazelnut in chocolate. In a first step, using hazelnut specific primers, it was possible to reach a limit of detection of 0.005% (50 ppm) of hazelnut in wheat dough. In a second step, reference mixtures of hazelnut in chocolate were used to compare different DNA extraction methods: CTAB, Wizard® Magnetic DNA Purification System for Food, NucleoSpin® Food and partial use of the Wizard® Plus Minipreps DNA Purification System kit. The protocol based on NucleoSpin® Food kit proved to be more reproducible and sensitive both in conventional PCR and real-time PCR amplification with TaqManTM probes, allowing a limit of detection of 0,005% (50 ppm) of hazelnut in chocolate. Concerning the detection of hazelnut in commercial chocolate samples, it was possible to detect, by both PCR techniques, traces of hazelnut in chocolates that were not labelled as containing hazelnut, but with the declaration of “may contain traces of…”. So, it can be claim that these chocolate samples were eventually contaminated with low amounts of hazelnut, during the production process. In general, the real-time PCR results confirmed the label statements, even those which were negative for hazelnut. The results of this study show that a reliable DNA extraction method is the basis for achieving adequate levels of sensitivity, for the detection and quantification of potential allergenic ingredients in complex food matrices, such as chocolate

Detection of adulterations in tradictional portuguese game meat products by polymerase chain reaction technique

Authenticity assessment and fraud detection in processed meat products have becn attracting an increased attention driven by public health, economic and legal cancems, and also for religiolls reasons. Currcntly, ooe af the major issues conceming adulterations in the meat indust:ry regards the fraudulent substitution af higher commercial valued meat species by less expensive oDes [1]. The manufacture af traditional meat products is a long-established practice in the Northeast of Portugal. One of the most appreciated products is called "Alheiras", which are traditional smoked fermented sausages, mainly produced with pork and poultry meat. In addition to the two Portuguese Alheiras with Protected Geographical Indication (pGI), other types of "Alheiras" are now available in the market, including the ones produced with game meat. Due to the game meat particular taste, intense Oavour and seasonality, it generally eommands higher prices compared to other meats [1]. Sinee game meat "Alheiras" should, totally or partially, include different types of game meat, they are particularly prone to fraudulent meat substitutions

Species identification and authentication of hare (Lepus) meat by the use of the mitochondrial cytb gene

Nowadays, consumers are increasingly concerned with issues of food safety and authenticity. In particular, game meat has been much appreciated by consumers for their exotic flavour and texture, low in fat and cholesterol as well as by the absence of steroids or other drugs. Food authenticity assessment is important in that it avoids unfair competition among producers and allows consumers to have accurate information about the products they purchase. Therefore, it is important to ensure that species of high commercial value declared are not replaced by other species of lesser value [I]

Authentication of a traditional game meat sausage (Alheira) by species-specific PCR assays to detect hare, rabbit, red deer, pork and cow meats

Alheira is a traditional meat product that is typical from the Northeast region of Portugal and much appreciated. It is a sort of sausage produced industrially or by small artisanal producers, having wheat bread and meats as main ingredients. Game meat Alheira (Alheira de caça) is considered one of the most attractive products since it should include different game meats. The aim of the present work was to identify the species of origin of meats added to game meat Alheira samples to verify their compliance with labelling. Species-specific PCR assays targeting mitochondrial genes of rabbit, hare, red deer, cow and pork were optimised and applied to industrial and artisanal samples. The assays revealed adequate specificity for each of the targeted species, with sensitivities of 0.01-0.1%. Results of the evaluation of 18 commercial samples identified several inconsistencies with labelling, namely the absence of declared game species (red deer, hare and rabbit) in ten samples and the presence of undeclared cow species in nine of the analysed samples. These findings indicate the occurrence of misleading labelling, suggesting the adulteration by substitution of game meats by cow meat to reduce production costs and the need to protect and valorise this kind of traditional food product.The authors acknowledge the financial support of the University of Porto/Santander Totta “Projectos pluridisciplinares 2010” and the Fundação para a Ciência e a Tecnologia (FCT) through grant no. PEst-C/EQB/LA0006/2013.info:eu-repo/semantics/publishedVersio

Identification of duck, partridge, pheasant, quail, chicken and turkey meats by species-specific PCR assays to assess the authenticity of traditional game meat Alheira sausages

Game meat Alheira (Alheira de caça) sausage is a traditional fermented product typical from the Northeast region of Portugal, having bread and meats (including game) as main ingredients. It is a particularly appreciated product by consumers that commands higher prices, especially in comparison with the common Alheira produced with pork and poultry meats. Following our previous work in which several mammalian game meat species were successfully identified in game meat Alheira sausages for authentication purposes, the present work aimed at identifying game bird's species for the overall assessment of labelling compliance. For that purpose, several species-specific PCR assays targeting mitochondrial DNA for the detection of game and domestic bird's meat, namely duck, partridge, pheasant, quail, chicken and turkey were developed, optimised and applied to commercial samples of game meat Alheira for their authentication. The assays revealed a high specificity and sensitivity to detect the addition of all evaluated species down to a level of 0.01% (w/w). PCR results indicated the existence of several inconsistencies with the labelled information, namely the absence of declared game species (duck, partridge and pheasant) and the presence of undeclared poultry meat, pointing out to adulterations owing to substitution of game by domestic meat species. Since this is considered a high-valued traditional product that should be valorised and protected, this work puts in evidence the need for inspection programs to enforce regulation. © 2014 Elsevier Ltd.The authors acknowledge the financial support of the University of Porto/Santander Totta “Projectos pluridisciplinares 2010” and the Fundação para a Ciência e a Tecnologia (FCT) through grant no. PEst-C/EQB/LA0006/2013.info:eu-repo/semantics/publishedVersio

Domain-size control by global feedback in bistable systems

We study domain structures in bistable systems such as the Ginzburg-Landau equation. The size of domains can be controlled by a global negative feedback. The domain-size control is applied for a localized spiral pattern

Detecção de adulterações em alheiras de caça: identificação de carne de aves com base em marcadores moleculares

A avaliação da autenticidade de produtos cárneos inclui diferentes aspectos, como a substituição de carnes com valor económico elevado, por outras de menor valor, e a presença de espécies não declaradas no rótulo [I]. Actualmente, para além da Alheira tradicional, produzida à base de carne de porco e/ou de aves (galinha e peru) são comercializadas Alheiras de caça, geralmente com preço mais elevado. Neste tipo de produtos processados, torna-se difícil a diferenciação das carnes utilizadas, pelo que são propícios a adulterações. Várias técnicas analíticas com base na detecção de proteínas (cromatográficas, electroforéticas e imunológicas) têm sido aplicadas na identificação de espécies em produtos cárneos. Devido à maior estabilidade do DNA, comparativamente com as proteínas, a sua utilização como molécula alvo através da técnica de reacção em cadeia da polimerase (PCR) apresenta-se como uma alternativa específica, rápida e sensível, mais adequada para a identificação de espécies em alimentos processados. Neste trabalho, propõe-se a identificação de espécies de aves (galinha, peru, faisão, perdiz e pato) em Alheiras de caça pela técnica de PCR. Prepararam-se misturas padrão contendo quantidades conhecidas de cada carne em estudo e procedeu-se à extracção de DNA pelo método Wizard [2]. O rendimento de extracção e a pureza dos extractos foram avaliados por espectrofotometria. Para a detecção de carne de perdiz e fa isão, utilizaram-se primers específicos para o gene mitocondrial 12S rRNA [1]. Para a detecção de peru, galinha e pato, foram desenhados primers específicos tendo como alvo o gene cytb. Os resultados demonstraram a especificidade e sensibilidade das reacções, permitindo detectar a adição de todas as espécies em carne de porco até ao nível de 0,01 %, à excepção do peru (0,1%). As metodologias propostas foram aplicadas com sucesso a 15 amostras comerciais de alheiras de caça, tendo-se detectado várias inconsistências na rotulagem, como a ausência de espécies de caça declaradas (faisão, perdiz e pato) e a presença de carnes (galinha e peru) não rotuladas

How seasonality and anthropogenic impacts can modulate the structure of aquatic benthic invertebrate assemblages

We studied a benthic invertebrate assemblage of a stream that passes through pristine, rural, suburban and urban areas of a municipality located in southeastern Brazil to investigate a possible relationship between this assemblage structure and urbanization. The environmental variables and fauna structure were analyzed in a spatial and temporal scale, sampling the four sites in a dry and wet season. We found a clear spatial pattern, with higher similarity between sites from rural and suburban area that presented intermediate environmental characteristics. The pristine site showed in both seasons the lowest values of alkalinity and fecal coliform. On the other hand, the site located in the urban area showed the lowest concentration of dissolved oxygen and higher of suspended solids, ammonia and fecal coliform. The extreme values of these three variables occurred in the wet season, probably related to the high rainfall values. The benthic invertebrate fauna structure followed the same longitudinal and seasonal pattern found for the environmental variables. The site in urban area showed the lowest richness, diversity and evenness, with a dominance of two groups resistant to adverse environmental conditions (Oligochaeta and Orthocladiinae) and absence of more sensitive groups (Coleoptera, Ephemeroptera and Trichoptera). The increase drag of the substrate and associated invertebrates can be responsible for the lower abundance and richness observed in the wet season. The environmental variables that best defined the differentiation between sites (dissolved oxygen, organic suspended solids and fecal coliform) related directly to urbanization effects, like dump effluents and removal of riparian vegetation. | Supporting Information Supporting Information </supplementary-material

Detection of partridge meat for the authentication of “alheiras de caça” using polymerase chain reaction

The manufacture or traditional meat products is a long-eslablished lradition in Northeastern region of Portugal, in particular the case of "Alheiras" Besides the traditional "Alheiras" mainly produced with pork and poultry meat, others are currently available in lhe market. which are produced with diffcrcnt game meats, such as "Alhciras de caça" Since this kind of mcat products are prepared using more expensive meats, they are prone to adulterations due to the economic profit that might result from the replacement or decrease of those high valued meat

Authentication of traditional game meat products by the use of species-specific PCR

Authenticity evaluation in meat products encompasses many issues, including the fraudulent substitution of higher commercial valued meats by cheaper meats and the presence of undeclared species. Due to its characteristic and intensive flavour and its healthier composition, game meats are considered as delicacy products and command higher prices compared to other meats, thus being susceptible targets for frauds. The manufacture of traditional meat products is a long-established practice in the Northeast of Portugal, being "Alheiras" one of the most appreciated products. "Alheiras" are traditional smoked fermented sausages, mainly produced with pork and poultry meat in a mixture with bread and spices. Currently, game meat "Alheiras" are also available as very attractive meat products and prone to adulterations. To allow accurate information for consumers and avoid unfair competition among producers, it is important to develop efficient methodologies to assess meat species identification and verify the compliance with labelling. This work aimed to develop analytical tools to assess authenticity of game meat "Alheiras", contributing to their valorisation. For this purpose, polymerase chain reaction (PCR) was the technique of choice for its specificity, fastness, accuracy and sensitivity. Meat species under study were game bird meat (partridge, pheasant and wild duck), chicken and turkey. Reference meat mixtures containing known amounts of each meat were prepared. DNA was extracted using the Wizard method. To specifically detect partridge (Alectoris spp.) and pheasant (Phasianus colchinus) species, specific primers targeting the mitochondrial 12S rRNA gene were used to obtain 141 bp and 113 bp DNA fragments (Rojas et al., 2009). To detect turkey (Meleagris gallopavo), chicken (Gallus gallus) and duck (Anas platyrhynchos), specific primers were designed targeting the cytb gene to amplify 144 bp, 129 bp and 111 bp fragments, respectively. The results showed the specific PCR detection forall species until the level of 0.01 %addition in pork meat, except for turkey (0.1% ). The proposed techniques were successfully applied to 15 commercial samples of game meat "Alheiras". Partridge meat was detected in one out of 5 samples, while pheasant was not detected in none of the 2 samples labelled as containing partridge and pheasant, respectively. Among 7 samples declaring duck meat, its detection was verified only in 4. In opposition to these results, the detection of chicken was obtained in 13 samples, from which only 3 had this indication on the label, and turkey, which was declared in only one sample, was identified in 5 "Alheiras". These preliminary results suggest clearly the omission of the game meat species under study and the predominant presence of undeclared chicken and turkey for its replacement. The conclusions seem to indicate the misleading labelling of game meat "Alheiras" and the need to valorise and protect this kind of traditional products