Percorrer por autor "Santos, Cristina Maria Gomes"
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- Authentication of traditional game meat products by the use of species-specific PCRPublication . Santos, Cristina Maria Gomes; Melo, Vítor S.; Mafra, Isabel; Amaral, Joana S.; Estevinho, Leticia M.; Oliveira, BeatrizAuthenticity 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.
- Avaliação da autenticidade de alheiras de caça por técnicas de biologia molecularPublication . Santos, Cristina Maria Gomes; Mafra, Isabel; Estevinho, Leticia M.O fabrico de enchidos à base de carne representa uma longa tradição na região transmontana, onde são produzidos, sendo muito apreciados pelas suas características organolépticas peculiares e elevado valor cultural. A alheira é um dos produtos tradicionais mais típicos de Portugal. Actualmente, para além da alheira tradicional, produzida à base de carne de porco e/ou de aves (galinha e peru) são comercializadas as alheiras de caça, geralmente com preço mais elevado. Neste tipo de produtos processados, é difícil a diferenciação das carnes utilizadas, pelo que são propícios a adulterações. Para isso, o recurso a técnicas de biologia molecular, em especial a Reacção em Cadeia da Polimerase (PCR), tem-se mostrado como uma alternativa específica, rápida, sensível e adequada para a identificação de espécies em produtos alimentares. A elaboração desta dissertação visou a avaliação da autenticidade de amostras comerciais de alheiras de caça. Para tal, foi necessária a optimização de técnicas de PCR qualitativa, recorrendo à preparação de amostras binárias contendo as espécies em estudo. Desta forma, foram utilizados primers específicos para a detecção dos genes mitocondriais cytb e rARN 12S. Dentro dos primers escolhidos, alguns foram propostos pela primeira vez neste trabalho, com especial destaque para a lebre. Os resultados demonstraram elevada especificidade e sensibilidade das reacções para as espécies em estudo, permitindo detectar a adição de faisão, perdiz, pato, coelho, vaca e lebre em carne de porco até ao limite de 0,01% e adição de veado em carne de porco até ao limite de 0,1%. No caso da detecção de galinha e peru, obteve-se um limite de detecção de 0,01% e 0,1%, respectivamente. No entanto, foi verificada alguma reactividade cruzada, pelo que estas duas técnicas deverão ser optimizadas com novos primers. As metodologias propostas foram aplicadas com sucesso a 18 amostras comerciais de alheiras de caça, tendo-se detectado várias inconsistências na rotulagem, nomeadamente a ausência de espécies de caça declaradas (faisão, perdiz, pato, veado, lebre e coelho) e a presença de carnes não rotuladas (vaca, galinha e peru). A lebre mereceu neste estudo um destaque importante por não terem sido encontrados estudos anteriores relativamente à identificação específica desta espécie. Pelo que, neste trabalho foram propostas duas técnicas de PCR para a detecção de lebre que permitiram atingir níveis da ordem de 0,01% de carne de lebre em carne de porco. Adicionalmente, por PCR em tempo real com a utilização do novo corante EvaGreen® atingiu-se o limite de detecção absoluto de 0,1 pg, tendo a nova metodologia proposta mostrado ser adequada para quantificação. The manufacture of traditional meat products is a long-established tradition in Northeastern region of Portugal, in particular the case of “alheiras”, which are very popular for its unique organoleptic characteristics. Besides the traditional “alheiras” mainly produced with pork and poultry meat, others are currently available in the market, which are produced with different game meats, such as “alheiras de caça”. Since this kind of meat 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 meats. Thus, the use of molecular biology techniques, especially Polymerase Chain Reaction (PCR), has become a reliable alternative, rapid in performance, sensitive and suitable for species identification in food. The preparation of this thesis aimed to evaluate the authenticity of commercial samples of “alheiras de caça”. For this purpose, it was necessary to optimize qualitative PCR technique, using the binary mixtures containing the species under study. Specific primers were also used for the detection of mitochondrial genes cytb and 12S rRNA. From the chosen primers, some were available on the literature while others were proposed for the first time in this work, with special emphasis to hare. PCR results revealed high sensitivity and specificity of the primers, allowing detecting the addition of pheasant, partridge, duck, rabbit, cow and hare in pork mixtures down to 0.01% and the addition of deer in pork down to 0.1%. The detection of chicken and turkey using the new designed primers enable positive amplifications until 0.01% and 0.1%, respectively. However, for these reactions it was also observed some cross-reactivity, indicating that the technique should be optimized using different primers. The proposed methods were successfully applied to 18 commercial samples of “alheiras de caça”, being detected several discrepancies in the labeling, including the absence of game species declared (pheasant, partridge, duck, deer, hare and rabbit) and the presence of meat species not labeled (cow, chicken and turkey). In this study, hare meat earned a prominent role due to the lack of reported works regarding specific identification of hare. With the techniques applied to hare detection it was possible to achieve a relative limit of detection (LOD) of 0.01% using qualitative PCR. By the use of real-time PCR with the new EvaGreen dye, an absolute LOD of 0.1 pg was reached, having the novel proposed methodology demonstrated its adequacy for quantification.