Identificação da origem botânica do mel por DNA barcoding

O mel e um produto natural muito apreciado pelas suas propriedades sensoriais, nutricionais e medicinais. Os méis monoflorais são produtos de valor acrescentado por serem considerados de elevada qualidade e com aroma e sabor bem definidos, sendo por isso susceptíveis a adulterações. Tal facto torna importante o desenvolvimento de novas metodologias para a avaliação da autenticidade e origem botânica. 0 método usado atualmente para a determinação da origem botânica baseia-se na analise melissopalinol6gica, que e morosa e requer técnicos especializados [1]. Os métodos de ADN apresentam-se como alternativas promissoras para a identificação de espécies em matrizes complexas e processadas.info:eu-repo/semantics/publishedVersio

Real time polymerase chain reaction for the quantitative detection

Soybean protein is reported to be the most widely used vegetable protein in the meat industry. Several characteristics of soybean protein such as the emulsifier properties, preventing the coalescence of fat during heating, and the increased capacity of water-holding improving the texture of the final product are reasons for its generalised use. However, as soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. Various methods have been proposed for the detection of soybean in food products, mainly based on the analysis of proteins, such as immunological assays, electrophoretic and chromatographic methods. Due to the higher stability of nucleic acids when compared to proteins and to their ubiquity in every type of cells, DNA molecules have been the target compounds for species identification in several recent works [1]. The aim of this work was to develop highly sensitive and fast DNA-based techniques as alternative to the currently used protein-based methods. For that purpose, binary mixtures of soybean protein in pork’s meat were prepared. In a previous stage of this project, qualitative PCR techniques were successfully applied in the species-specific PCR detection of soybean lectin gene in Frankfurt type sausages [2]. In the present work, we propose a novel approach for the quantitative detection of soybean in processed meat products by means of real-time PCR coupled with fluorescent TaqMan probes. The assays involved the amplification targeting an eukaryotic DNA fragment with specific primers and probe as reference gene for quantification. The amplification of soybean lectin gene was performed in parallel reactions using specific primers and probe. With the values of cycle threshold (Ct) a calibration standard curve was obtained using the Ct method, allowing the detection and quantification of soybean protein in pork’s meat in the proportions of 0.1% to 50%. The established real-time PCR technique was successfully applied in the confirmation of qualitative PCR results and in the estimation of soybean protein in commercial meat products

Assessing the variability of the fatty acid profile and cholesterol content of meat sausages

Eighteen different brands of meat sausages including pork, poultry and the mixture of both meats (pork and poultry) in sausages, were analysed for their nutritional composition (total fat, moisture, crude protein and ash), cholesterol content and fatty acid composition. As expected, the pork Frankfurter sausages presented a higher fat content compared to sausages that include poultry meat in their composition. A multivariate statistical analysis was applied to the data showing the existence of significant differences among samples. Regarding fatty acid composition, significant differences were verified in canonical variate plots when the samples were grouped by sausage type, suggesting that the fatty acid profile is strongly influenced by the type of meats, as well as other ingredients such as vegetable oil and lard, used in its formulation. The group of poultry Frankfurter sausages presented lower levels of SFA and higher levels of PUFA, which can point to a healthier profile compared to the pork and meat mixture sausages. Nevertheless, some poultry sausages showed a higher cholesterol content compared to the pork Frankfurters. The lowest mean cholesterol content was obtained for the group of pork Frankfurters, which somehow contradicts the consumers' idea that pork meat products should be avoided due to its high cholesterol levels.The authors acknowledge the grant no. PEst-C/EQB/LA0006/2011 to FCT - Fundação para a Ciência e a Tecnologia. Sónia Soares is grateful to FCT PhD grant (SFRH/BD/75091/2010).info:eu-repo/semantics/publishedVersio

A SYBR green real-time PCR assay to detect and quantify pork meat in processed poultry meat products

Species identification in meat products has grown in interest in recent years since these foodstuffs are susceptible targets for fraudulent labelling. In this work, a real-time PCR approach based on SYBR Green dye was proposed for the quantitative detection of pork meat in processed meat products. For the development of the method, binary meat mixtures containing known amounts of pork meat in poultry meat were used to obtain a normalised calibration model from 0.1 to 25% with high linear correlation and PCR efficiency. The method revealed high specificity by melting curve analysis, being successfully validated through its application to blind meat mixtures, which confirmed its adequacy for pork meat determination. The fully applicability of the method was further demonstrated in commercial meat products, allowing verification of labelling compliance and identification of meat species in processed foods

Improving DNA isolation from honey for the botanical origin identification

Honey is a natural product highly consumed due its known association with health benefits. Monofloral honeys are perceived as better quality products, being the most appreciated by consumers, thus attaining higher market values. Therefore efficient tools are needed as alternatives to the classical microscopic analysis presently used for the botanical origin identification of honey. In the present work, the use of DNA-based methods for the botanical species identification of honey is proposed. For this purpose, five DNA extraction methods (the kits NucleoSpin Plant (methods A and B) and DNeasy Plant Mini Kit, and the in-house CTAB-based and Wizard methods) combined with three different sample pre-treatments were applied to four honey samples (3 monofloral honeys of Calluna vulgaris, Lavandula spp. and Eucalyptus spp. and one multifloral honey). The 15 DNA extraction protocols were compared in terms of DNA integrity, yield and purity, as well as capacity of amplification targeting universal and adh1 specific genes of C. vulgaris. The results demonstrated the superior efficacy of the Wizard method in terms of DNA quality and amplification capacity, when combined with the sample preparation treatment with a mechanical disruption step of pollen to improve DNA yield. Although with considerable lower DNA yields, the CTAB and DNeasy methods were also successful because both were able to clearly amplify heather DNA from the monofloral heather honey.This work has been supported by FCT through grants PEst-C/ EQB/LA0006/2013 and NORTE-07-0124-FEDER-000069-Food Science. S. Soares is grateful to FCT PhD grant (SFRH/BD/75091/2010) financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

Polymerase chain reaction for soybean detection in heat processed meat products

Since vegetable proteins are considerably cheaper than muscle proteins, they are frequently used as meat extenders in order to reduce the cost of the final product. Due to several interesting characteristics, soybean is reported to be the most widely used vegetable protein in the meat industry. Nevertheless, soybean is included in the group of 12 ingredients potentially allergenic, which should therefore be labelled according to the Codex Alimentarius FAO/WHO and the European Commission (Directive 2003/89/EC). In fact, it has been described that amounts of soy bellow 0.1% and 1% (w/w) can lead to allergic reactions in sensitive consumers (1)

SYBR green I real-time polymerase chain reaction as a tool to detect poultry’s meat adulteration with pork’s meat

Nowadays, meat species adulteration in ground and comminuted products is being considered as a widespread problem in retail markets [1]. This problem encompasses many issues, such as adulteration by substitution with lower value meats, the presence of undeclared species and the fraudulent substitution of meat by lower price vegetable proteins. Another issue to be considered is related to religious practices since pork’s meat consumption is sometimes forbidden. Several techniques are currently used for meat species identification in complex mixtures, including different protein-based methods such as HPLC, ELISA and electrophoretic techniques. Nevertheless, these methods can be significantly less sensitive and difficulties can arise in the case of thermally processed foods. Due to the higher stability of DNA molecules compared to proteins, and to its ubiquity in every type of cell, they are currently preferred as target compounds for meat species identification. Moreover, the analysis of DNA coupled with polymerase chain reaction (PCR) presents a fast, sensitive and highly specific alternative to protein-based methods [2]. In the present work, the development of a real-time PCR technique for pork’s meat detection in complex matrices is reported. To achieve this objective, DNA was extracted from reference binary meat mixtures containing known percentages of pork’s meat. The real-time PCR approach was based on the specific amplification targeting the 18S rRNA mitochondrial gene for pork species detection and targeting a eukaryotic DNA fragment as a reference gene for quantification. The amplification products were monitored by using the fluorescent dye SYBR Green I associated with melting curve analysis to verify the specificity of obtained fragments. Under our experimental conditions, pork and eukaryotic detection systems produced fragments with 149 bp and 140 bp, and with melting temperatures of 83.5ºC and 87.5º, respectively. Calibration curves were obtained with the cycle threshold (Ct) values by using the Ct method. The detection and quantification of pork’s meat was achieved in the range of 0.1% to 25%, with a high correlation coefficient (R2=0.9943) and a PCR efficiency of 88.7%. The developed methodology was successfully validated using blind samples and applied to the quantitative evaluation of pork’s meat in different poultry processed meat products, including sausages, hamburgers and nuggets

Comparison of DNA extraction methods for the determination of botanical origin of portuguese honey

Honey can be c1assified based on the type of flowers used by the bees to collect pollen as unifloral honey, if arising predominantly from a single botanical origin, which generally represents more than 45% of the total pollen content, or as multi floral honey, if it is produced from various plant species A single plant origin should assure a product of better quality because it guarantees a specific and well-defined tlavor and aroma. For this reason, monotloral honeys generally attain higher commercially value, especially those labeled as PDO (Protected Designation of Origin) Thus, the botanical authentieation of honey is of high importanee. To date, mieroscopie analysis of pollen (melissopalynology) is the technique used to assess the origin of honey. However, this type of analysis is time consuming and dependent on the experienee and slcill of trained analysts. In contrast, DNA-based methods are less dependent on the analyst, highly specific and easily applied to laboratories with the suitable equipment [I]

Polymerase chain reaction for the detection of allergens: the case of soybean in processed meat products

In this work, a polymerase chain reaction (PCR) technique was used for soybean detection in processed meat products. The methodology was optimized using reference binary samples with different known percentages of pork meat and soybean protein, prepared in the laboratory. To evaluate the effect of thermal treatment, binary mixtures were submitted to thermal treatment at 121ºC for 5 min. The species-specific PCR targeting the lectin gene allowed the detection limit of 0.01 ng of soybean DNA in raw materials. The PCR method was successfully applied on 18 commercial Frankfurt sausages

Quantitative detection of soybean in meat products by a TaqMan real-time PCR assay

In the present work, we propose a normalised real-time quantitative PCR assay to determine the addition of soybean to meat products. The method proved to be a powerful tool for the quantification of soybean protein (dry basis) in the range of 0.01% to 6%, being successfully in-house validated. Its application was effective in the analysis of several meat products, indicating 2% of non-compliance with the food allergen labelling legislation, and some inconsistencies when comparing the declared with estimated amounts of soybean. This work highlights the importance of efficient tools to assess labelling statements of meat products, avoiding fraudulent practices.This work has been supported by Fundação para a Ciência e a Tecnologia (FCT) through grant no. PEst-C/EQB/LA0006/2013 and the University of Porto “Projectos Pluridisciplinares” IJUP2011-149. S. Soares is grateful to FCT PhD grant (SFRH/BD/75091/2010) financed by POPH— QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio