DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.

A todos aqueles que direta ou indiretamente contribuíram para que esta etapa fosse alcançada, o meu profundo agradecimento. Aos meus orientadores, à Vanessa e ao Frédéric, agradeço-vos por toda a confiança depositada e por todo o conhecimento que me transmitiram. A maneira como ambos pensam a ciência e a vossa capacidade multidisciplinar foram sem dúvida a inspiração que me orientou neste trabalho e me irá orientar para o futuro. À Doutora Ana Matias agradeço a oportunidade de me ter permitido desenvolver este trabalho no grupo Nutraceuticals & Bioactives Process Technology. Deixar também uma palavra de profundo agradecimento a todos os membros deste grupo pela ajuda e companheirismo prestado. Um obrigado especial à Carolina e à Ana Roda pelo vosso apoio. À Doutora Teresa Crespo e a todos os membros dos grupos Food Safety & Microbiology e Membrane Processes pela vossa disponibilidade e boa disposição. Ao Baixinho e ao Pedro, obrigado pela vossa presença e por todas as felicidades e tristezas partilhadas ao longo desta caminhada. Um profundo obrigado aos amigos de longa data Simão, Diogo, Pimentel, Louro, Salomé, Melo e David pelo vosso apoio e compreensão da minha ausência nos momentos mais atribulados. Aos grandes amigos de Coimbra, que mesmo longe se fizeram sentir sempre perto, obrigado Costa, Bibiana, Sandro, Cardoso e Daniel. Aos companheiros "lisboetas", obrigado Sofia, Serafim, Tiago e Adriana, que me fizeram sentir menos deslocado nesta aventura. Por fim, os meus últimos agradecimentos vão para a minha família. Aos meus pais, aos meus avós e ao meu irmão agradeço-vos profundamente por toda a ajuda na concretização desta etapa.

The potential of natural deep eutectic systems (NADESs) to efficiently extract astaxanthin (AXT) contained in crab shell wastes was evaluated. Different terpene-based mixtures were prepared and characterized. Aiming at maximizing the AXT recovery, we evaluated the effect of operating temperature and time on the extraction performance. As a proof of concept, this paper also highlights the potential of NADESs for AXT extraction from shrimp shells, mussels, and Haematococcus pluvialis. The biological potential of AXT-rich extracts; the AXT standard; and NADESs, their individual components, and equivalent physical mixtures was evaluated, including cytotoxicity, antiproliferative effects on human colorectal cancer cells, and antimicrobial potential against Staphylococcus aureus and Escherichia coli. Results showed that extractions with menthol:myristic acid (8:1) were able to match the AXT yield obtained by a Soxhlet extraction with acetone. Additionally, when using the same NADESs to recover AXT from the other biomasses under study, there was a 3-to 657-fold increase in yields when compared with the Soxhlet extraction. AXT-rich extracts obtained with NADESs showed antiproliferative and antimicrobial potential. This study suggests that NADESs can truly be used as alternative extraction media for the recovery of AXT from waste biomass and that these systems and respective extracts have the potential to be used as ingredients in industrial applications.

Despite the existence of various virulence factors in the Enterococcus genus, enterococcal virulence is still a debated issue. A main consideration is the detection of the same virulence genes in strains isolated from nosocomial or community-acquired infections, and from food products. The goal of this study was to evaluate the roles of two well-characterized enterococcal virulence factors, Fsr and gelatinase, in the potential virulence of Enterococcus faecalis food strains. Virulence of unrelated Enterococcus isolates, including dairy strains carrying fsr and gelE operons, was compared in the Galleria mellonella insect model. E. faecalis dairy strains were able to kill larvae and were as virulent as strain OG1RF, one of the most widely used for virulence studies. In contrast, Enterococcus durans and Enterococcus faecium strains were avirulent or poorly virulent for G. mellonella. To evaluate the role of fsrB and gelE in virulence of E. faecalis dairy strains, both genes were deleted independently in two strains. The DfsrB and DgelE deletion mutants both produced a gelatinase-negative phenotype. Although both mutations significantly attenuated virulence in G. mellonella, the DfsrB strains were more strongly attenuated. These results agree with previous findings suggesting the involvement of fsrB in the control of other cell functions relevant to virulence. Our work demonstrates that the presence of functional fsrB, and to a lesser extent gelE, in dairy enterococci should be considered with caution.