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- Characterization of Selected Wild Mediterranean Fruits and Comparative Efficacy as Inhibitors of Oxidative Reactions in Emulsified Raw Pork Burger PattiesPublication . Ganhão, Rui; Estévez, Mario; Kylli, Petri; Heinonen, Marina; Morcuende, DavidIn the present study, water, ethanolic, and methanolic extracts from seven selected wild fruits originally from the Mediterranean area, namely, strawberry tree (Arbutus unedo L., AU), azarole (Crataegus azarolus L., CA), common hawthorn (Crataegus monogyna L., CM), blackthorn (Prunus spinosa L., PS), dog rose (Rosa canina L., RC), elm-leaf blackberry (Rubus ulmifolius Schott, RU), and rowan (Sorbus aucuparia L., SA), were analyzed for the total amount and profile of phenolic compounds and for the in vitro antioxidant activity against the DPPH and ABTS radicals (study 1). The seven fruits showed different chemical compositions, which consequently led to different antioxidant potentials. Among the seven fruits initially analyzed, AU, CM, RC, and RU had the highest amount of phenolic compounds and displayed the greatest antioxidant activity in vitro. Extracts from these four fruits were tested as inhibitors of lipid oxidation in raw pork burger patties subjected to refrigerated storage at 2 °C for 12 days (study 2). The quantitative measurements of thiobarbituric acid reactive substances (TBA-RS), hexanal content, and color stability were used as indicators of oxidative reactions. The four selected fruits displayed intense antioxidant activity against lipid oxidation, which highlights the potential usage of these fruits as ingredients for the manufacture of healthy meat products. Among them, RC and AU were particularly efficient as their protective effect against lipid oxidation was more intense than that displayed by quercetin (230 mg/kg of burger patty).
- Diel vertical behavior of Copepoda community (naupliar, copepodites and adults) at the boundary of a temperate estuary and coastal watersPublication . Gonçalves, A.M.M.; Pardal, M.A.; Marques, S.C.; S. Mendes; M.J. Fernández-Gómez; M.P. Galindo-Villardón; U.M. AzeiteiroDespite a growing interest in diel vertical migration as a research topic, there are few studies in southern European marine coastal systems. This study determined the main structuring hydrological and physical factors at different temporal scales in copepod assemblage distribution patterns. Seasonal, tidal, lunar and diel vertical migrations accomplished by horizontal movements were examined on the main copepod fraction of the Mondego estuary, Portugal. Seasonal samples were conducted hourly at the mouth of the estuary, during diel cycles (25h), both over neap and spring tides, at the bottom and surface, using a 63μm and 335μm mesh size nets. Simultaneously, four sites inside the estuary were sampled during flood tide to evaluate and compare copepods species' distribution along the estuary. Species life cycles were also categorized. Spring-spring tide best expresses the stable part of copepod-environment dynamics. Acartia tonsa and Oithona nana were distributed mainly at the bottom during ebb tides. A clear resident estuarine performance was noticeable in O.nana proving the estuarine preferences of the species. Neritic species showed preferences by saline waters, whereas the resident species were found mainly at estuarine areas. Copepodites stages showed a similar distribution pattern as estuarine species, avoiding leaving the estuary. In contrast nauplii and Oithona plumifera showed higher densities at surface flood tides. Indeed, vertical migrations accomplished by horizontal movements were mainly influenced by depth and tidal cycles, whereas day and night were not ecologically significant.
- An empirical comparison of Canonical Correspondence Analysis and STATICO in the identification of spatio-temporal ecological relationshipsPublication . Mendes, Susana; Fernández-Gómez, M. José; Pereira, Mário Jorge; Azeiteiro, Ulisses Miranda; Galindo-Villardón, M. PurificaciónThe wide-ranging and rapidly evolving nature of ecological studies mean that it is not possible to cover all existing and emerging techniques for analyzing multivariate data. However, two important methods enticed many followers: the Canonical Correspondence Analysis (CCA) and the STATICO analysis. Despite the particular characteristics of each, they have similarities and differences, which when analyzed properly, can, together, provide important complementary results to those that are usually exploited by researchers. If on one hand, the use of CCA is completely generalized and implemented, solving many problems formulated by ecologists, on the other hand, this method has some weaknesses mainly caused by the imposition of the number of variables that is required to be applied (much higher in comparison with samples). Also, the STATICO method has no such restrictions, but requires that the number of variables (species or environment) is the same in each time or space.Yet, the STATICO method presents information that can be more detailed since it allows visualizing the variability within groups (either in time or space). In this study, the data needed for implementing these methods are sketched, as well as the comparison is made showing the advantages and disadvantages of each method. The treated ecological data are a sequence of pairs of ecological tables, where species abundances and environmental variables are measured at different, specified locations, over the course of time.
- Feeding habits of Solea senegalensis in earthen ponds in Sado estuaryPublication . Castelo Branco, Maria Ana; Arruda, Marco A.; Gamito, SofiaThe senegale sole, Solea senegalensis Kaup, 1858, is a commercially valuable fish and it is beginning to be cultured in a semi-intensive way in fish farms in southern European countries. The present study was initiated to investigate the diet of S. senegalensis under semi-extensive conditions. The feeding habits of sole were studied in two earthen ponds of a fish farm in the Sado estuary. In one earthen pond, artificial fish ration was given. S. senegalensis feeds on few prey items, its diet is mainly composed of insect larvae (Chironomus salinarus) and polychaeta (Hediste diversicolor). The diet composition of this species suggests feeding specialization, by consuming mainly annelids and insect larvae and by avoiding other items, extremely abundant in the environment, such as gastropods. In the water reservoir where ration was given, some fish consumed simultaneously benthic organism together with ration. However, benthic organisms seem to be the most important component of S. senegalensis diet.
- Immobilized heterologous Rhizopus oryzae lipase: A feasible biocatalyst for the production of human milk fat substitutesPublication . Tecelão, Carla; Guillén, Marina; Valero, Francisco; Ferreira-Dias, SuzanaThis work aims at evaluating the potentialities of a heterologous Rhizopus oryzae lipase (rROL) as a feasible biocatalyst, to be used as an alternative to commercial lipases, for the synthesis of Human Milk Fat Substitutes (HMFS). This enzyme was immobilized in three different supports (Accurel® MP 1000, Eupergit® C and Lewatit® VP OC 1600) and tested as catalysts for the acidolysis between tripalmitin and oleic acid, batchwise, at 60 ◦C, in solvent-free medium. The highest molar incorporations of oleic acid in the triacylglycerols were achieved with rROL in Lewatit® VP OC 1600 and Accurel® MP 1000, although the highest immobilization yields were observed with Eupergit® C. Quasi-equilibrium was attained after 6 h or 12 h reaction, with about 30 mol.% and 22 mol.% of oleic acid incorporation, when rROL immobilized in Lewatit and Accurel were used, respectively. Also, 23 h batch operational stability tests were performed: half-life times (t1/2) of 34.5 h and 64.0 h were achieved for rROL immobilized in Accurel® MP 1000 and in Lewatit® VP OC 1600, respectively. An important increase in the operational stability was observed for rROL immobilized in Lewatit after rehydration at the end of each batch (t1/2 = 202 h). Thus, the loss of activity may be explained by a progressive dehydration occurring along the reaction.
- Improved specific productivity in cephalexin synthesis by immobilized PGA in silica magnetic micro‐particlesPublication . Bernardino, Susana M. S. A.; Fernandes, Pedro; Fonseca, Luís P.There is a marked trend in pharmaceutical industry towards the replacement of classical organic methods by "green" alternatives that minimize or eliminate the generation of waste and avoid, where possible, the use of toxic and/or hazardous reagents and solvents. In this work the kinetically controlled synthesis of cephalexin by soluble and penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties was performed in aqueous media with PGME and 7-ADCA as substrates, at different concentrations of substrate, temperature, pH, enzyme to substrate ratio and acyl donor to nucleophile ratio. Excess acyl donor had a strong effect on cephalexin productivity. A PGME/7-ADCA ratio of 3 was considered optimum. A maximum specific productivity of 5.9 mmol h-1gbiocatalyst-1 at 160 mM 7-ADCA, 480 mM PGME and low enzyme to substrate ratio at 32.5 U mmol-1 7-ADCA was obtained with immobilized PGA in full aqueous medium, suggesting that diffusional limitations were minimized when compared with other commercial biocatalysts. A half-life of 133 h for the immobilized biocatalyst was estimated during cephalexin synthesis in the presence of 100 mM 7-ADCA and 300 mM PGME, in 50 mM Tris/HCl at pH 7.2 and 14°C. These results compare quite favorably with those previously reported for the kinetically controlled synthesis of cephalexin.
- Lipid composition and nutritional quality of intramuscular fat in Charneca-PDO beefPublication . Pestana, José M; Costa, Ana S. H.; Alfaia, Cristina M.; Costa, Paulo; Martins, Susana V.; Alves, Susana P.; Bessa, Rui J. B.; Prates, José A. M.This paper characterizes the intramuscular fat from longissimus lumborum (LL, relatively red) and semitendinous (ST, relatively white) muscles of Charneca beef from young bulls reared according to the protected designation of origin (PDO) specifications. The content of total lipids, total cholesterol, α-tocopherol and β-carotene, as well as the fatty acid composition, including the isomeric distribution of conjugated linoleic acid (CLA), was assessed. Charneca young bulls (n = 10) were raised on a semi-extensive production system, in which animals fed pasture plus concentrate during 15 months. The ST muscle was leaner and had higher percentages of PUFA, in contrast to the LL muscle, which presented higher percentages of SFA and MUFA. Thus, the ST muscle had a higher PUFA/SFA ratio than the LL muscle, although the ratio values of both muscles were inside the recommended figures for the human diet. In contrast, the contents of CLA isomers, total cholesterol, α-tocopherol and β-carotene, as well as the n-6/n-3 ratio, were not influenced by muscle type, thus suggesting no carcass variation for these compounds. In both muscles, the n-6/n-3 ratios were slightly above the dietary guideline for human diet, and the contents of α-tocopherol were very high, indicating a good lipid stability of Charneca-PDO beef. Overall, the results suggest that intramuscular fat of Charneca-PDO beef has good human health-related parameters, with small carcass variation, since the PUFA/SFA and n-6/n-3 ratio values are inside or very close to the recommended figures for the human diet, and the content of α-tocopherol is very high.
- Protein differential expression induced by endocrine disrupting compounds in a terrestrial isopodPublication . Lemos, Marco F. L.; Esteves, Ana Cristina; Samyn, Bart; Timperman, Isaak; van Beeumen, Jozef; Correia, António; van Gestel, Cornelis A. M.; Soares, Amadeu M. V. M.Endocrine disrupting compounds (EDCs) have been studied due to their impact on human health and increasing awareness of their impact on wildlife species. Studies concerning the organ-specific molecular effects of EDC in invertebrates are important to understand the mechanisms of action of this class of toxicants but are scarce in the literature. We have used a dose/response approach to unravel the protein expression in different organs of isopods exposed to bisphenol A (BPA) and vinclozolin (Vz) and assess their potential use as surrogate species. Male isopods were exposed to a range of Vz or of BPA concentrations. After animal dissection, proteins were extracted from gut, hepatopancreas and testes. Protein profiles were analysed by electrophoresis and differentially expressed proteins were identified by MALDI mass spectrometry. EDCs affected proteins involved in the energy metabolism (arginine kinase), proteins of the heat shock protein family (Hsp70 and GRP78) and most likely microtubule dynamics (tubulin). Different proteins expressed at different concentrations in different organs are indicative of the organ-specific effects of BPA and Vz. Additionally, several proteins were up-regulated at lower but not higher BPA or Vz concentrations, bringing new data to the non-monotonic response curve controversy. Furthermore, our findings suggest that some common responses to EDCs in both vertebrates and invertebrates may exist.
- Protein oxidation in emulsified cooked burger patties with added fruit extracts: Influence on colour and texture deterioration during chill storagePublication . Ganhão, Rui; Morcuende, David; Estévez, MarioThe influence of protein oxidation, as measured by the dinitrophenylhydrazine (DNPH) method, on colour and texture changes during chill storage (2. °C, 12. days) of cooked burger patties was studied. Extracts from arbutus-berries (Arbutus unedo L., AU), common hawthorns (Crataegus monogyna L., CM), dog roses (Rosa canina L., RC) and elm-leaf blackberries (Rubus ulmifolius Schott., RU) were prepared, added to burger patties (3% of total weight) and evaluated as inhibitors of protein oxidation and colour and texture changes. Negative (no added extract, C) and positive control (added quercetin; 230. mg/kg, Q) groups were also considered. The significant increase of protein carbonyls during chill storage of control burger patties reflect the intense oxidative degradation of the muscle proteins. Concomitantly, an intense loss of redness and increase of hardness was found to take place in burger patties throughout refrigerated storage. Most fruit extracts as well as Q significantly reduced the formation of protein carbonyls and inhibited colour and texture deterioration during chill storage. Likely mechanisms through which protein oxidation could play a major role on colour and texture changes during chill storage of burger patties are discussed. Amongst the extracts, RC was most suitable for use as a functional ingredient in processed meats since it enhanced oxidative stability, colour and texture properties of burger patties with no apparent drawbacks.
- Proteins in ecotoxicology – How, why and why not?Publication . Lemos, Marco F.L.; Soares, Amadeu M. V. M.; Correia, António C.; Esteves, Ana C.The growing interest in the application of proteomic technologies to solve toxicology issues and its relevance in ecotoxicology research has resulted in the emergence of "ecotoxicoproteomics". There is a general consensus that ecotoxicoproteomics is a powerful tool to spot early molecular events involved in toxicant responses, which are responsible for the adverse effects observed at higher levels of biological organization, thus contributing to elucidate the mode of action of stressors and to identify specific biomarkers. Ultimately, early-warning indicators can then be developed and deployed in "in situ" bioassays and in environmental risk assessment. The number of field experiments or laboratory trials using ecologically relevant test-species and involving proteomics has been, until recently, insufficient to allow a critical analysis of the real benefits of the application of this approach to ecotoxicology. This article intends to present an overview on the applications of proteomics in the context of ecotoxicology, focusing mainly on the prospective research to be done in invertebrates. Although these represent around 95% of all animal species and in spite of the key structural and functional roles they play in ecosystems, proteomic research in invertebrates is still in an incipient stage. We will review applications of ecotoxicoproteomics by evaluating the technical methods employed, the organisms and the contexts studied, the advances achieved until now and lastly the limitations yet to overcome will be discussed.