DPASA - Teses de Doutoramento
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- Composição de leite de vaca e bebidas vegetais tidas como alternativa ao leitePublication . Antunes, Irene Cristina da Silva; Quaresma, Mário Alexandre Gonçalves; Bexiga, José Ricardo Dias; Pinto, Carlos AugustoNos últimos anos, a substituição do leite de vaca (LV) por bebidas vegetais (BV) tem vindo a tornar-se cada vez mais popular. Esta tese tem como objetivo comparar os perfis nutricionais do LV e das BV, focando o seu conteúdo em macro e micronutrientes. Os resultados indicam que algumas BV, como a de soja e de côco, apresentam teores energéticos e de hidratos de carbono (HC) semelhantes aos do LV. Além disso, enquanto o perfil de HC do LV se limitou à lactose ou galactose-glicose, as BV apresentaram um perfil de HC mais complexo, contendo diferentes tipos e teores de HC como a rafinose, glucose, maltose, frutose, sacarose e sorbitol. Em relação à fração lipídica, o colesterol e os ácidos gordos (AG) trans foram quantificados apenas no LV, estando ausentes nas BV. O LV e a BV de côco apresentaram predominância de AG saturados (SFA), enquanto as BV de avelã e amêndoa foram ricas em AG monoinsaturados (MUFA) e as BV de soja, arroz e aveia destacaram-se pelos seus AG polinsaturados (PUFA). No entanto, é crucial analisar os SFA no LV individualmente, pois alguns não representam risco para a saúde. Relativamente aos micronutrientes, a BV de soja apresentou teores mais elevados de Ca, Mg e Cu em relação ao LV, juntamente com teores semelhantes de K e P. Por outro lado, o Se foi quantificado apenas no LV. Todas as BV apresentaram o mesmo teor de vitamina B12 que o LV, e a BV de soja foi o tipo de BV com maior teor na maioria das outras vitaminas. Embora muitas BV tenham sido fortificadas com micronutrientes, a sua biodisponibilidade é desconhecida. Dada a variabilidade nutricional das BV, é difícil identificar uma BV que seja nutricionalmente equivalente ao LV
- Strategies for the protection of Eicosapentaenoic acid (EPA) from the ovine rumen microbiota using Nannochloropsis oceanicaPublication . Vítor, Ana Cristina Martins de Miranda; Bessa, Rui José Branquinho de; Alves, Susana Paula Almeida; Huws, Sharon AnnMarine oils derived from microalgae are a rich source of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) for ruminants, with several productivity and environmental advantages over fish oil. Nannochloropsis oceanica is a single-celled marine microalga particularly rich in eicosapentaenoic acid (EPA). N. oceanica slurry biomass can be dried using different methods, having different impacts in the ability to preserve EPA in its interior. The aim of this thesis was to explore the in vivo EPA ruminal protection degree and tissues deposition in lambs that were fed diets supplemented with different N. oceanica dried biomasses. Scanning electron microscopy confirmed that N. oceanica cells in freeze-dried biomass were better preserved than in spray-dried biomass. In the rumen, the estimates of biohydrogenation extent (disappearance) of EPA also confirmed that freeze dried N. oceanica was more protected from biohydrogenation. A lower t10/t11-18:1 ratio was observed in animals fed N. oceanica compared to animals fed the control diet. Overall, no disturbances were found in fermentable parameters nor indicators of ruminal parakeratosis. Microbiome analysis revealed that t10-18:1 may be a more suitable indicator compared to t11-18:1, for determining specific bacteriome profiles in the rumen. Incorporating N. oceanica into the diets resulted in a higher content of n-3 LC PUFA in the meat and subcutaneous adipose tissue of the supplemented lambs, with EPA being the most significant contributor. It was also possible to conclude that brain EPA content was not responsive to dietary supplementation. However, the retina showed a significant response in lambs supplemented with N. oceanica. A novel ruminal-resistant n-3 LC-PUFA encapsulation method using N. oceanica extracted algaenan was developed and tested in vitro with yet inconclusive results regarding the protection of EPA
- Spirulina and Chlorella vulgaris as ingredients in diet of weaned piglets : a valorization approach using feed enzymesPublication . Martins, Cátia Falcão; Prates, José António Mestre; Freire, João Pedro Bengala; Almeida, André Martinho deABSTRACT - Worldwide swine production will face challenges in the coming years associated with feeding, mainly due to the high cost and environmental impact of importation of conventional feedstuffs. Therefore, the need for sustainable alternative feedstuffs is clear. Microalgae, due to their production systems, may be a sustainable alternative. However, their recalcitrant cell walls decrease nutrient bioavailability for pigs. To solve the association with feed enzymes, namely Carbohydrates-Active enZymes (CAZymes), has been equated. Arthrospira platensis (Spirulina) and Chlorella vulgaris are the two most studied microalgae, as a supplement, in swine feeding. Thus, in an initial approach, the aim of this Thesis was to evaluate the inclusion of 10% Spirulina in diets of post-weaned piglets, supplemented with two CAZymes (Rovabio® Excel AP or lysozyme) on growth performance, nutrient digestibility, health status, liver metabolism and meat quality traits. Growth performance was negatively affected by this dietary inclusion, with a significant increase in digesta viscosity and a lower protein digestibility. The use of lysozyme, in contrast to Rovabio® Excel AP, proved to be more efficient in the degradation of cell walls; however, the released proteins were not absorbed, as animal growth was impaired. Meat quality traits were not affected by the inclusion of Spirulina, individually or supplemented with feed enzymes, as well as no changes were observed on hepatic metabolism despite the improvement in hepatic antioxidant potential, especially in terms of total carotenoids. On a second approach, a post-weaning piglet trial was performed to determine the nutritional value of diets with 5% Chlorella vulgaris, supplemented with two CAZymes (Rovabio® Excel AP or a mixture of 4-CAZymes previously tested in vitro). Chlorella vulgaris inclusion negatively affected the digestibility of nutrients but without direct effects on growth performance, which can be explained by the compensatory mechanisms observed (greater development of the intestinal mucosa and predominance of beneficial bacteria on intestinal microflora). Meat quality traits were not affected by such dietary inclusion. Systematic antioxidant potential, immune response and hepatic lipid metabolism were improved in the groups fed with Chlorella vulgaris. In conclusion, the results indicate that microalgae have the potential to be exploited as ingredients in swine diets, providing meat nutritional enhancement and improving the health status of the animals. However, reducing the production cost and further knowledge of their nutritional value is necessary
- Óleo de soja e vitamina E : efeitos nos parâmetros produtivos e na qualidade da carne de vitelos de raça Holstein-FrísiaPublication . Silva, Rui Pedro Fortunato da; Lemos, José Pedro da Costa Cardoso de; Vinatea, Vicente JimenoNeste trabalho testámos o efeito da suplementação do alimento para vitelos em acabamento com óleo de soja e vitamina E sobre parâmetros produtivos e qualidade da carne. Utilizamos 54 vitelos de raça Holstein-Frísia, machos castrados com 6 meses de idade, distribuídos aleatoriamente por três tratamentos (um parque por tratamento com três réplicas), alimentados com alimento composto e palha ad libitum, durante 60 dias antes do abate. O alimento composto incluiu 4,7 % de óleo de palma saponificado, 4% de óleo de soja e 4% óleo de soja + 50mg/kg de Vit. E. Os alimentos foram formulados para serem isoenergéticos e isoproteicos. Os tratamentos óleo de Soja e óleo de Soja + Vit E apresentaram um ganho médio diário mais elevado (1,49 e 1,53 kg/dia frente a 1,35 para óleo de palma saponificado). A carne dos animais do tratamento óleo de Soja + Vit E apresentou maior percentagem de ácidos gordos 18:1trans (4,62 vs. 3,58 e 3,25 mg/100g carne, para óleo de palma saponificado e óleo soja), de 18:3 (0,24 frente a 0,19 e 0,22 para óleo de palma saponificado e óleo de soja). Nenhum dos tratamentos influenciou a força de corte. O painel de provadores não detetou off-flavours. O óleo de soja + Vit E é uma alternativa viável na alimentação vitelos em período de acabamento contribuindo para um maior GMD frente ao alimento OPS. A associação de óleo de soja e vitamina E influencia positivamente a cor da carne, contribuindo para uma carne mais clara e brilhante. A maturação é o fator determinante para diminuir a força de corte da carne. Num segundo ensaio também com 54 vitelos testou-se o efeito de concentrações crescentes de Vit E. Suplementação com 4% de óleo de soja + 50mg/kg de Vit. E, 4% de óleo de soja + 100mg/kg de Vit. E e 4% de óleo de soja + 200mg/kg de Vit. E. Os resultados não mostraram qualquer efeito nos parâmetros produtivos ou na qualidade de carne.
- Norovirus outbreaks in the portuguese armyPublication . João, António Eduardo Bruno Lopes; Alexandre, Maria de São José Garcia; Gonçalves, Carlos PenhaGastrointestinal infections have consistently been among the most frequent diseases and non-battle injuries, degrading operational effectiveness and force readiness in the military. Noroviruses appear as the most frequent agents causing acute gastrointestinal illness both in maritime and land theatres of operations, as well as in stationed troops or military training centers in many western countries. Nevertheless, little is known on the burden of norovirus gastroenteritis in the Portuguese military. In this work we set out to establish a gastroenteritis outbreak surveillance system (GOSS) to report disease cases to the Bromatology and Biologic Defense Laboratory during the five-year period, 2013-2017. During this period 14 gastroenteritis outbreaks were registered that affected a total of 410 military. Noroviruses were responsible for the majority of the outbreaks (8/14) and disease cases (210/410). Norovirus outbreaks occurred either in military exercises or in military bases and showed to have an impact on force readiness and operational effectiveness. In most cases the origin of the outbreaks was difficult to confirm but five (63%) were likely foodborne (including food handlers) or waterborne and three (37%) had predominant person-to-person transmission. Outbreaks were caused by both genogroup (G) I and II, but G II clearly outnumber those caused by G I (6/8) and case numbers (145/210). Norovirus GI was associated with waterborne outbreaks while GII was associated to foodborne and person-to-person transmission. Each outbreak was caused by a different genotype highlighting the high genetic diversity of the circulating noroviruses. Three of the identified genotypes (GI.9, GII.17 and GII.16-GII.2) were reported for the first time in Portugal. In conclusion, the present thesis identified norovirus as the most important etiologic agent of acute gastroenteritis outbreaks and the most frequent cause of food- and waterborne illness in the Portuguese Army. This work had contributed to change preventive measures and allowed the reinforcement of control actions that minimized the impact of norovirus outbreaks.
- High-Throughput production and characterization of Carbohydrate-Active enZYmes for animal nutritionPublication . Lopes, Vânia Alexandra da Silva Cardoso; Fontes, Carlos Mendes Godinho de Andrade; Brás, Joana Luís ArmadaThe biodegradation of plant cell wall (PCW) carbohydrates is performed by microbial enzymes that are generally referred to as CAZymes. In animal nutrition, it is now well established that the monogastric animals produce a limited repertoire of CAZymes and as such cannot use efficiently some dietary ingredients that sometimes display antinutritional properties. The dietary supplementation with exogenous CAZymes improves the nutritive value of diets and increases animal’s performance. In particular, this study demonstrated that 1,3-1,4-β-glucanases and not cellulases improve the nutritive value of β-glucan-containing diets for monogastric animals. In addition, it was revealed that exogenous enzyme supplementation with β-xylanases improved the nutritive value of diets incorporating wheat lots with high viscosity and low endogenous endo-1,4-β-xylanase activity. In contrast, when the wheat lot showed lower viscosity and higher levels of endogenous endo-1,4-β-xylanase activity, broiler response was clearly diminished. Moreover, the data revealed that xylo-oligosaccharides released by xylanases acting on cereal arabinoxylans display a pre-biotic and positive effect in broiler chicks. However, although we observe an exponential accumulation of genomic and metagenomic information, knowledge on CAZYmes with potential to be used in animal nutrition is limited. This work also aimed to develop high-throughput (HTP) methodologies to isolate and characterize potentially important enzymes for animal nutrition. Thus, 1476 recombinant enzymes were selected and produced recombinantly. The data revealed that 79% of recombinant proteins were produced in the soluble form in Escherichia coli. Factors, such as, organism of origin, gene production strategy, fusion with solubility tags, protein molecular weight and amino acids composition of primary sequences may be used to justify and predict levels of solubility. The establishment of a high-throughput pipeline for recombinant enzyme production was used to obtain a library of feruloyl esterases (FAEs) and glucuronoyl esterases (GEs), enzymes which remove the side chains and break crosslinks between hemicellulosic carbohydrates and lignin. Thus 480 putative FAEs and 20 GEs were produced and biochemically characterized. Following gene isolation, 372 FAEs and 11 GEs were produced in a soluble form in E. coli. Activity results showed that 50% of the enzymes produced retained significant levels of activity and stability. The library of innovative FAEs and GEs produced during this project will be used to develop a novel generation of enzymes for animal nutrition, in particular to exploit the release of cellulose and hemicellulose from lignin.
- Inclusion of Cistus ladanifer in ruminant diets : an approach to improve the nutritional value of edible fatsPublication . Guerreiro, Olinda Rosa Fragoso das Neves; Bessa, Rui José Branquinho de; Jerónimo, Eliana Alexandra Sousa; Duarte, Maria de Fátima PereiraCistus ladanifer is a shrub quite abundant in Mediterranean countries, that when fed to lambs has been associated with changes in rumen biohydrogenation (BH), increasing t11-18:1 concentration in abomasal digesta and meat. The thesis main motivation was to further knowledge regarding C. ladanifer plant and to explore its utilization in nutritional strategies to ruminal BH modulation. In the first two experiments, seasonal variation of C. ladanifer aerial parts, with two ages, was characterized for chemical composition, including proximate composition and total phenols and condensed tannins (CT) contents, for in vitro digestibility, antioxidant activity and fatty acid (FA) profile. Cistus ladanifer was considered a nutritionally unbalanced feed, however, it may be used in ruminant nutrition, but only associated with other feeding resources to complement its nutritional imbalances. In third experiment, C. ladanifer fractions effects were tested on in vitro ruminal BH. Condensed tannins fraction was the most active on ruminal BH modulation, leading to a higher t11-18:1 accumulation and higher disappearance of substrate polyunsaturated FA. Therefore, we intended to determine which CT amount can optimize t11-18:1 ruminal synthesis. However, all CT fraction levels led to a depression of microbial growth without effects on ruminal BH. Last experiment was designed to explore effect of two levels of C. ladanifer CT (1.25% and 2.5%) and two ways of CT supply (C. ladanifer aerial parts and C. ladanifer CT extract) on lamb growth performance, carcass composition, meat quality and FA composition of lamb fat. The highest amount of C. ladanifer aerial part (2.5% of CT) had detrimental effects on growth performance, whereas, C. ladanifer CT extract inclusion in diet (1.25% of CT) led to the highest t11-18:1 increase in fat. So, we can conclude that C. ladanifer CT extract may be a good approach to improve the nutritional value of the ruminant edible fats.
- Study of OCDD, 1,2,3,4,6,7,8–HPCDD, 1,2,3,6,7,8-HXCDD and 1,2,3,7,8-PECDD congeners of dioxin absorption in poultryPublication . Cardo, Miguel José Sardinha de Oliveira; Bernardo, Fernando Manuel d'AlmeidaThis thesis concerns a study which has integrated four main lines of investigation converging to a main goal – to contribute to the risk management of food crisis resulting from the contamination of the poultry chain with dioxins. Specifically, it includes: the development of a suitable analytical methodology to detect and quantify dioxins congeners in wood matrix; a study of the contaminant of biocides used for industrial wood treatments, the analyses of the pathway on how the contaminations are maintained and transferred throughout the poultry production chain in terms of level of contamination and respective chemical speciation and also depletion of the dioxins from poultry during production. The study of the dioxin contamination of different industrial wood preservatives, allowed to establish a clear correlation with the contamination profile of poultry and the previous contamination profile incidents found in wood shavings used as poultry litter. The study of the analytical methodology for wood shavings, adapted from the 1613B EPA method, showed recovery rates of labelled compounds ranging between 71.3% and 86.3%. These rates are considered acceptable, considering the reference method and also the criteria stated in the European Union legislation, for both food and feed. The study of contamination fingerprint along the poultry chain, allows to establish the relationship with the wood shavings, identified as the source of the dioxins. In muscle samples of poultry, the average concentration of OCDD was about 50% lower when compared to the profile of the wood shaving samples. On the other hand, the average relative concentration of 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD and 1,2,3,4,6,7,8-HpCDD in poultry muscle samples was 50% higher when compared to the litters. These findings suggest the existence of a transformation in vivo of the most chlorinated congeners. The study of depletion of dioxins in a holding of ducks presented an average daily depletion rate ranging between 1.43% and 4.35%, showing a clear reduction of contamination after the removal of the source of contamination. The depletion rates are factors that must be taken in consideration to assess risk and, indirectly, helpful to manage risk of dioxins in food.
- Modulation of ruminal biohydrogenation in sheep through dietary tannins or energy sourcesPublication . Costa, Mónica Mendes da; Bessa, Rui José Branquinho deIn the present thesis, four experiments were conducted to study how ruminal biohydrogenation pathways can be modulated through dietary inclusion of tannin sources and to acquire a better comprehension about the occurence of t10-shifted biohydrogenation pathways. In the first experiment, in vitro batch incubations with 100 g/kg dry matter (DM) of extracts of chestnut tannins (mostly hydrolysable tannins) and quebracho, grape seed or rockrose (Cistus ladanifer) condensed tannins, as well as a control treatment were incubated for 6 h with ruminal fluid from fistulated sheep and a dehydrated lucerne-based substrate with 60 g/kg DM of sunflower oil. Grape seed and, to a lesser extent, C. ladanifer led to a higher disappearance of 18:2n-6 with a consequent higher production of c9,t11-18:2 and t11-18:1 than chestnut, quebracho and control. There was no clear innibition of 18:0 production with any of the extracts comparing with control. In the second experiment, rumen fistulated sheep were fed tannin extracts from mimosa condensed tannins, chestnut hydrolysable tannins or their mixture (100 g/kg DM) in a complete diet with sunflower and linseed oils (40 g/kg DM), following a change-over design (3 treatments, 4 sheep and 4 periods). There was a variable inhibition of ruminal biohydrogenation and a lower “trans-/cis-18:1” ratio in bacterial fractions with mimosa than with chestnut. Mimosa led to a lower fermentative activity, as well as a lower abundance of Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio proteoclasticus and higher abundance of Selenomonas ruminantium with a lower bacterial biomass estimate of dimethylacetals than chestnut. In the third experiment, two rumen fistulated rams were housed in metabolic cages and adapted to a wheat-based diet with 41 g/kg DM of sunflower oil. During the first two weeks of trial, the t10-shift occurred temporarily in both animals but in different moments. These results were probably due to individual variability of rumen microbiota, since, for a selected period of the trial, a lower bacterial diversity was found for ram 1 compared to ram 2. Moreover, the t10-shift was associated with an increase of total trans-18:1 and a decrease of 18:0. There was no clear association of t10-shift with rumen pH or its expression in blood plasma. In the fourth experiment, 40 lambs were fed, for 6 weeks, with complete diets containing barley or barley completely replaced for dehydrated citrus pulp, dehydrated beet pulp or soybean hulls. All diets were supplemented with an oil blend (soybean:fish oils, 59:10 g/kg DM). Overall, the t10-/t11-18:1 ratio was above 3 in meat and subcutaneous fat, although soybean hulls increased t11-18:1 and c9,t11-18:2 comparing with the other treatments. Citrus pulp led to the lowest gene expression of fatty acid synthase, while that of stearoyl-CoA desaturase was inferior for soybean hulls and beet pulp.
- Divergent cellulosome architecture in rumen bacteria : structure and function studies in cohesin-dockerin complexes of Ruminococcus flavefaciensPublication . Gomes, Pedro Miguel Bule; Fontes, Carlos Mendes Godinho de Andrade; Najmudin, ShabirProtein-protein interactions play a vital role in many cellular processes as exemplified by the assembly of the cellulosome, a bacterial multi-enzyme complex that efficiently degrades cellulose and hemicellulose. Cellulosome assembly involves the high-affinity binding of type I enzyme-borne dockerins to repeated cohesin modules located on non-catalytic structural proteins termed scaffoldins. In addition, the complex is anchored into the bacterial surface through the binding of a scaffoldin type II dockerin to cell-bound cohesins. Initially, the architecture and organization of cellulosomes was thought to rely uniquely on type I and type II cohesin-dockerin interactions. It was recently suggested that cellulosomes from rumen bacteria are organized through different mechanisms involving a third type of cohesin-dockerin complexes. Thus, the genome of the major ruminal bacterium Ruminococcus flavefaciens FD-1 revealed a particularly elaborate cellulosome system that is assembled from a library of more than 200 different components through divergent cohesin-dockerin pairs. Providing structural insights for the specificity displayed by the increasing repertoire of cohesin-dockerin interaction is not only of fundamental importance but essential for the development of novel cellulosome based tools. The present work aimed to identify the molecular basis for the organization of R. flavefaciens cellulosome by dissecting the structural basis of cohesin-dockerin specificity in cellulosomes of rumen bacteria. The data revealed a collection of unique cohesin-dockerin interactions, supporting the functional relevance of dockerin classification in groups based on primary sequence similarity. In addition, R. flavefaciens cellulosome is assembled through a mechanism involving single but not dual-binding mode dockerins. This contrasts with the majority of the cellulosomes described to date where dockerins generally present two similar cohesin-binding interfaces, supporting a dual-binding mode. To illustrate this, the structures of two cohesin-dockerin complexes containing an Acetivibrio cellulolyticus dual-binding mode dockerin were solved. Finally, structural information was used to engineer a dockerin presenting a dual cohesin specificity, revealing the plasticity of the cohesin-dockerin platform to design novel protein-protein interactions.