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- Acinetobacter and public health : risks posed by strains isolated from foodsPublication . Carvalheira, Ana Isabel Teixeira; Teixeira, Paula Cristina Maia; Silva, Joana Gabriela Laranjeira daAcinetobacter spp. has emerged as a pathogen of a major public health concern due to their increased resistance to antibiotics and their association with a wide range of nosocomial infections. The aim of this work was to gain insight into the food-related ecology and epidemiology of Acinetobacter spp. and to compare food and clinical strains regarding biofilm production, resistance to desiccation and disinfectant susceptibility. As there is no standard procedure to recover Acinetobacter spp. from food, two selective enrichment media were evaluated for the recovery of low levels of these organisms. Enrichment in Dijkshoorn enrichment medium followed by plating on CHROMagarTM Acinetobacter medium was shown to be a reliable method. Using this procedure, Acinetobacter spp. were isolated from 77.9% of fruit (35/50) and vegetables (39/45) samples and from all the meat analysed (50). A high genetic diversity established by pulsed-field gel electrophoresis (PFGE) was observed among the isolates and based on the analysis of the partial sequence of rpoB,181 strains ecovered from fruits and vegetables and 156 strains recovered from meat samples were identified as members of eighteen and thirteen distinct species, respectively. Acinetobacter calcoaceticus and Acinetobacter johnsonii were the most common species (both with the frequency of 26.5%) recovered from fruit and vegetables, while Acinetobacter guillouiae (34.9%), A. johnsonii (15%) and A. bereziniae (12%) were the most common species recovered from meats. Acinetobacter spp. belonging to the A. baumannii group (11.0% from fuit and vegetables, 18.7% from meats), which is most frequently associated with nosocomial infections worldwide, were also recovered. Most of these strains were resistant to some of the antimicrobials recommended to treat Acinetobacter infections such as piperacillin¬tazobactam, ceftadidime, ciprofloxacin, as well as to colistin and polymyxin B, the last-resort drugs to treat infections caused by multidrug-resistant Acinetobacter. Overall, 29.8% of the isolates from fruit and lettuces, and 51.2% of the isolates from meats were classified as multidrug¬resistant (MDR), and 4.4% and 9.6% as extensively drug-resistant (XDR), respectively. The taxonomic status of six strains of Acinetobacter obtained from meats, was investigated, using a polyphasic analysis, since their partial rpoB sequence similarities to other Acinetobacter species with validly published names were lower than 95%. The species status of two groups was confirmed by comparative multilocus sequence analysis, including also the gyrB, recA and 16S rRNA genes, low (below 95%) whole-genome sequence (WGS) average nucleotide identity (ANI) values and low (below 70%) digital DNA¬DNA hybridization (dDDH) similarities between the WGS of the proposed type strains of each novel species and the representatives of the known Acinetobacter species. Phylogenomic treeing from core genome analysis supported these results as well as, the coherence of each new species¬lineage was supported by matrix-assisted laser desorption/ionization time¬of-flight mass spectrometry, differentiation of the species at the protein level by cellular fatty acids profiles and by unique and differential combinations of metabolic and physiological properties shared by each novel species. These strains represented two coherent lineages that were distinct from each other and from all known species, and the names Acinetobacter portensis sp. nov. (four strains: AC 877T = CCUG 68672T = CCM 8789T as type strain), and Acinetobacter guerrae sp. nov. (two strains: AC 1271T = CCUG 68674T = CCM 8791T as type strain) were proposed for these novel species. Control the dissemination of A. baumannii is challenging mainly due to its high adaptability to adverse environmental conditions. The biofilm formation ability in silicon and stainless steel, the resistance to desiccation on a stainless steel surface and the susceptibility to eleven commercial antimicrobial products was compared between food (10) and clinical strains (10). The clinical strains were selected based on their presumptive persistent and non-persistent status among 104 isolates recovered from patients in Hospital de São Marcos, Braga. Predominant clones of MDR A. baumannii repeatedly isolated in different years (2004 to 2007) or in different months (isolates recovered in 2014) were defined as persistent and strains recovered sporadically (with a PFGE pattern observed only once among all isolates) were defined as non-persistent. There were no significant differences between clinical and food strains since all the strains were able to form biofilm on silicon and stainless steel surfaces, exhibited desiccation resistance capacity ranging from 14 to 77 days and were susceptible to disinfectants at the recommended use concentrations. However, the biofiom-forming capacity of persistent strains was significantly higher than the non-persistant strains on both surfaces. The resistance to desiccation of persistent strains (mean survival time: 65.8 days) was also significantly longer than that of the non-persistent strains (mean survival time: 35.8 days). Therefore, these factors may contribute to their maintenance in the hospital setting. A high intra¬species variability in susceptibility to disinfectants was observed for A. baumannii and there was no correlation between the efficiency of disinfectants and the origin of the isolates. Moreover, no correlation between antibiotic resistance and biofilm production, resistance to desiccation and disinfectant susceptibility was found. Therefore, food products may be a potential vehicle of spread in the community and clinical environments of Acinetobacter strains resistant to several antibiotics, able to produce biofilm and to survive to desiccation, which may led to nosocomial and community¬acquired infections in susceptible individuals.
- Acorn starch extraction and modification by emerging technologiesPublication . Castro, Luís Manuel Gomes de; Pintado, Maria Manuela Estevez; Saraiva, Jorge Manuel Alexandre; Alexandre, Elisabete Maria da CruzNative to the Northern Hemisphere, the trees of the genus Quercus spp. are found between the temperate and tropical latitudes of the Americas, Asia, Europe, and North Africa. These oaks are evergreen, semi-deciduous, or deciduous trees and produce small fruits called acorns. According to the National Institute of Statistics, these trees represent 35% of the forest area in mainland Portugal, where 85% of cork oaks and 92% of holm oaks are found in the Alentejo, while 94% of the remaining species are concentrated in the North and Center regions. For this reason, large quantities of acorns are produced, and it is estimated that the annual production is 401,585 tons. Despite being a fruit rich in polyphenols and gluten-free (resistant) starch, about 55% of the acorns produced are underused. Currently, as starch has multiple applications in the food and non-food industry, the exploitation of acorns as a source of starch would be advantageous. According to the literature, alkaline extraction is the methodology that allows obtaining the highest extraction yields in starch with the highest purity and with the most relevant properties. However, these starches cannot be used as food additives in the European Union due to a lack of approval. Thus, the main objective of this thesis was to value acorns within the scope of the principle of sustainable development through the creation of high-added value and zero-waste ingredients. Different species of acorns (Q. pyrenaica, Q. robur, and Q. ilex) were collected to study the effect of different dehulling methods on their nutritional and phytochemical composition. Each specie was dehulled separately by three different methods: manual, thermal (by thermal shock), and drying. It was found that the carbohydrate content was not significantly affected, but there was a loss of resistant starch of 76, 42 and 62% after thermal dehulling and a loss of 35, 27 and 25% after dehulling by drying compared to manual dehulling for Q. pyrenaica, Q. robur, and Q. ilex, respectively. As Q. robur was the specie with the highest resistant starch/total starch ratio (0.89), as well as the highest phenolic contents (19.3±1.3 mg GA Eq./g DF), hydrolyzable and condensable tannins (73.2±4.6 mg TA Eq./g DF and 0.103±0.01 mg TA Eq./g DF, respectively) comparatively to Q. pyrenaica and Q. ilex, the manually dehulled acorn of the Q. robur specie was selected in the following tests. Then, the optimization of the starch extraction was conducted at room temperature using water as extraction solvent by high hydrostatic pressure (HP) (pressure levels between 0.1 and 500 MPa and extraction times between 5 and 20 min) and pulsed electric field (PEF) (field strengths between 0.1 and 20 kV/cm and times between 35 and 85 μs) technologies. Due to the presence of large tannin content, which are anti-nutritional compounds of great importance in leather production, their extraction was also optimized, as well as other phenolic compounds, to obtain antioxidant extracts with high added value. Regarding the HP extraction, the optimal extraction condition was found at 333 MPa for 17.4 min with a desirability of 79.5%. The starch yield was 35.4±1.1% (w/w DF) and the extracts had a phenolic and hydrolyzable tannin content of 23.1±0.3 mg GA Eq./g DF and 54.1±0.1 mg TA Eq./g DF, respectively, and an antioxidant activity measured by ABTS of 53.6±0.6 mg TX Eq./g DF. As for the PEF extraction, the optimal extraction condition was obtained at 0.1 kV/cm for 63.3 μs with a desirability of 86.5%. The starch yield was 34.5±0.4% (m/m DF) and the extracts had a phenolic and hydrolyzable tannin content of 27.0±1.2 mg GA Eq./g DF and 56.7±0.6 mg TA Eq./g DF, respectively with antioxidant activity of 59.1±1.9 mg TX Eq./g DF. Condensed tannins were not detected in any of the extracts. Compared to alkaline extraction, the results observed under optimal conditions represent an increase in phenolic yields, tannins, and antioxidant activity by 2, 900, and 4% by HP and an increase by 24, 1012, and 15% by PEF, respectively. Thus, these technologies make it possible to obtain aqueous extracts with a higher tannin content than alkaline extraction, thus valuing these compounds. However, it was the PEF extraction technology that made it possible to obtain extracts with a higher content of polyphenols (phenolics and tannins) and with greater antioxidant activity than HP. Concerning starch, although the extraction yields obtained by HP and PEF under optimal conditions were lower than those verified in the respective alkaline extractions (49.7±0.5 and 48.9±1.2% w/w DF for HP and PEF, respectively), these starches are clean-label and can be used more safely as an additive and/or ingredient in human food than alkaline starches. As the extraction yields under optimal conditions were like each other, the impact of technologies on starch properties was studied and the properties were compared to commercial corn starch to try to identify the most advantageous extraction technology. Regarding the starch obtained by HP under optimal conditions, pressurization preserved the type of polymorphism of the acorn starch and did not significantly alter the relative crystallinity and the onset gelatinization temperatures (i.e., it preserved the hydrogen bonds). However, the significant increase in the amylose/amylopectin ratio by 97%, motivated by the cleavage of the amylopectin chains, led to changes in the properties of starches. Pressurization led to a decrease in the solubility and swelling power of the starches relative to the control and led to an increase in the resistance of the gels to deformation. However, no differences were observed in the in-vitro digestibility, pseudo-plastic behavior, or resistance to flow after pressurization. Regarding the starch obtained by PEF under optimal conditions, the amylose and amylopectin contents were not altered, as well as the onset gelatinization temperatures (hydrogen bonds). Although the type of polymorphism was not changed, there was a decrease in relative crystallinity of 17%. PEF treatment did not alter the starch properties concerning the control starch (solubility, swelling power, in-vitro digestibility, and rheology). Compared to commercial corn starch, acorn starches showed lower gelatinization temperatures and enthalpies, better pseudo-plastic behavior, lower in-vitro digestibility, greater resistance to complex flow, and lower resistance to deformation. Overall, acorn starches also showed higher solubility and swelling power up to 80 °C than commercial starch, which encourages the use of acorn starch as an additive in fermented yogurt and dairy products. As extraction by HP and PEF led to obtaining acorn starches with different properties, a dairy product was developed to try to identify the most suitable starch for this purpose. The effect of replacing commercial corn starch with acorn starch extracted by HP and PEF under optimal conditions on the nutritional composition, functional and sensory properties, and shelf-life of chocolate puddings was studied. Acorn starch improved the rheological properties of puddings without negatively affecting their nutritional composition, internal structure, or in-vitro digestibility. The sensory analysis revealed that 61% of tasters preferred the puddings made with acorn starch over commercial corn starch (control). As for the shelf life, acorn starch led to color stabilization and improved texture of the puddings after 28 days of storage at 4°C compared to the control without compromising the microbiological safety. Of the acorn starches used, PEF-extracted acorn starch led to the greatest improvement in pudding texture. In addition, the chocolate pudding made with PEF-extracted acorn starch also had a higher acceptability index than the HP-extracted starch. Thus, PEF-extracted acorn starch seems to be the most suitable for pudding production. In its entirety, the work described in this Doctoral thesis marks important steps in the valorization of acorns in Portugal, that is, in the valorization of its polyphenols and starch. This work will also contribute to the sustainability and transformation of the acorn in a circular economy context, as it demonstrates the potential of using PEF technology, as a sustainable alternative to alkaline extractions, in obtaining products with high added value and of great economic interest.
- Aerobic granular sludge as a source of extracellular polymeric substances and the potential of the technology combined with bioaugmentation to treat industrial wastewaterPublication . Oliveira, Ana Sofia Teixeira; Castro, Paula Maria Lima e; Amorim, Catarina Raquel Leite; van Loosdrecht, MarkA variety of organic pollutants reach wastewater treatment plants (WWTP), often associated with high salinity levels, making their treatment challenging. Aerobic granular sludge (AGS) technology is thought to protect the microbial communities from stress due to the high content of extracellular polymeric substances (EPS). There is an increasing need to turn WWTP more efficient, with a range of opportunities for resource recovery to integrate them into the circular economy concept. The work described in this thesis aimed to explore AGS biomass as a source of EPS and to understand the variations of EPS production facing different stressors, namely 2-fluorophenol (2-FP) and salinity. Particular attention was given to the microbial communities, diversity and function, of the investigated systems. The recovery of EPS from AGS represents an opportunity for valorization of surplus biomass. AGS from a full-scale WWTP treating urban wastewater was regularly collected for 4 months to assess variability in EPS composition and in granular morphology. Variations in the EPS composition occurred with time, with proteins and humic acids as the main EPS components and polysaccharides and DNA as minor constituents. An extra purification step led to the recovery of a purer EPS form with a rather homogeneous composition however the yield of each EPS component decreased, especially for polysaccharides. Yield and product homogeneity are key features for downstream application of the recovered EPS. The effect of intermittent short-term loadings of 2-FP and low to moderate salinity wastewater on the performance and EPS production of an AGS system was studied. Ammonium removal was highly inhibited by stressors, recovering when 2-FP feeding ceased. Phosphate removal, initially disturbed by exposure to stress conditions, recovered when stressors were still present. EPS composition and concentration in the granules decreased from 133.3 to 33.7 mg/g VSS of AGS during the first phases of stress but its production recovered to 176.1 mg/g VSS of AGS even in the stressor’s presence. The nutrient removal recovery after exposure to stressors and the increased EPS production response support the robustness of AGS systems to deal with intermittent stressful conditions. EPS recovered from AGS were used as an immobilizing agent for Rhodococcus sp. FP1, a 2-FP degrading strain. The produced EPS granules exhibited 2-FP degrading ability of 100%, retaining its original activity up to 2 months storage. Moreover, the EPS granules were used to bioaugment an AGS reactor intermittently fed with low to moderate saline wastewater amended with 2-FP. After bioaugmentation, complete 2-FP removal occurred and phosphate and ammonium removal (previously impaired by 2-FP load) improved from 14 to 46% and from 25 to 42%, respectively. After bioaugmentation, strain FP1 was detected up to 3 days in the reactor effluent by qPCR and eleven bacterial isolates able to degrade 2-FP were retrieved from the AGS. Maintenance of cell viability through storage and improvement of bioreactor.
- Association of micro and nanocarriers with thin films for buccal delivery of bioactive moleculesPublication . Castro, Pedro João Neves Miranda de; Pintado, Maria Manuela Estevez; Sarmento, Bruno; Madureira, RaquelVisando o aumento da biodisponibilidade dos compostos bioactivos administrados por via oral (com especial ênfase para a absorção bucal), o plano de trabalhos deste programa doutoral visou a optimização de formulações de filmes orais e de micro- e nanopartículas que, conjugados constituam sistemas inovadores com atividade sinérgica. As moléculas bioactivas seleccionadas para estudar o comportamento e eficácia das formulações optimizadas foram a cafeína e dois péptidos presentes na proteína do soro do leite com actividades antihipertensora (sequência: KGYGGVSLPEW) e relaxante (sequência: YLGYLEQLLR). O processo de optimização das formulações foi iniciado pela seriação e comparação preliminar dos excipientes para a produção de filmes e micro/nanopartículas. No primeiro estudo realizado, foram optimizadas e comparadas duas formulações de filmes (usando-se os polímeros carboximetilcelulose sódica e gelatina tipo A) como veículos para libertação oral de cafeína. Verificou-se, através da análise por espectroscopia no infravermelho por transformada de Fourier com reflectância total atenuada (FTIR-ATR), que a estrutura química da cafeína não fora alterada durante o processo de produção dos filmes. Concluiu-se também, através do ensaio de dissolução estabelecido pela Farmacopeia Americana (USP), que os filmes produzidos com gelatina tipo A permitiram uma libertação mais lenta da cafeína ao passo que os filmes de carboximetilcelulose apresentaram um perfil de libertação imediata. Em concordância, o valor de permeabilidade aparente da cafeína, determinada através do ensaio de permeabilidade ex vivo, através de excisões de intestino delgado de origem porcina, verificou-se superior quando esta foi veiculada pelos filmes de carboximetilcelulose, comparativamente com os filmes de gelatina tipo A. O tempo de desintegração de ambas as formulações mostrou-se, contudo, demasiado alto para formulações orodispersíveis, não ocorrendo desintegração completa após 30 segundos. Ainda na sequência da escolha do polímero com melhores características para integrar a composição de filmes orais, uma nova formulação contendo goma-guar foi optimizada por desenho factorial. Os filmes de goma-guar apresentaram características mecânicas e físico-químicas superiores às verificadas para os filmes de carboximetilcelulose e gelatina, tomando-se como factores de decisão a capacidade de absorção de água, a erosão em saliva artificial e o tempo de desintegração apresentados pelos filmes de goma-guar. Procedeu-se também à optimização (por desenho fatorial) de uma formulação de micropartículas de alginato que garantissem, em conjunto com os filmes de goma-guar, uma libertação controlada de cafeína, assim como uma maior biodisponibilidade da mesma. A associação de micropartículas de alginato aos filmes de gomaguar – GfB - não induziu alterações das características químicas da cafeína, de acordo com o verificado por FTIR-ATR, nem toxicidade para as linhas celulares usadas para mimetizar as mucosas bucal (TR146) e intestinal (Caco-2/HT29-MTX), de acordo com os resultados obtidos pelo ensaio de viabilidade celular MTT (Brometo de 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazólio). Adicionalmente, os perfis de libertação e permeabilidade in vitro (através das linhas celulares TR146 e Caco-2/HT29-MTX cultivadas em camada) e ex vivo (através de epitélio intestinal de origem porcina) mostraram-se mais lentos que os observados para as micropartículas de alginato, filmes de goma-guar ou com a solução controlo de cafeína. A formulação GfB promoveu o aumento do contacto efectivo entre a cafeína e o epitélio bucal, oferecendo uma permeação mais completa ao longo do tempo. De forma a incrementar também a biodisponibilidade do péptido KGYGGVSLPEW com actividade anti-hipertensora, as micropartículas de alginato foram substituídas por nanopartículas de ácido poli(láctico-co-glicólico) – PLGA - por estas oferecerem uma eficácia de associação superior, assim como um maior potencial de permeação das membranas biológicas, dado o tamanho de partícula ser significativamente inferior. A formulação de nanopartículas de PLGA foi optimizada por desenho factorial. O sistema compreendido pelas nanopartículas de PLGA associadas aos filmes de goma-guar (GfNp) não comprometeu a viabilidade das linhas celulares TR146 e Caco-2/HT29-MTX às concentrações testadas. O sistema desenvolvido promoveu a libertação e permeabilidade controladas do péptido, através das células TR146 e Caco-2/HT29- MTX cultivadas em camada, comparativamente com os filmes e nanopartículas isoladamente, assim como com a solução de péptido livre (controlo). Contudo, a permeabilidade aparente verificou-se superior para a formulação GfNp, comparativamente com as restantes formulações. Estes resultados deveram-se ao contacto íntimo entre o péptido e o epitélio absorptivo, promovido pela formulação GfNp. Verificou-se ainda, através da realização do ensaio in vitro da capacidade de inibição da enzima conversora da angiotensina I, que o péptido transportado por GfNp apresentava a maior actividade anti-hipertensora após ser sujeito à simulação do tracto gastrointestinal, comparativamente com o péptido transportado pelas nanopartículas ou filme, isoladamente, ou com a solução de péptido livre. O sistema previamente optimizado para a libertação do péptido antihipertensor foi também usado de forma a incrementar a biodisponibilidade do péptido relaxante alfa-casozepina (sequência: YLGYLEQLLR). Através do ensaio MTT, foi possível concluir que nenhuma das formulações comprometeu a viabilidade da linha celular TR146 e da co-cultura Caco-2 /HT29- MTX. Por isso, a permeabilidade do péptido, sujeito às condições do tracto gastrointestinal simulado, através dos modelos in vitro bucal e intestinal foi estudada. Verificou-se que a associação de nanopartículas de PLGA com filmes de goma-guar promoveu um aumento da permeabilidade face às nanopartículas e filmes não conjugados, assim como com o péptido em solução (controlo). Estes resultados estão correlacionados com o incremento da mucoadesão conferida pela associação das nanopartículas de PLGA com os filmes de goma-guar, verificada através da análise da adesividade e trabalho de adesão à língua de vaca. Validada a efectividade das formulações para a libertação e permeabilidade de cafeína e péptidos bioactivos, foram realizados estudos preliminares de modo a verificar a estabilidade da formulação GfB e a compreender a opinião de potenciais futuros consumidores face aos produtos desenvolvidos. As formulações foram sujeitas a condições de degradação acelerada (i.e. 40 ºC e 75% de humidade relativa) de acordo com a International Conference of Harmonization (ICH), não se verificando alterações químicas da cafeína em nenhum dos tempos de amostragem (imediatamente após a preparação da formulação e após 3, 6 e 9 meses) através da análise do espectro obtido por ATR-FTIR, assim como dos tempos de retenção em HPLC-UV. Verificou-se ainda um aumento significativo do conteúdo em água de GfB ao longo dos tempos de amostragem. Por fim, um estudo por focus group e um estudo de análise sensorial com um painel naive permitiram compreender a adequabilidade dos sabores propostos, assim como a tolerância à acidez e amargor por parte do consumidor. Observou-se uma ligeira tendência para a aceitação do sabor a menta e alguma tolerância ao amargor e acidez quando a menta foi usada na formulação. Os sistemas para libertação oral de compostos bioactivos, desenvolvidos e optimizados no âmbito desta tese, induziram melhorias significativas no comportamento farmacocinético in vitro dos compostos veiculados. De facto, a associação de filmes orais com micro- ou nanopartículas pode representar um novo sistema de libertação que ofereça maior efectividade e adesão por parte do consumidor/utente.
- Bacterial diversity and antibiotic resistance from the water source to the tapPublication . Moreira, Ivone Cristina Vaz; Rodrigues, Célia Maria Manaia; Nunes, Olga Cristina PastorWater is one of the most important habitats for bacteria in the environment. The continuous flux in the urban water cycle carries water through many places, dragging bacteria and numerous chemical contaminants. This makes of water one of the most important vehicles, not only for the dissemination of the chemical substances, but also for the dissemination of organisms and, consequently, the respective resistance genes in the environment. The main goal of this study was to investigate if drinking water production and distribution could represent a hotspot for the proliferation, selection or incoming of antibiotic resistant bacteria, and the likelihood of these organisms to reach the final consumer, via tap water. In order to meet this objective, the study was planned aiming the tracking of bacterial communities and individual isolates from the source to the tap. Firstly, the abundance and diversity of bacteria in raw, treated and final (tap) water was characterized using culture-dependent and culture-independent (16S rRNA-DGGE) approaches. Both approaches showed that the water treatment reduced the bacterial counts, diversity and cultivability, promoting also a shift in the cultivable bacterial community from predominantly Gram-negative to predominately Gram-positive bacteria. Nevertheless, this effect was reverted, and in tap water Gram-negative bacteria became predominant. Moreover, in tap water total and cultivable bacteria counts were higher than in the disinfected water collected from the distribution system. These results suggest the occurrence of bacterial regrowth and/or biofilm formation over the distribution system or at tap level. Although changes in the bacterial community structure over the water circuit were observed, the predominant phylum detected, by 16S rRNA-DGGE, was the same in all the sampling points – Proteobacteria (mainly of classes Alpha, Beta and Gamma). Culture-dependent and culture-independent approaches were compared to assess which groups might be overlooked by cultivation procedures. In order to have a clear evidence of the bacterial groups which could be overlapped using those procedures, culture-dependent and two culture-independent (16S rRNA gene based DGGE and 454 pyrosequencing) methods were compared for their ability to survey the bacterial diversity of a sample. Such a comparison showed that although the different methods detected the same predominant phyla, different bacteria were targeted. Thus, besides the previous expectation that culture-independent methods would detect more bacterial groups than cultivation methods, it was also concluded that both approaches target different bacterial populations. Based on the study of the bacterial diversity, mainly of cultivable bacteria, and in the literature available, two of the most relevant taxonomic groups detected in drinking waters, due to the widespread distribution and/or abundance, were further studied. Thus, Sphingomonadaceae and Pseudomonas spp. isolated from the source to the tap were studied for species diversity, intra-species variability and potential to spread antibiotic resistance. Although members of the same species were detected in different sampled sites, the same genotype was never detected in raw water and in tap water. According to these results, the hypothesis that bacteria detected in tap water had origin in the water source had to be rejected. Other hypotheses, namely the occurrence of regrowth in water pipelines or taps or an external contamination downstream the sampled sites in the distribution system, emerged from this study. Additionally, the analysis of the antibiotic resistance profiles confirmed that both Sphingomonadaceae and Pseudomonas spp. are potential reservoirs of antibiotic resistance. Nevertheless, clear evidences of horizontal gene transfer were not obtained in this study. Indeed, antibiotic resistance patterns were mainly species-, rather than site- or strain-related, suggesting the importance of vertical resistance transmission in water bacteria. Some antibiotic resistance phenotypes were observed in tap water but not upstream. Examples of this situation were the resistance phenotypes to ampicillin-sulbactam, piperacillin plus tazobactam-pyocyanin, imipenem, ceftazidime, cefepime, gentamicin or tobramycin in Sphingomonadaceae, or to streptomycin and rifampicin in Pseudomonas spp. Cultivation-independent methods show invariably that most of the bacteria in a community are unknown, which means that were never cultivated, characterized and integrated in a validly named taxonomic group. Bacterial taxonomy can have a contribution to gradually narrow the tranche corresponding to the unknown bacteria. In this study a new species name Bacillus purgationiresistens sp. nov. was proposed, based in a single isolate recovered from treated water. Drinking water was confirmed as a potential hotspot for the spreading of antibiotic resistant bacteria, with emphasis on the transfer environment-humans.
- Bioactive compounds from nannochloropsis oculata grown under modulated stress : from production to validationPublication . Sousa, Sérgio Daniel da Cruz e; Gomes, Ana Maria Pereira; Carvalho, Ana Paula Taboada da Costa SantosNannochloropsis oculata is a microalga widely recognized as a potential source of polyunsaturated fatty acids, namely the omega-3 eicosapentaenoic acid (EPA), which beneficial health effects have been demonstrated. In order to maximize the EPA amount that may be obtained from a culture, different strategies may be pursued, such as increasing the microalgas’ biomass EPA content and the efficiency of its extraction process. Based on the above rationale, the main goal of the current thesis was to apply different stress conditions to N. oculata culture, which would increase EPA content, but without hindering growth. The strategy employed entailed the application of stress in a modulated sinusoidal fashion, to stimulate the response, although providing the microalga the possibility to adapt and not cease its growth. Two abiotic factors were independently utilized (temperature and light intensity), and conditions were alternated between optimum and stress levels, in multiday cycles. The factors were applied at lower-than-optimum values (5 and 10 ºC; 30 and 50 μmol photons/m2/s for temperature and light intensity, respectively), and it was found that the approach was successful at achieving the desired goals. While temperature stress (10 ºC) presented the best results, with an 158% EPA content increase, light intensity (30 μmol photons/m2/s) also increased EPA content significantly by 126%. Moreover, the increases in EPA content originated from individual cells contents and not just from overall biomass increase, and these were obtained in a relatively short period of time, which is of utmost importance regarding its industrial viability. Eicosapentaenoic acid is conventionally extracted with hazardous organic solvents. Therefore, an additional line of research was undertaken, aiming at extracting EPA by using less hazardous solvent mixtures (SM), namely hexane:isopropanol (Hxn:2-PrOH; 3:2) and diethyl ether:ethanol (Et2O:EtOH; 2:1). The use of alternative technologies, namely high hydrostatic pressure (HHP) and moderate electric fields (MEF), was also assessed as adjuvants to increase the extraction efficiency of the SM. Concerning SM, Et2O:EtOH was able to increase 1.3-fold the EPA yield of the conventional Folch SM. Regarding the technologies, neither increased extraction yields when used independently; however their sequential combination was able to increase EPA yield by 162%. When associated, Et2O:EtOH and the combined technologies (HHP - 200 MPa, 21 °C, 15 min – followed by MEF processing at 40 °C, 15 min) enabled to extract higher amounts of EPA from N. oculata wet biomass, which is extremely relevant for the industries as no expensive drying step is required. Moreover, it is of dire importance to provide such alternatives to the “classical” extraction processes and solvents, as the industries require higher yields and lower environmental impact in their downstream processing. Since the biological potential of a microalga (or an extract thereof) is intrinsically dependent on its composition, the impact of the stress modulation and the SM utilized for extraction of lipid fraction were studied by characterization and assessment of biochemical and biological activities. Concerning the lipid extracts, it was revealed that the one obtained from the stressed culture presented the best results, namely in terms of anti-steatosis (hepatic lipid accumulation < 8% than that of steatosis-induced cells) and anti-inflammatory activities (decreased expression of interleukine (IL)-6, IL-10, and interferon- in LPS-stimulated RAW 264.7 macrophages). Moreover, both extracts presented oxygen radical absorbance capacity (within the range of 49- 50 μmolTrolox equivalent/mgextract), no metabolic inhibition of several cell lines (Caco-2, HT29-MTXE12, Hep G2, 3T3-L1, and RAW 264.7), and inhibition of triacylglycerols hepatic accumulation (glycerol release ca. 2-fold higher than in non-treated cells). Overall, the results showed that the modulated stress did indeed enhance the potential beneficial effects of the N. oculata lipid extracts on human health, as well as maintain their safety. Once the lipid fraction was extracted from the stressed culture, remaining biomass may still contain other compounds with interesting biological activities. Hence, by applying a biorefinery concept, the defatted biomass was enzymatically hydrolyzed with cellulase Celusoft Supreme for 3 h, followed by the protease NewPro, for 6 h, both at 53 ºC, after which the soluble fraction of hydrolysate (SFH) was characterized in terms of peptide and polysaccharide profiles, and assessed regarding its biochemical and biological activities. The results showed that the defatted biomass may still be utilized as a resource, since the SFH presented antioxidant capacity (3.2 μmolTrolox equivalent/mgFDB), antidiabetic (19.4% inhibition of α-glucosidase) and immunosuppression potential (NO production by LPS-stimulated Raw 264.7 cells decreased to < 10% that of the control), anti-inflammatory activity (decreased expression of IL-6, IL-8 and tumor necrosis factor-α by Caco-2 cells in LPS-induced inflammation) and antimicrobial activity, particularly against Gram-positive bacteria. In conclusion, it was demonstrated how modulated stress may enhance the production of bioactive compounds from N. oculata (specifically EPA) and new strategies to efficiently extract them were identified, as well as their biological potential was unveiled. The strategies presented herein for production of N. oculata with increased EPA content, and extraction thereof, may constitute solutions for the food and nutraceutical industries, allowing them to obtain EPA faster and with higher yields, concomitantly decreasing the process environmental impact. Ultimately, that will increase the economic viability of obtaining EPA from N. oculata.
- Biotechnology of microalgae : study of the influence of biochemical and culture parameters on the production of biomass and bioactive compoundsPublication . Raposo, Maria Filomena de Jesus; Morais, Rui Manuel Santos Costa de; Bernardo, Alcina Maria MirandaA biotecnologia das microalgas está a ganhar cada vez mais importância, havendo já um número significativo de estudos publicados. Contudo, a grande maioria dos trabalhos realizados continua a ser com macroalgas. A maior parte dos estudos com macro- e microalgas têm, contudo, uma base empírica, relacionada com os organismos congéneres que são as plantas vasculares. No trabalho apresentado nesta Tese revemos inicialmente algumas das aplicações das microalgas, em especial as de origem marinha. Após uma resenha sobre os polissacarídeos (capítulo 1.1), focamo-nos na aplicação (potencial) dos biocompostos na saúde humana, em geral (capítulo 1.2) e nas doenças cardiovasculares, em particular (capítulo 1.3) e ainda em doenças crónicas como as relacionadas com o stress oxidativo, a diabetes ou o Parkinson, devido às suas propriedades antioxidantes. Em seguida, aprofundamos o conhecimento sobre os polissacáridos algais (capítulo 2). Neste capítulo, revemos as características bioquímicas deste tipo de polímeros e mostramos algumas das vastas aplicações dos polissacáridos produzidos pelas microalgas (capítulo 2.1). Mostramos ainda quão abrangente pode ser a utilização destes compostos, quer em aplicações médicas (capítulo 2.2), quer em nutrição, devido à sua riqueza em fibras (capítulo 2.3). Sabendo que duas das espécies produtoras de elevadas quantidades de carotenoides são a Dunaliella salina e a Haematococcus pluvialis, desenvolvemos um primeiro trabalho experimental em que os respectivos meios de cultura foram enriquecidos com as fito-hormonas cinetina (kin) e ácido diclorofenoxiacético (2,4-D) (capítulo 3.1). A influência positiva de 2,4-D foi bastante significativa, dado que a adição de 1.0 mg /l meio de cultura de H. pluvialis, sózinha ou em conjunto com kin (1.0 mg /l), aumentou a produção de biomassa em 320% e 290% respectivamente. Uma ainda menor concentração de 2,4-D (0.5 mg /l) induziu, em D. salina (a 15% salinidade, NaCl m/v), um aumento de 410% no número de células. Num trabalho experimental posterior submeteu-se a Haematococcus a condições de stress (privação de nutrientes e luminosidade elevada) para indução da carotenogénese. A biomassa foi depois recolhida e procurou-se a melhor forma de manter a estabilidade dos carotenoides produzidos e uma taxa de degradação mais reduzida. As melhores condições de preservação verificaram-se quando se secou as algas por atomização (temperatura de entrada de 180ºC e temperatura de saída de 110ºC) e armazenamento dos pós a -21ºC em azoto. Desta forma conseguiu-se recuperar 90% do pigmento astaxantina (capítulo 4). Quando se manipulou o meio de cultura de Porphyridium cruentum, verificou-se que a adição de sulfato (21 mM em sulfato) aumentou a produção do exopolissacárido (EPS), e o conteúdo em proteína (52 mM e 104 mM em sulfato) e sulfato (104 mM) no EPS. Contudo, as características físico-químicas do polímero não sofreram alterações. Este EPS mostrou possuir capacidade antiviral, em especial contra o vírus Vesicular estomatitis (capítulo 3.2). Duas experiências diferentes permitiram mostrar que as microalgas têm também aplicação na área ambiental (capítulo 5). Verificou-se que a flora autóctone de um efluente de cervejeira pode ser utilizada para remoção de grande parte do azoto e fósforo, mas também para remover uma parte significativa da carga orgânica dessa água residual. Devido à qualidade da proteína obtida — na qual se observou um aumento de vários aminoácidos essenciais — e do perfil lipídico da biomassa — tendo-se verificado um aumento de alguns ácidos gordos como o ácido eicosapentaenóico (ou EPA) —, a biomassa final pode ser recolhida e eventualmente utilizada em rações para animais, ou ainda para produção de biodiesel (capítulo 5.1). Numa última experiência testou-se a influência da utilização das microalgas, tanto sozinhas como em consórcio com rizobactérias promotoras do crescimento, no melhoramento dos solos. Os pós das algas Chlorella vulgaris e/ou dos consórcios obtidos por atomização foram também incluídos em microcápsulas revestidas por maltodextrina (MD) e goma-arábica (GA) ou gelatina (G). Os consórcios de Chlorella:Stenotrophomonas encapsulados em MD:G e MD:GA 1:1 (m/m) foram os que induziram um melhor crescimento, em termos de biomassa seca, tanto da raiz, como da parte aérea das plantas utilizadas neste estudo (capítulo 5.2). Resumindo, pensamos que, com estes trabalhos, contribuímos para o aumento do conhecimento em biotecnologia de microalgas e teremos talvez alertado para o grande potencial das microalgas, especialmente as de origem marinha, devido à sua grande variedade e riqueza da sua biomassa em compostos bioactivos.
- Characterisation of staphylococcus aureus circulating in PortugalPublication . Castro, Ana Isabel Rodrigues Pereira de; Teixeira, Paula Cristina Maia; Silva, Joana Gabriela Laranjeira daStaphylococcus aureus is a pathogen responsible for skin infections and invasive diseases such as meningitis or pulmonary infection and staphylococcal food poisoning (SFP). The main goal of this study was to increase knowledge on S. aureus circulating in Portugal mainly concerning their virulence characteristics and antimicrobial resistance. According to EFSA-ECDC, Portugal is considered to have one of the highest levels of methicillin-resistant S. aureus (MRSA) from bacteraemia in Europe. The frequency of occurrence of S. aureus from different origins was determined. Swab samples were collected from hands and nose of health care professionals and food handlers - noses and hands are niches where S. aureus are likely to be present in humans - and particularly noses of children (3 to 6 years). Presence of S. aureus in food samples was also determined. Collected isolates were further characterized phenotypically and genomically. Nasal carriage of S. aureus was higher in children (48.6%) compared to health care professionals (39.6%) and food handlers (19.8%). The occurrence of S. aureus on the hands of health care professionals and food handlers was 8.9% and 11.1%, respectively. Globally, higher S. aureus occurrence rates were obtained for nasal carriage. The first case of Methicillin-Resistant Staphylococcus aureus (MRSA) was reported in 1961. Since then, it has been considered the leading cause of nosocomial infections, responsible for causing serious morbidity and mortality rates, worldwide. Globally, the occurrence of MRSA strains was higher in hospital health care professionals; no MRSA strains were detected in food handlers, children presented a carriage of 9.7% of MRSA and low occurrence rates were detected in the analysed food samples (between 0.68 and 5.5 %). Other virulence factors have been reported for S. aureus strains including antibiotic resistance and presence of enterotoxins, Panton-Valentine Leukocidin (PVL), haemolysins, exfoliative toxins and numerous factors involved in invasion of host cells among others. In the present study, resistance to oxacillin, penicillin and ampicillin and to antibiotics of classes other than betalactams, namely ciprofloxacin, gentamicin, rifampicinn, vancomycin, tetracycline, erythromycin, nitrofurantoin and chloramphenicol was investigated. As expected, high levels of resistance to ß-lactams were observed. Tested strains showed low resistance rates to gentamicin, chloramphenicol and rifampicin. Regardless of the source of isolates, resistance to erythromycin was evident. Enterotoxin genes sea - sej and tst were evaluated by multiplex PCR. Although sea is considered the most prevalent enterotoxin gene reported all over the world, in the present study this was not verified. tst gene was detected in children, hospital health care professionals and food handlers. PVL genes were analysed in all the MRSA strains collected from various origins, but only one food strain showed the presence of these genes. Typing by SCCmec has been performed and revealed the presence of type IV and V. SCCmec type IV is associated worldwide with PVL positive strains, but which was not verified in this study. PFGE typing was performed on S. aureus strains isolated from hands and from nose of the same individual of the health care professionals and food handlers; 60 and 30% of the individuals, respectively, presented the same S. aureus strain on hands and nose.
- Chemiomics : a systematic chemistry approach to unravel the interface pathways between oxidation and Maillard mechanism responsible for flavour modulation during wine storagePublication . Monforte, Ana Rita Araújo da Silva Monteiro; Ferreira, António César da Silva; Maaten, Sara Isabel da Fonseca Selgas Martins Van Der; Bro, RasmusWine is a widely consumed beverage with a high commercial value, which price can increase with aging for a certain type. However, the knowledge of the chemical processes occurring during aging that result in a specific chemical/sensory profile remains limited. This lack of knowledge and understanding significantly limits the ability to improve product quality and consistency. For that reason, unravelling the chemical changes occurring during aging that are responsible for wine flavour, constitutes a critical task when one attempts to address issues related with authenticity and sensory quality. These considerable chemical modifications are related in particular with the formation of aroma active substances such as Strecker aldehydes. Depending on the storage conditions, namely related with dissolved oxygen, and availability, quality and quantity of antioxidants as well as precursors concentrations of key odorants the chemical profile of wines changes. In that regards these work presents several studies in order to evaluate the impact of such factors in the formation of Strecker aldehydes, in particular phenylacetaldehyde (Chapters 3, 4, 5 and 6) and other molecules (Chapter 7 and 8). In chapters 3, 4, 5 and 6, studies were performed in wine model solutions and the results demonstrated that the presence of metals, are relevant in promoting the Strecker degradation through phenolics oxidation pathways at wine pH. Also, this route is the major pathway for phenylacetaldehyde formation. For the first time it was demonstrated that the presence of glucose inhibits the formation of phenylacetaldehyde in both wine model systems and in white wine. By quinone quantitation it was shown that glucose affects directly their concentration, which suggest that in white wine glucose has an antioxidant effect by inhibiting o-quinones formation. In order to study the phenomenon in high complex systems, in chapters 7 and 8, aged white wines were studied.
- Chiral pharmaceuticals in the environment : enantiomeric fraction and biodegradation studiesPublication . Ribeiro, Ana Rita Lado Teixeira; Castro, Paula Maria Lima; Tiritan, Maria Elizabeth; Afonso, Carlos Manuel MagalhãesThe importance of the stereochemistry in medicinal chemistry and in pharmacology is well recognized and the diverse behaviour of enantiomers in the presence of chiral entities is fully documented. Therefore biodegradation in biotic media, as occurs in secondary treatment of wastewater treatment plants (WWTPs) can be enantioselective. The significance of chirality in the environment is comparable to the magnitude in medicinal chemistry and pharmacology since enantiomers of chiral contaminants can significantly differ in environmental fate as well as in effects. The need to evaluate the occurrence and biodegradation processes of single enantiomers of chiral pharmaceuticals (CPs) in the environment is imperative by enantiomeric fraction (EF) monitoring of biodegradation processes, wastewaters and surface waters. Assessing EF is only possible through enantioselective validated methods, which are currently scarce. The work described in this thesis comprises the development of new enantioselective analytical methodologies to accurately measure the EFs of CPs in environmental matrices and/or during biodegradation processes, especially for application to monitoring studies and designed biodegradation. The enantioselective analytical methods developed allowed the studies of biodegradation of several CPs in mineral growth medium (MM) inoculated with activated sludge; with the bacterial strain Labrys portucalensis F11; and in wastewater effluents. Additionally an enantioselective method for EFs quantification of multi-residue CPs was developed to monitor effluents of WWTPs and laboratory-scale bioreactors. Thus, this thesis allows increasing the knowledge on EF variation during biodegradation studies and also in environmental matrices. A vancomycin-based chiral stationary phase ChirobioticTM V column (4.6 mm internal diameter (i.d.)) was used to develop and validate two enantioselective analytical methods by High Performance Liquid Chromatography with Fluorescence Detector (HPLC-FD), allowing the analysis of enantiomers of alprenolol (ALP), atenolol (ATE), metoprolol (MET), propranolol (PHO) and fluoxetine (FLX) in biodegradation assays using MM inoculated with activated sludge. Biodegradation of the racemic mixtures was assessed in MM inoculated with activated sludge. The developed HPLC-FD methods under polar ionic mode had several advantages such as a simple sample preparation method; short chromatographic run; low flow rate; no need of solvents for sample preparation or derivatization and the use of an easy to operate, selective and sensitive detector. Biodegradation by activated sludge had slightly higher rates for the (S)-enantiomeric forms of ALP, PHO and MET, with the enhanced biodegradation by acetate as another growth substrate. Biodegradation of ATE and FLX was non-enantioselective. The effect of simultaneous supplementation decreased the biodegradation rates of all enantiomers perhaps due to a competitive mechanism. An enantioselective analytical method by HPLC-FD was optimized and validated using the ChirobioticTM V column (4.6 mm i.d.) to analyse individual enantiomers of FLX and the metabolite norfluoxetine (NFLX). This method preconcetrated 50 mL of wastewater samples through solid phase extraction (SPE) with Oasis® Mixed mode Cation eXchange (MCX) cartridges and allowed the simultaneous analysis of FLX and NFLX enantiomers under reversed mode using an eco-friendly mobile phase constituted by ethanol and ammonium acetate buffer. The method demonstrated to be easy to operate, selective, sensitive and easy to adapt to other matrices. Biodegradation of racemic FLX in microcosms mimicking aquatic environments (WWTP effluents) was followed by the validated method during 46 days with no observation of enantioselectivity in degradation process. The biodegradation of each enantiomer of FLX separately was also evaluated in MM inoculated by the bacterial strain Labrys portucalensis F11. Degradation of the two enantiomers with preferential degradation of the (R)-enantiomer was observed, with no observation of enantiomerization during the monitorization. The metabolite NFLX was not detected during degradation. An enantioselective SPE-HPLC with tandem Mass Spectrometry method was developed with a triple quadrupole mass analyser using the ChirobioticTM V column with 2.1 mm i.d. in reversed mode to quantify enantiomers of seven CPs belonging to different therapeutic classes (ALP, bisoprolol (BSP), MET, PHO, FLX, venlafaxine (VNF) and salbutamol (SBT)) and one metabolite (NFLX). This method used effluent of a laboratory-scale bioreactor for matrix validation and Oasis® MCX cartridges to preconcentrate 250 mL of water samples, being useful for monitoring wastewater effluents and also effluents of laboratory-scale bioreactors. The limits of detection were between 0.65 and 11.5 ng L-1. The use of a selective and sensitive detector allowed the unequivocal identification and quantification of the target enantiomers in complex matrices. The effluents of three municipal WWTPs in Portugal were analysed. Enantiomers of FLX, VNF, BSP, MET and PHO were found. VNF and FLX were found with EFs different from 0.5.