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- Exploiting the use of a liquid waveguide capillary cell for spectrophotometric determinations in flow-based systemsPublication . Páscoa, Ricardo Nuno Mendes de Jorge; Rangel, António O. S. S.; Tóth, Ildikó V.In this thesis, the use of a liquid waveguide capillary cell (LWCC) was exploited for the spectrophotometric determination of several analytes in different types of water. With the purpose of in-line sample handling, different flow approaches were used for the development of simple, robust, cheap and automated analytical procedures. The first procedure was based on a sequential injection analysis (SIA) system for the determination of iron in coastal waters. With the goal of reaching low levels of iron, a LWCC was coupled to the system. This procedure used a doubled-line SIA system to improve mixing conditions between sample and reagents. The detection was based on a colorimetric reaction and two different reagents (ferrozine and 1,10-phenanthroline) were tested. The absorbance measurements were carried out at the wavelengths of 512 and 562 nm for the detection of iron-1,10-phenathroline and iron-ferrozine complex, respectively. An interference study was performed for both reagents. The developed method was also applied to natural waters (river, well, ground, potable and sea waters) and then compared with the reference procedure. One certified reference water sample was used to test the accuracy of the developed method. The objective of the second work was to determine iron at lower levels than the previous work and, as a consequence, to measure the levels of iron in ocean waters. With this in mind, a LWCC and a pre-concentration resin were coupled to a multisyringe flow injection analysis (MSFIA) system. Two different pre-concentration resins (Chelex 100 and NTA Superflow) were tested and evaluated. The determination of iron was also based on a colorimetric reaction and two reagents were tested, ferrozine and ammonium thiocyanate. The reactions were monitored at the wavelengths of 480 and 562 nm for the detection of iron-ammonium thiocyanate and iron-ferrozine complex, respectively. The accuracy was assessed using a certified reference water sample. A multi-parametric system for the spectrophotometric determination of zinc and copper at low levels in waters was the third work of this thesis. To attain this objective, a LWCC was coupled to a MSFIA system. The developed procedure for both analytes was based on a colorimetric reaction with zincon reagent at different pH values and monitored at 620 nm. Zincon reagent reacts only with copper at pH 5 and with copper and zinc at pH 9. An interference study for both determinations was carried out. The developed work was also applied to natural waters and three certified reference water samples. Subsequently, a multi-pumping flow system (MPFS) coupled with a LWCC was developed for the determination of titanium. This determination was based on the colorimetric reaction of titanium with chromotropic acid and the absorbance measurements were carried out at 425 nm. An interference study was performed in order to evaluate possible interferences. The developed procedure was applied to natural waters as well as to sunscreen formulations (the results were compared with the reference procedure). The accuracy was assessed with one certified lake sediment. The development of a spectrophotmetric method for bromate determination in waters at trace levels was the last work of this thesis. With this objective, a LWCC was coupled to a MPFS. The proposed methodology was based on a colorimetric reaction and two different colour reagents were tested, chlorpromazine and trifluoperazine. The lack of repeatability detected in this approach led to the development of a FIA approach in order to find out the reasons of this occurrence.
- Production of glycerol and 1,3-propanediol from renewable ressources : study of new biocatalysts and process strategiesPublication . Mendes, Filipa Cristina Soares; Vasconcelos, Isabel M.1,3-Propanediol can be biologically produced from glycerol by several microorganisms, namely clostridia. However, no natural microorganism is able to synthesize 1,3-propanediol from glucose. Different strategies have been tried to accomplish this conversion and set up a flexible process operating either on glycerol or glucose. In this work the production of 1,3 - propanediol from glucose and sugar cane molasses was investigated in a two-step process using two recombinant microorganisms. The first stage of the process was the conversion of glucose or other sugar into glycerol by a Saccharomyces cerevisiae strain. To increase glycerol production, different tpi1Δ mutant strains of S. cerevisiae, resulting from genetic engineering strategies, focused on the redirection of metabolic fluxes and overexpression of the key enzymes of the glycerol formation pathway, were tested. Best results were obtained with the mutant strain S. cerevisiae HC42 which exhibits a glycerol yield of 0.46 g.g-1 glucose and a productivity of 3.1 mmol glycerol.h-1.g-1 dry mass on 20 g.l-1 of glucose. Genomic analysis of this strain revealed that 384 genes had significantly changed in response to the genetic modifications introduced; also, the genetic strategy followed led to an intracellular glycerol concentration 10-fold higher than in the parent strain. To solve this issue, the overexpression of FPS1 was exploited in S. cerevisiae HC42. A novel mutant strain FM62 was obtained; however no significant differences on glycerol intracellular accumulation and production were reported. The strain HC42 was not able to grow on high glucose concentrations. In order to increase the glycerol production, the strain was adapted to high glucose concentrations (> 200 g.l-1). The adapted strains FH100 and FH200 were able to grow on 100 and 200 g.l-1 glucose leading to final glycerol concentrations of 49.2 g.l-1 and 60.0 g.l-1 respectively. FH100 was also cultivated on sugar cane molasses media; the production of glycerol increased with the initial total sugars concentration and 47.1 g.l-1 of glycerol were produced when the strain was grown on 101.3 g.l-1 total sugars. The second stage of the process was carried out by the engineered strain Clostridium acetobutylicum DG1 (pSPD5) able to convert glycerol to 1,3- propanediol. This two-step strategy led to a flexible process, resulting in a 1,3- propanediol production and yield that depended on the initial sugar concentration. Below 56.2 g.l-1 of sugar concentration, cultivation on molasses or glucose showed no significant differences. However, at higher molasses concentrations glycerol initially produced by yeast could not be totally converted into 1,3- propanediol by C. acetobutylicum and a lower 1,3-propanediol overall yield was observed. Best results were obtained with an initial glucose concentration of 103 g.l-1, leading to a final 1,3-propanediol concentration of 25.5 g.l-1, a productivity of 0.16 g.l-1.h-1 and 1,3-propanediol yields of 0.56 g.g-1 glycerol and 0.24 g.g-1 sugar, which is, to our knowledge, the highest value reported for a two-step process. For an initial sugar concentration (from molasses) of 56.2 g.l-1, 27.4 g.l-1 of glycerol were produced, leading to 14.6 g.l-1 of 1.3-propanediol and similar values of productivity, 0.15 g.l-1.h-1, and overall yield, 0.26 g.g-1 sugar.
- Physiological bases of texture and color changes in fresh-cut fruit using pear and melon as model systems : implications for the development of technologies and processesPublication . Gomes, Maria Helena Martins Teixeira; Almeida, Domingos Paulo Ferreira de
- Development of sequential injection enzymatic and bead injection assays in lab on valve formatPublication . Vidigal, Susana Maria Socorro de Matos Peixoto; Rangel, António O. S. S.; Tóth, Ildikó V.The development of bioengineering and biotechnology has promoted the interest for faster and more reliable methods for monitoring biochemical processes. Flowbased systems stand out as the most effective in the automation and miniaturization of these methods. A significantly reduction on the analysis time and dispensing skilled labour is achieved, thus becoming an important tool for implementation and enforcement of these assays. The main objective was the development of new methodologies for the miniaturization of biochemical analysis system using a sequential injection lab-onvalve (SI-LOV) system. In this context, analytical methodologies were developed based on enzymatic assays for the detection of enzyme substrates, for measurements of enzyme activity, or for enzymatic inhibition studies. Under this program, some enzymatic methodologies were developed: determination of ethanol in alcoholic beverages; determination of hydrogen peroxide in cleaning solutions for contact lenses, with and without dilution in line; and determination of the activity of peroxidase in vegetable extracts. The developed method for the enzymatic assay of ethanol in beverages (Chap.3) was based on the conversion of ethanol to acetaldehyde by alcohol dehydrogenase (ADH), using spectrophotometric detection. The quantification of hydrogen peroxide (H2O2) (Chap.4) and peroxidase activity (Chap.5) was based on the reaction of oxidation of 2,2’-Azino-bis(3-Ethilbenzothiazoline 6-sulfonic acid) (ABTS) with hydrogen peroxide in the presence of peroxidase (HRP). Another major goal was to explore the potential of the SI-LOV systems to execute solid phase spectrophotometry, demonstrating its suitability for handling complex samples. This process, in the form of bead injection, was implemented inside the flow system. For this purpose, we used the resin "NitriloaceticAcid (NTA) Superflow", capable of retaining metal ions, which can be derivatized and measured by spectrophotometry (solid-phase spectrophotometry). These methods were used for the quantification of iron and protein content in wine samples. The developed method for the quantification of iron in wine samples (Chap. 6) was based on the reaction of Fe3+ with SCN-, where the Fe3+ ions were retained at the surface of the NTA beads. The total iron content was achieved by performing the inline oxidation of the Fe2+ to Fe3+. The methodology for total protein content in white wine samples (Chap.7) was based on the Lowry method. The NTA beads were initially charged with Cu2+ to complex with the proteins present in the sample. The quantification was achieved by the colour reaction of the proteins with the Folin Ciocalteau reagent. As for detection system, UV/Vis spectrophotometry was used in all of the developed methodologies.
- Valorisation of codfish (Gadus morhua L.) salting processing wastewater through the extraction of high added value compoundsPublication . Ferraro, Vincenza; Castro, Paula Maria Lima; Pintado, Maria Manuela EstevezApesar do desenvolvimento de outros métodos de preservação, a salga do bacalhau (Gadus morhua L.) continua a ser uma realidade devido a um conjunto de factores, entre os quais a simplicidade e o baixo custo do processo bem como as características sensoriais do produto final, muito apreciadas pelos consumidores. Ao longo do processo de salga o bacalhau incorpora sal até 20% do seu peso e liberta concomitantemente cerca de 22% da sua água fisiológica; assim, aproximadamente 200 litros de água residual salgada são gerados para cada tonelada de bacalhau fresco. Esse efluente é actualmente tratado como resíduo tóxico devido ao alto teor de cloro, que pode atingir valores de concentração de cerca de 160 g/L. A libertação desta água trás como consequência alterações significativas na composição e na estrutura do tecido muscular do bacalhau, levando à perda de compostos bioativos importantes, entre os quais aminoácidos livres, peptídeos e proteínas, nutrientes que, embora não essenciais, podem ser benéficos em certas circunstâncias. Assim, a recuperação de compostos orgánicos e do sal marinho de grau alimentar utilizado no processo de salga pode contribuir para a gestão integrada da água residual em questão. Com este objetivo, o perfil químico da água libertada foi avaliado. No final do período de salga o conteúdo de matéria seca na água libertada atinge o valor de ca. 10 g/L. A concentração de aminoácidos livres aumentou de 3.5 g/L a 6.5 g/L em 6 dias, devido a fenómenos de proteólise. Creatina, ácidos aspártico e glutâmico, arginina, glicina, metionina, lisina, taurina e triptofano, foram os aminoácidos livres predominantes e cuja libertação demonstrou-se obedecer a uma cinética monomolecular ou de pseudo-segunda ordem, dependendo do aminoácido. Embora em menor escala, a concentração de proteínas miofibrilares também aumentou com o tempo – de 3 para 3.7 g/L – fenómeno este ainda atribuível à proteólise. A análise do azoto total e as suas fracções mostraram que, no final do processo de salga, 36.6% (w/w) de azoto total corresponde a peptídeos curtos com até 20 residuos e a aminoácidos livres, 14.7% (w/w) aos peptídeos com mais de 20 residuos e os restantes 48.7% (w/w) às proteínas. A concentração total de aminas biogénicas na água no final do processo de salga foi de ca. 100 mg/kg. Proteínas, peptídeos a aminoácidos foram recuperados com sucesso por meio de um processo de sorção em batch e após um pré-tratamento com etanol de grau alimentar de forma a reduzir a concentração de sal da água, cuja elevada concentração, ca. 4.3 M, demonstrou afectar negativamente o mecanismo de adsorção dos aminoácidos. A presença do sal em certas concentrações demonstrou-se no entanto ser positiva, devido ao efeito da força iónica sobre a adsorção de aminoácidos. A resina polimérica comercial Amberlite XAD16, uma resina neutra e não-polar, foi seleccionada para prosseguir com estudos de adsorção. Aminoácidos livres, proteínas e peptideos foram adsorvidos no mesmo estágio. O processo de recuperação foi efectuado recorrendo a solventes de grau alimentar. Foi efectuada uma análise paramétrica do processo de adsorção examinando o efeito de diferentes parâmetros, nomeadamente temperatura, pH, percentagem de etanol adicionado a água residual, agitação, força iónica da solução, quantidade de adsorvente. O volume de solvente eluente e a temperatura foram os parâmetros avaliados na etapa de desadsorção. Os resultados mostraram que o processo de adsorção é controlado pela temperatura e pela força iónica, a qual neutraliza os efeitos do pH, e que a desadsorção é controlada pela temperatura e pela natureza do solvente eluente. A acetona resultou como o melhor solvente para desadsorção de aminoácidos livres, com um rendimento de recuperação de aminoácidos hidrofobicos e neutros de 100 %. As proteínas foram desadsorvidas por uma solução básica de hidróxido de sódio em água ao 4% (w/v), com um rendimento de recuperação de 100 %. Os aminoácidos livres extraídos da água residual mostraram actividade antioxidante em geral e de proteção do DNA contra a oxidação em particular. Para os mesmos aminoácidos, a biodisponibilidade in-vitro foi estudada usando celulas Caco-2, medindo a taxa de transporte paracelular e a resistência elétrica transepitelial para verificar a integridade das células do epitelio intestinal. Os resultados mostraram que todos os aminoácidos livres extraídos permearam através da monocamada celular intestinal, embora em taxas diferentes e dependendo da sua concentração inicial; o transporte foi superior a 90 % para todos os aminoácidos livres, excepto a creatina, cujo transporte não passou de 6 %. A presença de sal na solução teve um papel positivo sendo o cloreto de sódio entre os mais importantes osmólitos de aminoácidos em seres humanos.
- Ethylene oxide sterilisation of medical devices : development of mathematical models for prediction of ethylene oxide diffusion and microbial lethalityPublication . Mendes, Gisela Cristina da Cunha; Silva, Cristina Luísa Miranda; Brandão, Teresa Maria Ribeiro da SilvaO óxido de etileno (EO) é um agente de esterilização dominante na indústria dos dispositivos médicos, devido à sua efectividade e compatibilidade com a maioria dos materiais. Considerando o crescimento exponencial do mercado de dispositivos médicos sensíveis, complexos e sofisticados, assim como de conjuntos de procedimento customizados (que combinam uma grande diversidade de produtos e gama de polímeros) utilizados em actos médicos e cirúrgicos específicos, o EO emerge como o método de esterilização de eleição. A optimização deste processo constitui um desafio devido ao facto da competitividade do mercado global exigir custo-efectividade, flexibilidade e redução do tempo de ciclo necessário à colocação dos produtos no mercado, sem prejuízo da segurança e assegurando o cumprimento dos requisitos reguladores. A esterilização por EO é um processo multi-paramétrico complexo, que exige uma fase final de arejamento dos materiais. A eficácia destes processos é influenciada por diversas variáveis (e.g. temperatura, humidade, concentração do agente, material alvo), pelo que a sua optimização exige o conhecimento da influência das condições impostas e das suas relações com os produtos. A modelização matemática do processo de esterilização e arejamento permite a definição das condições óptimas para morte microbiana e desgaseificação, respectivamente. Tal permite a redução dos tempos de ciclo e/ou concentração de EO, assim como a comparação de diferentes processos de esterilização. Deste modo é possível contribuir para o desenvolvimento de processos com eficiência e flexibilidade acrescidas e a libertação paramétrica da esterilização surge assim cientificamente apoiada. Nesta dissertação foi estudada a influência das condições de processo na esterilização de dispositivos médicos por EO. Este é um processo implementado na empresa Bastos Viegas, S.A., desde 2005. Foram estudados os efeitos e interacções da temperatura, concentração de óxido de etileno e humidade relativa na inactivação do Bacillus subtilis, var. niger (ATCC 9372), o microrganismo de referência usado no controlo do processo. As experiências foram realizadas em câmaras de esterilização, com uma carga de campos cirúrgicos. Aplicou-se um planeamento experimental factorial 23 para avaliação dos efeitos das três variáveis (de acordo com limites comuns de condições operacionais) na letalidade da esterilização por EO. A inactivação do B. subtilis apresentou um comportamento sigmoidal e um modelo baseado em Gompertz foi ajustado com sucesso aos dados experimentais. Características importantes das curvas, tais como atraso inicial e taxa de inactivação foram considerados parâmetros do modelo. A temperatura e a concentração de óxido de etileno foram as variáveis que afectaram significativamente esses parâmetros, pelo que experiências adicionais foram realizadas de forma a incluir o efeito dessas variáveis do processo. Foi desenvolvido um modelo matemático para previsão da morte de B. subtilis expresso em função da temperatura e concentração de EO. Uma vez que a letalidade demonstrou estar directamente relacionada com a concentração de agente esterilizante, compreender a efectividade da esterilização por EO suscita obter a permeabilidade dos materiais ao gás, assim como o conhecimento sobre a dinâmica deste processo. A análise em tempo real da concentração de EO no espaço-livre do esterilizador tem-se tornado prática comum e o desafio neste momento é prever o seu valor no interior da carga. Foi desenhado, concebido e desenvolvido um equipamento para determinar a difusividade e a solubilidade de EO, e a permeabilidade de diversos materiais ao gás, pela metodologia de tempo de atraso. A operação deste equipamento é baseada no princípio de medição de uma mudança transiente de pressão em condições de volume e temperatura constantes (abordagem pressão-variável) e permite a determinação do tempo de atraso e do fluxo de EO em estado estacionário através de diversos materiais. As experiências foram executadas com um material de campo cirúrgico, a uma temperatura típica de esterilização, i.e. 48 ˚C, e a uma pressão de 3,2x105 Pa. As propriedades de transferência do EO no campo cirúrgico foram usadas para modelizar a sua cinética numa carga industrial do mesmo material. A segunda lei de Fick demonstrou ser adequada na descrição do processo de transferência, o que foi validado pela utilização de dosímetros que integraram a concentração de EO durante o tempo de exposição. A etapa de arejamento é importante para reduzir os resíduos de EO a um nível seguro e neste contexto, prever a desorção do EO é uma importante ferramenta para a optimização global da esterilização por EO. A cinética de desorção de EO de diferentes materiais esterilizados foi avaliada numa gama de temperaturas de arejamento entre 1,5 e 59,0 °C. Os dados experimentais seguiram um processo de difusão Fickiano e as difusividades foram estimadas para dois materiais médicos têxteis e dois poliméricos. Os resultados apresentados nesta dissertação contribuem para uma melhor compreensão da dinâmica do processo de esterilização por EO e por conseguinte, para uma optimização e controlo eficiente deste processo.
- 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.
- Modulation of aroma volatiles and phytochemical quality of fresh-cut melon (Cucumis melo L.) by oxygen levels, 1-methylcyclopropene and lysophosphatidylethanolameinePublication . Amaro, Ana Luísa Leite Fernandes; Almeida, Domingos Paulo Ferreira deCurrent fresh-cut technologies are effective in the effort of maintaining visual quality during the fresh-cut fruit supply chain. However, studies on extended shelf life based on appearance attributes or microbial stability have neglected the understanding of the effect of processing technologies and conservation techniques in the aromatic, nutritional, and functional properties of processed fruit. Three technologies were evaluated for their effects on quality of fresh-cut melon, with emphasis on aroma volatiles: oxygen levels, the ethylene action inhibitor 1-methylcyclopropene (1-MCP), and lysophosphatidylethanolamine (LPE), an inhibitor of phospholipase D (PLD). Modified atmosphere packaging (MAP) is widely used in fresh-cut fruit, with the objective of extending shelf-life. Nevertheless, MAP requirements and recommendations for fresh-cut fruit target mostly color and texture maintenance, neglecting the effects on aroma production. In this thesis, the quality attributes and aroma production of fresh-cut melon were monitored during MAP storage. Cantaloupe and honeydew melon cultivars were processed into cubes and stored at 5 ºC under high passive MAP or reduced controlled atmosphere (CA; 5 kPa O2 + 10 kPa CO2, balance N2). Oxygen levels during storage had a greater impact on aroma than on other quality attributes during storage of fresh-cut climacteric and non-climacteric melon. Cantaloupe and honeydew melon flavor-important volatiles were differently affected by oxygen levels inside the packages. Also, storage time had a significant effect on volatile production of fresh-cut cantaloupe and honeydew melons. 1-MCP application to whole fruit, its effects on quality, ethylene production and perception have been the subject of many studies. In contrast, fewer studies have focused on 1-MCP application to fresh-cut fruit. For its known inhibitory effect on ethylene production, we anticipated 1-MCP to have beneficial effects on some quality attributes of fresh-cut melon, while altering aroma profile. The effect of 1-MCP application in quality and synthesis of flavor-important volatile compounds in melon was evaluated during storage of fresh-cut cantaloupe. Cantaloupe melons were processed and treated with 1.0 μL L-1 of 1-MCP, for 24 h at 5 ºC, packaged in vented plastic clamshells and stored under normal atmosphere at 5 ºC for 9 days. Treatment with 1-MCP did not affect the main quality attributes of fresh-cut melon, but affected nine of the flavor-important volatiles, particularly those with isoleucine and phenylalanine as precursors. 1-MCP affected fresh-cut cantaloupe production of propyl acetate, 2-methylbutyl acetate, methyl butanoate, methyl 2-methyl butanoate, methyl hexanoate, 2-methylbutyl alcohol, phenethyl alcohol, benzyl alcohol and heptanal. LPE is reported to inhibit PLD activity, reduce ethylene production, and extend shelf-life of many horticultural commodities. Therefore, LPE could provide a novel treatment to improve the quality of fresh-cut fruit, via maintenance of membrane integrity and reduction of the wound response. The effect of LPE on PLD activity, quality and volatile production of fresh-cut melon was investigated during storage. Charentais-type melons were processed into cubes and vacuum-infiltrated with 200 mg L-1 of LPE, packaged in plastic clamshells and stored at 5 ºC for 9 days. LPE was not effective in maintaining overall quality of fresh-cut melon but induced a reduction of aldehydes production in the first three days of storage. LPE inhibited PLD activity in the first day after processing, which may have induced the reduction of aldehydes observed at day three of storage of fresh-cut melon. In conclusion, oxygen levels significantly affect the volatile profile of fresh-cut melon, and there may be opportunity to optimize oxygen recommendation for aroma preservation. 1-MCP and LPE altered the volatile profile of fresh-cut melon but had minimal or no effect on other quality attributes.
- Exploiting the capacity of Labrys Portucalensis strain F11 for biotransformation of fluoroaromatic compoundsPublication . Moreira, Irina Susana Sousa; Castro, Paula Maria Lima; Afonso, Carlos; Carvalho, FátimaEnvironmental contamination with toxic chemicals of human origin is a threat to ecosystems and public health. Thus, it is extremely important to understand and explore the removal of these contaminants from different environments through biodegradation. The main objective of the work described in this thesis was the exploitation of the potential of Labrys portucalensis F11 – a bacterial strain previously isolated by its ability to degrade fluorobenzene (FB) - for biotransformation of fluorinated aromatic compounds of different complexity. Typically, contaminated environments are not contaminated with a single pollutant but with mixtures of pollutants. In particular the presence of organic compounds and metals in the same ecosystem, or appearing simultaneously in wastewater streams, is very common. In this study, the effect of three metals with different biological importance - iron, copper and silver - on the degradation of FB by strain F11 was evaluated. At a concentration of 1 mM, iron proved to be beneficial for bacterial growth without adversely affecting the biodegradation of up to 2 mM of FB. The presence of 1 mM of copper and silver inhibited the degradation of FB and led to the accumulation of intermediate metabolites, catechol and 4-fluorocatecol suggesting inhibition of the catechol 1,2-dioxygenase, which is a key enzyme of the metabolic pathway of FB. The degradation of compounds with chemical structures similar to FB, namely chlorobenzene (CB) and difluorobenzenes (DFBS), by L. portucalensis was investigated. Strain F11 was able to cometabolise CB in the presence of FB or when previously induced by this compound. Total degradation of 0.5 mM of each substrate was observed when both were added to the culture medium. Strain F11 was capable of degrading CB when the expression of enzymes is induced by FB however CB was not able to induce the enzymes for its own degradation. For DFBS, strain F11 proved to be able to degrade 0.5 mM of 1,3-DFB as sole carbon source, and to degrade 1,4-DFB (0.5 mM) in cometabolism with FB (0.5 mM). Strain F11 was unable to degrade 1,2-DFB and this compound inhibited the degradation of FB. These results reinforce the importance of the nature, number and position of the substituents in the molecule for enzyme expression and subsequently conversion of the target compounds. Fluoxetine (FLX) is a fluorinated chiral drug, which contamination, toxicity and persistence in the environment have been well documented in the past years. L. portucalensis F11 showed to be able to degrade both enantiomers of this compound as the sole carbon source (up to 9 μM) and in the presence of a conventional carbon source, sodium acetate (up to 89 μM of FLX). Degradation extents of at least 80% of total FLX were obtained. At the lowest FLX concentration tested (2 μM) as single carbon source, degradation was complete and fluoride release was stoichiometric. The degradation was shown to be enantioselective, with preferential degradation of R-FLX in relation to S-FLX. The transient formation of norfluoxetine (NFLX) as an intermediary metabolite was detected. With the objective of finding the genes responsible for the expression of FB dioxygenase, a genomic library consisting of 960 clones was constructed from the DNA of L. portucalensis F11. This library can be used for future work, such as the generation and confirmation of sequencing data, for comparative genomic studies or to search for other genes of interest. It is important to note that this strain has shown extraordinary capabilities of degradation of toxic compounds, and as such it would be very interesting to further study the genes that confer these capabilities. A partial nucleotide sequence of the gene cluster involved in FB degradation was determined. Sequencing results revealed the presence of four open reading frames, namely the gene coding for 1,2-catechol dioxygenase, and three genes encoding a ringhydroxylating dioxygenase (alpha and beta subunit of the dioxygenase component and the oxidoreductase component). Alignment of the deduced aminoacid sequences with sequences of others ring-hydoxylating dioxygenases revealed a high degree of similarity (≥80% identity) to the components of (halo)benzoate dioxygenases. The conserved amino acid residues that are involved in cofactor binding were also identified in the protein sequence. Recombinant strains carrying the putative FB dioxygenase genes were tested for expression. The SDS-PAGE analysis revealed that most of the expressed protein was on the pellet fraction and not on the soluble form, which could be due to improper folding of the enzyme components. Decrease of substrate concentration was observed in bioconversion experiments but the product formed was not detected/ identify. Overall, strain F11 revealed to be capable of degrading a vast range of fluorinated compounds with different complexity and as such can be a potential strain to devise biotechnological solutions for biotransformation processes.
- Hazards and control of risks in artesanal meat products in PortugalPublication . Campelos, Maria Isabel Pereira da Silva; Teixeira, Paula; Gibbs, Paul; Hogg, Tim"Salpicão de Vinhais" and "Chouriça de Vinhais" are traditional dry-fermented smoked meat sausages produced in Vinhais, a small region of Trás-os-Montes. The scientific knowledge of this sausage variety is limited. This work aims to collect scientific data that could, partially, fill the gaps in knowledge regarding these products, analyse the data and develop a risk-based study, according to an internationally accepted framework and finally, to explore the effect of hypothetical risk management measures on the safety of consumers, regarding traditional dry fermented meat smoked sausages. Seventy seven samples of ―Salpicão‖ and ―Chouriça de Vinhais‖ were purchased from producers, local markets and retail stores. Their microbiological and physical chemical characteristics were analyzed. The same analyses were performed on the products during the production processes. The raw materials and ingredients were also analyzed. Regarding the pathogenic flora, Staphylococcus aureus, spores of sulphite-reducing clostridia, Escherichia coli 0157:H7, Yersinia spp. and Salmonella spp., were not detected in any of the samples analyzed; Listeria monocytogenes was detected in 14.3% of the samples. The manufacturing process, namely fermentation, ripening/drying and smoking reduced the numbers of pathogen and hygiene indicator microorganisms. According to the Commission Regulation (EC) 2073/2005, 39% of the contaminated samples of ―Salpicão‖ and ―Chouriça de Vinhais‖ were able to support growth of L. monocytogenes. A quantitative microbiological risk assessment using a probabilistic model was developed. Considering the Portuguese population, with data obtained in this work, the calculated risk of listeriosis, for the intermediate age sub-population was 0.1297 cases per year, for the perinatal sub-population, 1.3695 cases per year, and elderly sub-population 0.1995 cases per year. This means that Traditional Dry Fermented Smoked Sausages constitute a low risk for the Intermediate Age and Elderly population (less than 1 case per annum) and an intermediate risk for the Perinatal populations, considering this last group of consumers as adult pregnant women (1-10 cases per annum). The effect of several putative risk management actions such as the adoption of a Performance Objective of 10 CFU/g, 1 CFU/g and 0.04 CFU/g reduced the risk of listeriosis for all sub-groups, at the end of shelf life, by 25.5%, 58.4% and 58.6%, respectively. The effect of bacteriocinogenic strain of Pediococcus acidilactici HA6111-2, previously isolated from ―alheira‖, was studied on both ―Salpicão‖ and ―Chouriça‖, in a challenge test against a cocktail of Listeria innocua, at pilot scale. A reduction of the numbers of Listeria innocua was achieved in all batches, being the biphasic equation the model that gave the better fit. The use of a bacteriocinogenic strain added to the batter reduced the risk of listeriosis for all sub-groups by 26.7%; obtaining a final product with aw below 0.92, supposedly below the growth limits of L. monocytogenes, reduces the risk by 8.6% in all sub-groups of population; the combination of the bacteriocinogenic strain and the limit of aw 0.92 reduces the risk by 41.0%. Traditional dry fermented smoked sausages present low to medium risk to the health of consumers. The use of more restrictive Performance Objectives during processing and/or distribution will result in a further reduction of risk. A more rigorous control of final product water activity and the use of a bacteriocinogenic bioprotective culture during production may contribute significantly to reducing the risk of listeriosis in consumers of these Traditional Dry Fermented Smoked Sausages (TDFSS).