ESB - Teses de Doutoramento / Doctoral Theses
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- 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.
- Contributions to flow techniques and mass spectrometry in water analysisPublication . Santos, Inês Carvalho dos; Rangel, António Osmaro Santos Silva; Mesquita, Raquel Beatriz Ribeiro deIn this thesis, the use of different flow systems was exploited along with the use of different detection techniques for the development of simple, robust, and automated analytical procedures. With the purpose to perform in-line sample handling and pretreatment operations, different separation units were used. The main target for these methods was waters samples. The first procedure was based on a sequential injection analysis (SIA) system for carbon speciation (alkalinity, dissolved inorganic carbon (DIC), dissolved organic carbon (DOC), and CO2) in bathing waters. The determination of alkalinity was based on the reaction with acetic acid and corresponding color change of bromcresol green (λ = 620 nm). The DIC, DOC, and CO2 determination was based in the color change of bromothymol blue (BTB) indicator (λ = 660 nm) after the diffusion of the gaseous CO2 through a hydrophobic membrane in a gas diffusion unit (GDU). For the DIC determination, an in-line acidification prior to the GDU was needed. For DOC determination, an in-line UV photo-oxidation of the sample was performed to convert all carbon forms to CO2. A multi-reflective flow cell combined with a LED was used as detection system enabling the minimization of the schlieren effect. The developed method was successfully applied to inland bathing waters. The results were in agreement with the reference procedure. The objective of the second work was the use of a SIA system to determine iodide using potentiometric determination and iodate using a spectrophotometric detection. Aiming to overcome the matrix interferences and cope with low analyte concentration levels, the standard addition method was used. The incorporation of a mixing chamber in a lateral port of the selection valve for a flow-batch approach enhanced the mixture between sample, standards and reagent. The accuracy of the method was assessed by comparing the obtained results with a reference procedure and using recovery tests. The developed method was effectively applied to bathing waters and seaweed extracts. The development of a multi-parametric system for cadmium and lead determination was the objective of the third work of this thesis. A SI-LOV method with spectrophotometric detection was used. Dithizone was chosen as the colour reagent as it forms a coloured complex with both metals at different pH conditions. Cadmium determination was attained at alkaline conditions while lead determination was attained at mid alkaline conditions. The developed method was successfully applied to marine port waters and the results were in agreement with the reference procedure. Subsequently, a solid phase spectrophotometry method was developed for cadmium, zinc, and copper determination in freshwaters. The NTA resin was retained at the flow cell of the SI-LOV. After analyte retention, the determination was based on the colorimetric reaction between the metal ions and dithizone. Different retention and reaction pH were used to individually determine zinc and copper. Cadmium concentration was calculated using a two equation system. The results obtained were in agreement with the reference procedure. Afterwards, a flow injection analysis system was developed for the determination of bromate in drinking waters. With the goal of reaching low levels of bromate, a LWCC was coupled to the system. The spectrophotometric determination was based on the oxidation of chlorpromazine by bromate in an acidic medium, resulting in the formation of a coloured radical product. The accuracy of the developed method was assessed with recovery studies. The objective of the sixth work was to develop a cheaper and simpler method to monitor the biodegradation of ciprofloxacin and ofloxacin. A monolithic column was coupled to a flow injection analysis (FIA) system to separate both analytes. A spectrophotometer was used as detection system (295 and 275 nm). The method was successfully applied to monitor the biodegradation of both compounds by the strain Labrys portucalensis F11. Subsequently, a new MS-based noncovalent binding determination method was developed. For that purpose, a continuous stirred tank reactor (CSTR) was used as a flow injection device for exponential dilution of an equimolar host-guest solution over time. By combining an exponential dilution model with a previously established equimolar binding model, binding constants for host-guest complexes were determined with a single injection. This methodology was applied for the determination of binding constants between vancomycin and Ac-Lys(Ac)-Ala-Ala tripeptide stereoisomers. The determination of anionic surfactants using an ESI-MS/MS method was the objective of the eighth work. Different di-positive cationic reagents were studied using Scan, SIM and MS/MS methods and the obtained detection limits (DLs) were compared. The developed method was effectively applied for the determination of perfluorooctanesulfonic acid, perfluorooctanoic acid, sodium dodecyl sulfate, stearic acid and dodecylbenzenesulfonate in different water samples. The ninth work consisted in the rapid identification of bacteria in potentially contaminated water using MALDI-TOF MS. The possible application of the identified bacteria to bioremediation was evaluated by determining their ability to degrade toluene and chloroform.
- Development of a functional orange juice powder by spray dryingPublication . Barbosa, Joana Inês Bastos; Teixeira, Paula Cristina MaiaThe concept of functional food products has acquired a huge dimension with the enormous number of people concerned about consuming foods with health benefits. An innovative non-dairy functional food would be very attractive, not only to these consumers but also to specific niches in the market (e.g. lactose intolerants and vegetarians). The aim of this work was to develop a powdered functional orange juice. For this, cultures with probiotic characteristics were used and the necessary conditions to obtain the dried product by spray drying were defined. Two cultures were selected, a commercial probiotic, Lactobacillus plantarum 299v and an isolate from a food matrix, Pediococcus acidilactici HA-6111-2. Safety and the presence of some functional characteristics of P. acidilactici were investigated and compared with those of the commercial probiotic. None of the bacteria produced any of the virulence factors investigated; they did not exhibit significant resistance to antibiotics and reductions lower than one logarithmic cycle were observed after exposure to simulated gastro-intestinal tract conditions. It was also verified that both bacteria survived during the spray drying process and subsequent storage for 60 days. Powdered orange juice was initially obtained in a Büchi Mini Spray Dryer at constant feed temperature (ºC), flow rates of feed (mL/min), drying air (%) and compressed air (L/h) and varying inlet and outlet air temperatures (ºC) as well as the ratio of total soluble solids (orange juice): drying aid (10DE maltodextrin or gum Arabic). At selected inlet and outlet air temperatures of 120 ºC and 65 ºC, respectively, and ratio of soluble solids: drying aid of 0.5: 2, high drying yields and powders with low water activities (aw) were obtained. No cell inactivation was observed for each culture after incorporated into orange juice and spray dried. Their survival during storage at 4 ºC was higher than at room temperature under uncontrolled aw. Pediococcus acidilactici was more resistant than L. plantarum, especially during storage at 4 ºC, with logarithmic reductions lower than 1 log-unit during a period of at least seven months. To enhance survival of spray dried bacteria during storage, different pre-spray drying conditions were tested: i) different sugars were added to the culture media used (cell growth in the presence of lactose, followed by glucose resulted in the highest survival) and ii) cells were exposed to different sub-lethal stresses (only survival of L. plantarum cells during storage at room temperature was increased). For both cases, powders obtained were stored under different conditions of temperature, light exposure and aw. Generally, survival was enhanced at 4 °C, aw of 0.03 and absence of light. Cell viability during passage through simulated gastro-intestinal tract conditions was also investigated at the end of the storage at 4 ºC; the viability of both cultures was not enhanced by prior exposure to sub-lethal stresses. Survival of cultures in orange fruit powders obtained by spray-, freeze- and convective hot air drying was investigated during drying and subsequent storage. Cell inactivation was only observed during convective hot air drying (about 2 logarithmic cycle reduction). Minimal reductions were observed for P. acidilactici for most conditions of storage while for L. plantarum only during storage at 4 ºC. Taking into account the initial number of cells obtained after each drying process, the techniques which allowed survival of an increased number of cells after storage, were spray- and freeze drying. Better dissolution, color and vitamin C retention were also obtained when these techniques were used. Despite the scale-up and validation at industrial scale being still necessary, spray drying at pilot scale allowed producing a new functional orange juice powder with probiotic characteristics. The bacteria used demonstrated to be good candidates for the development of this product.
- A fundamental study on physical properties and stability in food systems : the relationship with molecular dynamicsPublication . Pereira, Joana de Freitas Salgado do Fundo; Silva, Cristina L. M.; Quintas, Mafalda A. C.Food systems physical properties and stability are critical for delivering safe and healthy food to the consumers, and thus this is a theme that attracts food scientists for a long time. Recently, literature suggests that stability can only be fully grasped if food molecular dynamics and structure are taken into consideration, i.e. an appropriate understanding of the behaviour of food products requires knowledge of its composition, structure and molecular dynamics, through the three-dimensional arrangement of the various structural elements and their interactions. Food systems behaviour is strongly dependent on the water molecular dynamics. Understanding changes in water location and mobility represents a significant step in food stability knowledge, once that water “availability” profoundly affects the chemical, physical and microbiological quality of foods. Nuclear magnetic resonance (NMR), through the analysis of nuclear magnetisation relaxation times, has been presented as a powerful technique to investigate water dynamics and physical structures of foods. It provides information on molecular dynamics of different components in complex systems. The application of this technique may be very useful in predicting food systems physicochemical changes, namely texture, viscosity or water migration. The research leading to this thesis focused on two main food systems: i) films from biological sources, for their interest as model matrices and potential for food industry; and ii) fresh-cut fruit, due to its complexity and significance in food markets. Films from biological sources, particularly chitosan, present several applications including biodegradable packaging and edible coatings for shelf-life extension. As model food systems, films from biological sources are partially crystalline, partially amorphous, and easily reproducible materials. From a fundamental perspective, foods are mainly edible and digestible biopolymers that are also partially crystalline/partially amorphous. Despite of the wealth of information on literature, a systematic approach to understand the contribution of film forming solutions (FFS) on chitosan films physical properties, as well as the knowledge on its molecular dynamics to such properties, are still uncommon. In this thesis, the relevance of FFS composition on films properties is highlighted through the monitoring of solutions with different polymer/plasticiser ratios. Also the molecular dynamics, evaluated through NMR methodology, was analysed and compared with the films physical properties. Results demonstrated the influence of solutions polymer/plasticiser concentrations on both thermomechanical and water related properties. Chitosan concentration in solutions affected consistency coefficient, and this was related with differences in films water retention and structure. Plasticiser quantities used in FFS are responsible for films compositions, while polymer/plasticiser ratio determined the thickness and thus the structure of the films. NMR allows understanding the films molecular rearrangement, demonstrating that water is also an important component in these matrices and performs differently when compared with the plasticiser. A relationship between water and plasticiser dynamics and films macroscopic properties was also observed. Fruits are high water content products with a complex cellular structure, where water can be present in both intra and extra cellular spaces. Fresh-cut fruit, due to processing, has high metabolic rates with faster physiological and biochemical changes and microbial degradation, which results in product’s colour and texture alterations. The second part of this thesis focused on fresh-cut fruits, pear and melon, which were chosen for their significantly different composition and structure. Fresh-cut fruit was monitored during 7 days of refrigerated storage conditions. Relevant quality parameters, such as colour and firmness, were analysed. Water activity (aw) and water molecular dynamics (T2), measured by a NMR technique, were also assessed throughout storage. Results demonstrated that processing and storage affected quality parameters, as was expected, but also system’s water molecular dynamics. Throughout storage, it was possible to find relationships between the molecular dynamics and the quality parameters. These relationships were different for the two studied fruits, and the role of microstructure on food stability could be observed. These studies highlight the significance and impact of molecular dynamics on physical properties and stability of foods, and also the usefulness of NMR methodology as a tool to evaluate food physical properties and stability. Therefore, NMR could provide a novel instrument to improve the knowledge of food systems, even when complex.
- 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).
- Physiological and molecular mechanisms of stomatal functioning in plants grow at high humidityPublication . Carvalho, Dália Rosa Alves; Carvalho, Susana Maria Pinto de; Vasconcelos, Marta W.; Heuvelink, EpStomata from several plant species developed at high relative air humidity (RH ≥ 85%) may become malfunctional, leading to excessive water loss in conditions of high evaporative demand. In ornamental plants, including rose, this results in a low postharvest longevity. The factors underlying a poor stomatal functioning under high RH are not yet fully understood, but the magnitude of this effect is genotype dependent which broadens the possibilities for plant breeding. The aim of this thesis is to analyze the physiological and molecular mechanisms of stomatal malfunctioning in plants cultivated at high RH, focusing on abscisic acid (ABA) regulation and its effects on stomatal closure. The identification of genomic regions associated with the control of water loss is also a subject of this study. Rosa × hybrida was used as the model system since in this species there are cultivars with a contrasting sensitivity to high RH. Stomatal movements are to a large extent regulated by ABA concentration ([ABA]), which is determined by its metabolism within the leaf, as well as by its import from the roots. The role of root-to-shoot ABA signaling in inducing stomatal closure and its role in inducing genotypic differences in stomatal functioning was investigated in ten genotypes grown at contrasting RH conditions. It was found that xylem sap [ABA] did not explain genotypic differences in stomatal functioning, since sensitive and tolerant genotypes to high RH had a similar estimated [ABA] in the root xylem sap and a similar ABA delivery rate and [ABA] in the leaf petiole sap. Elevated air movement (MOV) and moderate salinity are two environmental stress factors known to induce stomatal closure. In two different studies, we combined a high RH level with high MOV or with a moderate salinity throughout plant growth in order to test if these physiological stresses would counteract the negative effect of high RH on stomatal functioning due to an increased [ABA]. Indeed, stomata developed at high RH with additional MOV or moderate salinity closed faster in response to leaf desiccation when compared to plants grown at high RH without either of the imposed stresses. While salinity enhanced stomatal functioning due to an increase in leaf [ABA], high MOV improved stomatal responsiveness due to a higher stomatal sensitivity to ABA. The two parents and the 108 offspring of the K5 tetraploid cut rose population grown at high RH were phenotyped for stomatal responsiveness to desiccation, showing large genotypic differences [i.e., relative water content after 4h of leaflet desiccation (RWC_4h) varied between 7 and 62%]. Three QTLs (two major and one putative minor) explained 32% of the genotypic variability and low RWC_4h proved to be a good proxy for eliminating the offspring with shorter vase life. These particular findings contribute to speeding up genotype selection using marker-assisted selection programs for breeding cultivars with more responsive stomata after cultivation at high RH, minimizing the negative impacts associated with excessive water loss during postharvest. The transcriptional analysis of nine ABA-related genes (involved in ABA biosynthesis, oxidation and conjugation) and two non-ABA related genes (involved in water stress response) in four contrasting genotypes selected from the K5 tetraploid cut rose population revealed that stomatal responsiveness to desiccation is a polygenic trait forming a highly complex regulatory network acting towards tolerance to high RH. The large majority of the studied genes had a relevant role on stomatal functioning (NCED1, AAO3, UGT75B2, BG2, OST1, ABF3 and Rh-APX) while three others showed a minor contribution (CYP707A1, CYP707A3 and BG1) and DREB1B did not contribute to the tolerance trait. Overall, this study has helped identifying major factors responsible for the reduction of potential vase life of cut roses due to uncontrolled water loss, and represents a major step forward in developing future tools to mitigate this phenomenon. In summary, it highlights that: (1) roses have a large genotypic variation in terms of stomatal functioning; (2) xylem sap [ABA] does not explain genotypic differences in stomatal functioning; (3) additional MOV or moderate salinity enhances stomatal functioning due to higher sensitivity to ABA or higher leaf [ABA], respectively; (4) three QTLs can explain 32% of the genotypic variability in stomatal functioning of the K5 tetraploid cut rose population in response to desiccation; (5) multiple genes form a highly complex regulatory network acting together towards the genotypic tolerance to high RH.
- Potential of Ectomycorrhizal Fungi as drivers of plant establishment on soils contaminated with fluorinated organic compoundsPublication . Franco, Albina Cristina Ribeiro; Castro, Paula Maria Lima; Oliveira, RuiEctomycorrhizal (ECM) fungi can play an important role on forest ecosystems. It is crucial to understand and explore the capacity of ECM fungi to improve plant development and successful establishment on disturbed soils. The main objective of the work described in this thesis was to explore the potential of ECM fungi found to persist in a disturbed area to tolerate and/or degrade fluorinated organic compounds, and also to assess the tolerance and symbiotic efficiency of ECM fungi - Pine on contaminated soils. The diversity and persistence of ECM fungi in a Pinus pinaster plantation established with plants inoculated at the nursery with selected ECM fungi and with non-inoculated plants was investigated. Nursery inoculated plants promoted plant growth in the field, with different ECM communities found to be present on the inoculated and non-inoculated plants. The inoculated fungi Laccaria sp., Rhizopogon sp., Suillus bovinus, and Pisolithus sp. persisted on the inoculated plants after 2 and 5 years of field establishment. Laccaria sp., Rhizopogon sp., and Pisolithus sp. were also detected within the non-inoculated trees but, with the exception of Pisolithus sp., the fungi did not correspond to the same fungal species detected on the inoculated plants. The results reinforced the benefits of using nursery inoculated plants on forest reclamation processes. The potential of P. tinctorius, a widespread fungus on forest and found to persist in plants after field establishment in disturbed areas, to tolerate and/or degrade organic pollutants was further assessed. P. tinctorius was able to tolerate and degrade 2- and 3-Fluorophenol (FP) at different extents in glucose supplemented liquid medium. 3-Fluorocatechol (FC) and 4-FC were detected as degradation metabolites. Metabolic intermediates compatible with fluoromuconates were identified, indicating that cleavage of the aromatic ring occurred. To our knowledge, this is the first time the degradation of mono-FP by an ECM fungus is reported. P. tinctorius was also capable to tolerate and degrade trifluoroacetate, at different extents, in solid and liquid medium supplemented with glucose. Defluorination was not found and a volatile compound with a mass similar to fluoroform was detected from cultures of the fungus. However, further confirmation of this compound is still needed. The results evidence the role ECM fungi play in the degradation of fluorinated organic compounds, enhancing their potential contribution to rhizoremediation technology. The effect of benfluralin, a persistent herbicide, on the development of P. pinea seedlings and colonisation by P. tinctorius and S. bellinii was performed. Benfluralin had an inhibitory effect on plant growth and ECM colonisation. Inoculation with P. tinctorius promoted plant development and nutrient uptake in benfluralin spiked soil, suggesting that the fungus may have conferred seedlings protection against benfluralin toxicity. The same was not observed on the seedlings inoculated with S. bellinii. Therefore, the use of P. tinctorius - P. pinea symbiosis could enhance plant establishment in pesticide contaminated soils. Furthermore, how benfluralin affected P. pinea seedlings previously mycorrhized with P. tinctorius and S. bellinii was also assessed. Mycorrhized P. pinea seedlings revealed higher plant growth in the presence of benfluralin, although the herbicide affected ECM colonisation and nutrient uptake, even after establishing the symbiosis. Fungal role on plant antioxidant response in the absence and presence of benfluralin was also evaluated. Inoculation with S. bellinii led to higher lipid peroxidation in the presence and absence of benfluralin, suggesting that this increase could be related to the fungus symbiosis and not to benfluralin. In plants inoculated with P. tinctorius, lipid peroxidation and H2O2 decreased, indicating that the fungus conferred root protection against benfluralin. Superoxide dismutase and catalase activity increased on noninoculated seedlings exposed to benfluralin, but the same was not observed in the inoculated seedlings. Therefore, the antioxidant response to abiotic stress depends of the plant-fungus species, and of the level of stress. The results corroborated the findings from the previous study, demonstrating that P. tinctorius-P. pinea association represents a possibility for increasing plant’s protection and growth when exposed to contaminated soils. Overall, P. tinctorius revealed to be capable to tolerate and degrade fluorinated organic compounds, as well as to potentiate plant growth on contaminated soils. As such, P. tinctorius could confer plants with an important advantage as a reconversion strategy for soils contaminated with organic pollutants.
- Preservation technologies towards improvement of fresh whole tomato fruits qualityPublication . Pinheiro, Maria Joaquina da Cunha; Silva, Cristina Luisa Miranda; Gonçalves, Elsa Margarida Nunes LealO tomate (Solanum lycopersicum) é um dos frutos mais consumidos no mundo e com um papel relevante na saúde do consumidor devido à sua riqueza em vitaminas, licopeno, fibras e compostos fenólicos. Os desafios que se colocam aos produtores e indústria de tomate em fresco são o de manter a qualidade e prolongar o seu período de vida útil. Tecnologias de pós-colheita alternativas e emergentes, tais como o tratamento térmico (WHT), ultrassons (US), termossonicação (TS) e radiação ultravioleta (UV-C), têm sido testados em diferentes frutos e vegetais de forma a minimizar as perdas de qualidade sensorial e nutricional com reduzida utilização de aditivos químicos. Nesta dissertação, foram demonstradas as vantagens da aplicação destas tecnologias alternativas na manutenção da qualidade do tomate em fresco (cv. Zinac) durante o período logístico de armazenamento. A avaliação da qualidade do tomate foi realizada através de parâmetros físico-químicos (cor CIELab, textura, pH, teor de sólidos solúveis, perda de peso), sensoriais (cor, índice de deterioração e aceitabilidade global), bioquímicos (atividade enzimática da peroxidase: POD, e pectinametilesterase: PME), bioativos (conteúdo fenólico total e capacidade antioxidante), e carga microbiana (mesófilos, bolores e leveduras). Inicialmente foi realizado um estudo de avaliação do impacto de diferentes temperaturas de armazenamento (2º, 5º, 10º, 15º e 20 ºC) na conservação da qualidade do tomate inteiro. Os parâmetros de qualidade foram descritos através do modelo de conversão fracionário e o efeito da temperatura de armazenamento foi descrito com sucesso pela lei de Arrhenius. A partir dos resultados experimentais, a temperatura ótima de armazenamento (10 ºC) foi encontrada para o cv. Zinac. O modelo obtido representa uma ferramenta preditiva útil para estimar a qualidade do tomate durante a cadeia de distribuição do produto em fresco. Os efeitos do tratamento térmico (WHT) na gama de temperatura de 40–60 ºC, durante diferentes períodos de tempo (2–60 min), foram avaliados no tomate em fresco. Os resultados permitem concluir que o WHT influencia de forma significativa (P<0.05) os parâmetros físico-químicos. Contudo, os tratamentos a 40 ºC_30 min e 50 ºC_2 min, demonstraram uma melhor conservação da cor e textura, bem como a redução da carga microbiana inicial. A influência de dois estados de maturação do tomate (turning e pink) face à condição ótima de WHT (40 ºC_30 min) e ao tratamento convencional de descontaminação que utiliza cloro (HIPO), foi avaliada ao longo de 15 dias de armazenamento. Os resultados demonstraram que o WHT conduz a uma conservação dos parâmetros de qualidade analisados mas a sua eficácia é dependente do estado de maturação dos frutos. A metodologia de resposta de superfície (RSM) foi utilizada para avaliar o efeito do tratamento de US (na gama de potência: 10-100%, tempo de tratamento: 1-19 min) e período de armazenamento (1–15 dias), na qualidade do tomate. Os modelos preditivos dos parâmetros de cor a* e hº, e o conteúdo de fenólicos totais foram significativos, (P<0.05) apresentando bons coeficientes de correlação. Tratamentos à frequência de 45 kHz, 80% de potência e durante 30 min, resultam no atraso da deterioração do fruto armazenamento a 10 ºC. Os efeitos de TS na qualidade do tomate foram avaliados através de RSM, utilizando como variáveis independentes: temperatura (32–48 ºC), tempo de tratamento (13–47 min) e período de armazenamento (1–15 dias). Três condições de termossonicação foram selecionadas (32 ºC_13 min, 35 ºC_20 min e 40 ºC_30 min) e analisadas durante 15 dias a 10 ºC. O tratamento de TS (40 ºC_30 min) demonstrou benefícios no atraso das alterações dos parâmetros de qualidade analisados. O impacto da radiação UV-C a 5 condições de tratamento (0.32 – 4.83 kJ.m-2 a 254 nm) foi analisado durante o período de refrigeração do tomate. A radiação UV-C a 0.97 kJ.m-2 para além de afetar a carga microbiana inicial do tomate contribui ainda para a sua redução significativa comparativamente à amostra controlo no final do armazenamento. Do estudo de validação das condições otimizadas: WHT, US, TS e UV-C (aplicado ao tomate fresco armazenado a 10 ºC, comparado com as amostras controlo (HIPO)), concluiu-se que todos os tratamentos alternativos estudados resultaram na diminuição da velocidade de deterioração em termos de desenvolvimento da cor vermelha e da firmeza. Além disso, o tratamento de UV-C foi o que permitiu um aumento dos compostos fenólicos e uma maior redução da carga microbiana, comparativamente aos restantes tratamentos. Os resultados deste trabalho podem ser utilizados para conservar/prolongar a qualidade do tomate inteiro durante o período pós-colheita, fornecendo a todos os interveniente da cadeia, matéria-prima de elevada qualidade com o menor desenvolvimento microbiano durante um período mais extenso.
- Role of plants and microorganisms in the management of heavy-metal polluted areas as an approach for land restorationPublication . Moreira, Helena Maria Gomes; Castro, Paula Maria Lima; Rangel, António Osmaro Santos Silva; Marques, Ana Paula Guimarães CorreiaPhytoremediation is a promising alternative to conventional soil clean-up methods. However, the current knowledge on plants’ suitable for such purposes is still limited. Furthermore, the potential of the combined use of rhizosphere microbiota, such as plant Growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) in phytoremediation approaches is still underexploited. Sites contaminated with heavy metals become unprofitable and require years of proactive decontamination measures for suitable restoration. Using economically valuable plant species can contribute to capitalize these otherwise wasted areas, with the advantage of decreasing the risks posed by heavy metal contamination. In this thesis, an industrialised area (Estarreja, Portugal) known for its zinc (Zn) contamination was examined in a one year-round screening in search for native plant species with potential for phytomanagement actions. This survey revealed that Zn levels in the plants ranged from 34 to 2440 mg kg varying according to plant structure and species. Five species showed high accumulation of Zn in the shoot, and bioconcentration factors and metal translocation factors greater than 1. Others excluded Zn from the shoot tissues and stored the metal at the root zone. In spite of the potential shown by several indigenous species for use in phytoremediation, the biomass of the plants thriving at the site was too low to make them suitable for land valorisation. Given its worldwide agricultural use, economical value (namely for bioenergetics purposes), high biomass and metal resistance, maize ( Zea!mays!L.) presents a high potential for use in phytomanagement actions in heavy-metal contaminated areas. Furthermore, when associated with this crop species, microorganisms like PGPR or AMF can favour its growth, further contributing to capitalise affected areas. Therefore, maize combined with rhizospheric microbiota was used in experiments grown in spiked soils and soils collected from a mine (Panasqueira, Portugal) and from an industrially contaminated area (Estarreja) for: i) assessing the effects of selected PGPR in the growth and metal uptake in sterile soils spiked with up to 30 mg Cd kg and up to 1000 mg kg Zn, and in soils collected from Panasqueira and Estarreja (sterile and non-sterile); ii) evaluating if PGPR effectiveness in promoting maize growth and metal accumulation is inoculums-size dependent; iii) determining the joint effect of PGPR and AMF in growth and metal accumulation in maize plants. PGPR inoculation in soils spiked with Cd and Zn contributed to the increase of biomass and metal accumulation of maize roots. However, it led to a reduction in the accumulation of metals in the shoot of plants grown in Cd-contaminated soils, and to an increase of the same parameter in Zn-contaminated soils. These differences suggest that the inoculation outcome is metal-type dependent. Bacterial inoculation also increased maize biomass in sterilized soils from Estarreja. Moreover, it contributed to an increase of Zn accumulation in the shoots and in the activity of antioxidant enzymes (CAT and SOD). However, PGPR inoculation heightened Zn accumulation in the roots of maize grown in non-sterile soil. The different PGPR strains applied in sterile mine contaminated soil revealed distinct effects on plant biomass and elongation, independently of inocula size. However, Cd accumulation in plant tissues was positively related to PGPR inoculum size, pattern that was not observed for Zn. In non-sterile mine soil, the PGPR induced an increase in plant biomass. Further, AMF R. irregularis acted synergistically with PGPR to significantly increase biometric parameters as well as improving P accumulation. However, there was a significant decrease in Zn accumulation in plants for all microbial treatments. The results obtained from the different experiments suggested that maize inoculated with PGPR and AMF has high potential for application in soil remediation strategies, effectively enhancing metal bioavailability, plant growth and metal accumulation/stabilization. Furthermore, the inoculum size revealed to be important for the plants’ metal accumulation abilities without significantly compromising their biomass, and should be taken into account in the planning of such strategies.
- Selection of an antimicrobial culture to be used in the prevention of neonatal listeriosisPublication . Borges, Sandra Cristina Ferreira; Teixeira, Paula Cristina Maia; Silva, Joana Gabriela LaranjeiraThe capacity of lactic acid bacteria to produce acidic products and/or secrete antimicrobial compounds is important in the impairment of vaginal colonization by pathogens. Vaginal pH is normally acidic, varying between 3.5-4.5; an increase in vaginal pH (5.0 to 6.5) can be associated with colonization by pathogenic microorganisms. The main goal of this study was to select an antimicrobial culture to be used in the prevention of vaginal colonization of Listeria monocytogenes during pregnancy, and consequently, prevent neonatal listeriosis. The survival and biofilm formation of 20 isolates of L. monocytogenes in simulated vaginal fluid at normal vaginal pH (4.2) and at higher pH values (5.5 and 6.5) was investigated. This pathogen was inhibited by the normal vaginal pH but survives when pH increases. All isolates tested were biofilm producers at different pH values. Streptococcus agalactiae is an important cause of neonatal infection and maternal colonization. Therefore, its behavior in simulated vaginal fluid was also analyzed. As with L. monocytogenes, S. agalactiae (n=10) survived longer at higher pH values than at normal vaginal pH. All S. agalactiae isolates were also biofilm producers. Therefore, since L. monocytogenes and S. agalactiae can survive at higher vaginal pHs, fetuses/neonates from women having increased vaginal pH values during pregnancy, may be at higher risk of neonatal infection. Biofilm production increases the probability of occurrence of neonatal infection. The application of vaginal probiotics could have the potential for preventing vaginal Listeria colonization in pregnant women and consequently reduce neonatal infections. Thirty-five isolates of Pediococcus spp. showed antimicrobial activity against L. monocytogenes, by production of a bacteriocin, but did not inhibit S. agalactiae isolates. Pediococcus spp. isolates demonstrated the ability to survive in simulated vaginal fluid at pH 4.2. Based on the higher bacteriocinogenic activity and survival in simulated vaginal fluid, one isolate of Pediococcus spp. was selected and characterized to evaluate its safety before use as vaginal probiotic. Pediococcus pentosaceus SB83 did not show the presence of virulence factors such as the production of gelatinase, lipase and DNase, hemolytic activity, nor the presence of virulence genes (i.e. surface adhesin, aggregation protein, cytolysin and extracellular metallo-endopeptidase). No relevant antibiotic resistance traits were detected. Pediococcus pentosaceus SB83 produced biofilms at different pH values (4.2, 5.5 and 6.5) in simulated vaginal fluid, which could also serve as a protective layer against colonization by pathogenic bacteria. The bacteriocin produced by P. pentosaceus SB83, designated as bacteriocin SB83, also showed inhibitory activity against Enterococcus faecalis and Enterococcus faecium, but did not inhibit vaginal lactic acid bacteria. Bacteriocin SB83 is resistant to several conditions, including conditions in the vaginal tract (pH and components of vaginal fluid). The bacteriocinogenic activity of P. pentosaceus SB83 against L. monocytogenes was evaluated in simulated vaginal fluid at pH 6.5, since this is the ideal pH to the L. monocytogenes survival and proliferation. There the inhibitory effect of the bacteriocinogenic culture was assessed in suspension, as lyophilized powder and in tablets. Suspensions of P. pentosaceus SB83 (1010 CFU/mL) reduced the pathogen (10 CFU/mL) 5after only 2 h of exposure to below the detection limit; the lyophilized bacteria after 24 h of contact and in tablet form, P. pentosaceus SB83 lost the antimicrobial activity. The pH of simulated vaginal fluid decreased in all the tested conditions. Since P. pentosaceus SB83 lose its antimicrobial activity in tablet form, it could be therefore used in the form of lyophilized powder, which may be administered intra-vaginally, for instance as a washing solution. This formulation was selected to evaluate the anti-listerial activity during 12 months of storage. During storage at room temperature, lyophilized bacteria totally inhibited the pathogen (below the detection limit) only during one month; after this time, there was a decrease in the cell counts of P.pentosaceus SB83 and, consequently, in antimicrobial potential. During storage at 4 ºC, P. pentosaceus SB83 showed antimicrobial activity throughout the time of storage investigated. The bacteriocin produced by P. pentosaceus SB83 after storage at 4 ºC, remained active at least during 12 months, however a slight decrease in antimicrobial activity occurred between 9 to 12 months. Therefore, the best formulation of P. pentosaceus SB83 is as a lyophilized powder stored at 4 ºC. These in vitro results prove a concept for the use of P. pentosaceus SB83 as a vaginal probiotic, to prevent vaginal colonization by L. monocytogenes in pregnant women.