R - Teses de Doutoramento / Doctoral Theses
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Browsing R - Teses de Doutoramento / Doctoral Theses by Sustainable Development Goals (SDG) "12:Produção e Consumo Sustentáveis"
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- Exploring new sustainable solutions based on chitosan and cellulose crystals towards the preventive conservation of cultural heritagePublication . Silva, Nádia Suati Caetano da; Costa, Patrícia Raquel Fernandes de Melo Moreira da; Pintado, Maria Manuela Estevez; Madureira, Ana Raquel Mendes Ferreira MonteiroOutdoor sculptures made of stone and mortar are a significant part of the worldʼs cultural heritage. Consequently, their conservation is of great importance, mainly due to their vulnerability to deterioration. Deterioration occurs as a combination of factors that cause alterations and devalue the artworks over time, including those resulting from biodeterioration mechanisms triggered by the growth and activity of microorganisms. Developing sustainable strategies to slow down the proliferation of microorganisms is necessary to reduce the negative environmental and human health impacts of currently used toxic biocides. Hence, this thesis aims to contribute to developing low-toxicity and more sustainable antimicrobial coatings for the preventive conservation of stone heritage, particularly outdoor sculptures. Firstly, an evaluation of the biocontamination and microbiological composition of five outdoor sculptures, made of different materials and located in the district of Porto (Portugal), was performed. Two methodologies were tested to collect samples from the sculptures in a non-invasive and non-destructive way: cotton swabs (classical approach) and poly(2-hydroxyethyl methacrylate) cryogels (alternative method). The quantification and determination of the cell viability of the microorganisms collected were performed by flow cytometry, which proved to be an analytical technique of interest for microbiological studies of cultural heritage. Both methods were effective, although higher concentrations of microorganisms were collected with swabs, while data visualisation of viable and non-viable cells was clearer with cryogels. Consequently, swab samples were taken to characterise the epilithic bacterial and fungal communities of the sculptures. High-throughput sequencing revealed great taxonomic diversity and species richness, including in well-preserved sculptures. Fungal diversity was lower than that of the bacterial communities. Proteobacteria was the core taxa common to all the sculptures. Ascomycota were also detected in all the sculptures and Basidiomycota were a significant part of the microbiomes in granite, where an abundance of pigment-producing microorganisms was also found. Additionally, colourimetry and adenosine triphosphate quantification assays quickly identified contaminated areas of the sculptures. The next stage of the work dealt with the preparation and characterisation of chitosan formulations, to be used as antimicrobial coatings to inhibit the growth of microorganisms in sculptures. The first set of formulations consisted of chitosan solutions containing citric acid and different concentrations of sodium tripolyphosphate (CHGCA-TPP), which were polymerised into films for their characterisation. Fourier transform infrared (FTIR) spectroscopy confirmed the establishment of cross-linking interactions, and the films exhibited partial wettability (40.81-31.44°), solubility (43.64-55.14%) and swelling (123.36-75.17%) in water, but allowed water vapour exchanges due to their high permeability (140.055-372.575 g m-2 d). The films reduced the growth of microorganisms that commonly colonise stone heritage: Staphylococcus aureus (0.87-1.58-log reduction), Bacillus cereus (ca. 1-3-log reduction), Rhodotorula spp. (between 0.73-1.27-log reduction and complete inhibition) and Penicillium chrysogenum (11.92-21.48% inhibition). Another set of formulations was prepared to enhance the properties of the CHGCA-TPP formulations. A screening was conducted with chitosan solutions containing microcrystalline cellulose (MCC) or cellulose nanocrystals, and the chosen formulation was supplemented with oregano essential oil (OEO) at 1% and 2%. Cross-linking interactions and incorporation of OEO were confirmed by FTIR analysis of the films, which remained poorly soluble (15.74-16.85%), with low percentages of swelling (16.10-28.22%) and relatively low wettability (> 70°). These formulations are an improvement on the CHGCA-TPP ones, including their antimicrobial action, since the films containing 2% OEO completely inhibited the growth of S. aureus, Rhodotorula spp. and P. aeruginosa, induced ca. 60% inhibition of P. chrysogenum and reduced the growth of B. cereus. Lastly, the two most promising formulations (CHGCA-TPP-a and 2-MCC-OEO-2) were tested in granite, limestone and marble samples. Both formulations polymerised on the stones’ surfaces, as confirmed by scanning electron microscopy and FTIR spectroscopy, but formed irregular coatings. The CHGCA-TPP-a coating reduced the wettability of granite and limestone, while 2-MCC-OEO-2 did the same in a more pronounced way and in all stone types. Neither coating caused visible colour changes when the formulations were applied with a brush. In vitro antimicrobial assays with stone samples inoculated with selected strains showed that CHGCA-TPP-a inhibited the growth of B. cereus, Rhodotorula spp. and P. aeruginosa in granite after 7 days to ca. 4 to 5 log10 (CFU mL -1 ). In contrast, 2-MCC-OEO-2 reduced the concentrations of viable cells in all stone types to ca. 3 to 5 log10 (CFU mL -1 ) depending on the strain. In situ antimicrobial assays, where samples were placed in an outdoor setting, showed that CHGCA-TPP-a did not have an inhibitory effect under the conditions tested, with concentrations of viable cells equal to or higher than those of uncoated slabs, regardless of the mode of application of the formulation (deposition with a micropipette or spreading by brush) or the number of treatments over the assay period. However, multiple applications of 2-MCC-OEO-2 with a micropipette over time reduced the concentrations of viable cells in granite and marble, but the same occurred only in limestone when the coating was applied with a brush. In conclusion, this work added to the knowledge of the biocontamination and diversity of the microbiomes of outdoor sculptures, in particular of the Porto district in Portugal, which can help outline future conservation strategies. The chitosan formulations tested, especially those with MCC and OEO, are a potential base for further development and optimisation of novel low-toxicity antimicrobial coatings towards more sustainable preventive conservation strategies of stone heritage.
- Functional proteins and peptides obtained from fish by-productsPublication . Ghalamara, Soudabeh; Pintado, Maria Manuela Estevez; Silva, Sara Nunes da Costa e; Ferreira, Carla Maria Carvalho Gil Brazinha de BarrosMarine processing industries face significant challenges due to excessive by-products, which contribute to both environmental and economic issues. By-products from fish processing, which can constitute between 30% and 70% of the fish's total weight, exacerbate these challenges. Activities like washing, thawing, cooking, and fishmeal production generate more wastewater, intensifying the industry's environmental impact. This research explored using fish by-products like sardine cooking effluent and codfish bloodwater (CFBW) in a sustainable, zero-waste approach. The functional proteins and peptides extracted from fish by-products enhanced their value. The research evaluated their properties and potential food uses, aligning with circular bioeconomy principles. Fish by-products were fractionated using membrane technology at laboratory and pilot scales, aligning with zero-waste and biorefinery approaches. Ultrafiltration (UF) membranes were used to extract bioactive peptide-enriched fractions from sardine cooking effluent and CFBW at the laboratory scale. The process involved analyzing the selective permeation of small peptides (<1 kDa) using different membranes. The membranes effectively rejected (>10 kDa) of proteins and peptides but had relatively low rejection of <1 kDa peptides, with UP010 from CFBW achieving a 2% rejection rate and GH from sardine cooking effluent operating at minimum pressure (1 bar) achieving a 23% rejection rate. Peptides from CFBW using MW and UP010 membranes demonstrated potent antioxidant activity (high ABTS+ and ORAC values). However, the peptide fractions from sardine cooking effluent using the GH membrane did not enhance antioxidant activity. Nevertheless, the peptide fractions from CFBW (UP010 membrane) and sardine cooking effluent (GH membrane, 1 bar pressure) inhibited E. coli growth. Peptide-enriched fractions from CFBW were successfully obtained using a pilot-scale process involving microfiltration (MF), UF, and reverse osmosis (RO). The process aimed to fractionate CFBW into protein- and peptide-rich fractions. Chemical and biological characterization revealed that CFBW and pretreated CFBW consisted primarily of proteins and peptides. UF membrane fractions had lower protein content but higher ash levels. The UF retentate with a MWCO of 50 kDa, followed by RO, displayed the highest antioxidant values, indicating the presence of potent antioxidants. Additionally, the UF permeate obtained with a MWCO of 50 kDa from the MF-PFG showed antimicrobial activity against E. coli. A pilot-scale integrated membrane process was developed to obtain protein- and peptiderich fractions from sardine cooking effluent. The process used UF, nanofiltration (NF), and RO combined with enzymatic hydrolysis. Specifically, the RO retentate fractions of NF retentate were derived from diluted sardine cooking effluent, diluted hydrolyzed UF retentate, and diluted non-hydrolyzed UF retentate. The process involved UF, nanofiltration (NF), and RO combined with enzymatic hydrolysis. Specifically, the RO retentate fractions of NF retentate fractions were derived from water-diluted sardine cooking effluent (sardine cooking effluent-NF-RO), water-diluted UF retentate hydrolyzed (UF-H-NF-RO), and water-diluted UF retentate non-hydrolyzed (UF-NH-NF-RO). UF-H-NF-RO showed enhanced antioxidant and antimicrobial activities. The UF-NH-NF-RO peptide fraction displayed significantly enhanced functional properties in terms of WHC, FBC, emulsifying properties, and foaming properties at pH 4. In vitro digestion analysis showed this fraction also had the highest antioxidant activity, and none of the fractions exhibited cytotoxicity. Finally, foamy fish sauces (FFSs) were developed and evaluated using protein/peptide and lipofish fractions obtained from centrifugation of sardine cooking effluent. The control sample (CS) used a butter-based sauce emulsified with lecithin. An alternative lipofish sauce (LS) substituted unsalted butter with fish oil and included lecithin as the emulsifier. Three other formulas, namely lipofish-sardine cooking effluent-NF-RO-sauce (LSS), lipofish-UFH-NF-RO-sauce (LHS), and lipofish-UF-NH-NF-RO-sauce (LNHS), replaced unsalted butter with fish oil and incorporated a combination of protein/peptide fractions and lecithin. Despite minor physicochemical differences, the sauce formulas showed improvements compared to the control, including enhanced fatty acid (FA) content and profile, slightly reduced viscosity, improved foaming capacity, and enhanced foam stability. In vitro digestion analysis demonstrated high recovery of FAs, with the formula LNHS exhibiting the highest efficacy in scavenging ABTS radicals, indicating strong antioxidant properties. Furthermore, the FFS received remarkable acceptance from the trained panelists, who highly praised its texture, aroma, color, and flavor. This study's findings on fish by-products have significant implications for sustainable development in the fish processing industry. The research showcased the potential to obtain functional ingredients through eco-friendly strategies, preserving their bioactivity. These outcomes promote responsible and sustainable fish processing, reducing waste and maximizing by-product utilization.
- IMPULSE : IMpact of a PULSE-based partial replacement diet on metabolome and healthPublication . Ferreira, Helena Alexandra Gonçalves; Pinto, Elisabete Cristina Bastos; Vasconcelos, Marta Wilton Pereira Leite de; Gil, Ana Maria Pissarra CoelhoThis doctoral project was conceived in the context of urgent global challenges related to food security and environmental sustainability, as a response to the need for transitioning to plantrich dietary patterns. The project aimed to explore the feasibility and impact on human organisms of partially substituting animal protein-rich foods with plant-based protein-rich foods, specifically pulses, in omnivorous diets. Identified gaps in scientific evidence regarding the health effects of pulses, especially as protein alternatives, prompted a comprehensive exploration combining classical nutrition research tools with molecular biology and metabolomics techniques. A quasi-experimental dietary intervention was designed involving a single group of volunteers. Non-vegetarian individuals aged 18 to 45 were provided with a pulse-based vegetarian meal on weekdays for eight consecutive weeks in Porto, Portugal. Sociodemographic data, health-related information, and lifestyle details were recorded for each volunteer. A post-meal satisfaction questionnaire assessed participants' satisfaction with each meal using a nine-point hedonic scale. Untargeted 1 H nuclear magnetic resonance (NMR) metabolomics was applied to plasma, urine, and fecal samples (totaling 248 samples), and quantitative polymerase chain reaction (qPCR) molecular biology was used on fecal samples (34 samples) to obtain holistic and integrated information on the impact of pulse diets on the metabolome and intestinal microbiota. Additionally, three-day food diaries, anthropometric parameters, and blood biochemical analyses were obtained for each individual, contributing to a comprehensive data collection. Data analysis, including comparison of different parameters at the intervention's start and end, utilized various statistical tests for proportion and mean comparisons, and multivariate analysis. Significant differences were considered when the p-value was below 5%. The meal satisfaction analysis highlighted the significant role of cooking methods in determining food preferences, with greater satisfaction for stewed dishes and lower ratings for boiled dishes. Peas received lower appreciation, likely reflecting them being less preferred as protein alternatives. Moreover, most participants were able to meet national and international guidelines for pulse consumption without exacerbating nutritional inadequacies for the tested macro- and micronutrients, except for vitamin B12. The intervention demonstrated potential benefits in reducing blood lipids, with decreases in total and LDL cholesterol levels, supporting cardiovascular health benefits associated with pulse-rich diets. Using NMR spectroscopy, a dynamic metabolic response to pulse consumption was identified in plasma and urine samples, emphasizing the modulation of protein and energy metabolism, as well as the identification of potential intake markers. Correlations between bacterial quantification and fecal metabolome suggest possible metabolic patterns of some of the 8 studied bacterial groups. The results highlight the potential benefits and challenges of adopting pulse-based diets but suggest that pulses would be readily accepted in dietary habits if prepared and presented pleasantly. While positive impacts on cardiovascular health and metabolic activity are evident, the results emphasize the need to avoid nutritional deficiencies, especially in vitamin B12, when substituting animal-origin foods with pulses. Metabolomic analysis highlighted the dynamic nature of the response to pulse consumption, and its combination with molecular biology revealed the potential impact of these foods on the intestinal microbiota. Through a multidisciplinary approach, this study elucidated the potential of pulse-based diets in promoting individual well-being and sustainable food choices. These results need confirmation in experimental studies involving a larger number of participants and a control group, but they identify potential biomarkers and advance the understanding of the broader effects of pulses on human health. Future work should further optimize pulse-based dietary recommendations and explore innovative strategies to enhance the adoption of sustainable dietary patterns on a larger scale.
- PULSE UP : promoting pulses in the portuguese agri-food system through children's dietsPublication . Vieira, Evla; Vasconcelos, Marta Wilton Pereira Leite de; Gomes, Ana Maria Pereira; Gil, Ana Maria Pissarra CoelhoPlant-based eating patterns are healthy and can decrease environmental impacts. When discussing sources of dietary plant protein, it is essential to consider the dry-harvested legume crops, known as pulses. Pulses are farm-to-fork actors that promote sustainability and healthy diets. When integrating cropping systems, pulses reduce greenhouse gas emissions per unit area. Also, if animal protein is replaced in places of overconsumption, pulses can potentially mitigate the pressure on natural resources. Despite the critical function in food systems’ sustainability, the area growing pulses in Europe declined in the last sixty years, and consumption is around 2 to 8 kg per person-year when the Food and Agriculture Organization (FAO) recommendation is 15 to 25 kg per person-year. In the face of this low consumption and given all benefits, identifying strategies and policies for promoting pulses in Europe's agri-food system should be among policymakers' top priorities. However, implementing changes requires many alterations in several sectors. Thus, taking specific measures directed to priority groups, such as children, may produce more assertive results. This thesis’s main objective is to develop and validate practical and viable solutions to promote pulses in the Portuguese agri-food system through children’s diets. First, a critical literature review on the measures that should stimulate pulses in children's diets was proceeded. Considering the feasibility and impact, two actions were selected to be developed. The chosen actions were to enrich a child's food with a pulse and pulse promotion in the school food environment. Regarding enriching children's diets with pulses, the first study helped select the pulse that is more suitable for developing the product. Next, a children’s yogurt enriched with lupin was developed and characterized in nutritional, rheological, and sensorial terms, and environmental impact was demonstrated. Also, metabolomic alterations were presented. Concerning pulse promotion in the school food environment, a guidance material for preschool teachers on promoting the consumption of pulses was prepared, and an online focus group was carried out to characterize the consumption of pulses in selected kindergartens and evaluate the material produced. The results in this thesis indicate that the enrichment of children's yogurts with lupin and the promotion of pulses in kindergartens are viable measures that can increase the demand and consumption of pulses. Enrich children’s yogurt with lupin is an alternative to produce a nutritious and perhaps functional product while it may drive a local pulse-based value chain. Additionally, promoting pulses in kindergartens by local procurement and actions that stimulate preschoolers’ consumption can favor domestic production with social and economic inclusion and help increment the habit of pulse consumption in the Portuguese population.
- Tradition, science, and innovation : bioactive substances to mitigate microbiological risks of innovative alheirasPublication . Moreira, Inês Gonçalves de Azevedo; Teixeira, Paula Cristina Maia; Barbosa, Joana Inês Bastos; Albano, Helena da Conceição PereiraAlheira, a well-known delicacy produced in the north of Portugal, is a valuable part of our country's gastronomic heritage. Traditional alheiras, produced mainly with shredded pork and poultry meats, traditional wheat bread, olive oil, pork fat, and spices, are well-studied products and in the past years, some new formulations have emerged. Consumer preferences are constantly changing, so the food industry must proactively develop new products. In addition to traditional fermented sausages, mainly made with meat, other products are being developed to offer alternatives in line with consumer trends to reduce meat consumption. Vegetarian, vegan, and flexitarian diets are on the rise and sausages made from a variety of ingredients, such as codfish, mushrooms, tofu, soya, or vegetables, and other meat analogues are appearing worldwide. The aim of this study was to characterize these products, as little is known about their microbiological and chemical characteristics, and it becomes essential to determine their potential role in food safety. Classical microbiological and chemical analysis were therefore carried out on a universe of 21 alheiras, 14 of which were considered innovative and seven traditional. Enterobacteriaceae, Enterococcus, Lactic Acid Bacteria (LAB), yeasts, and moulds were the prevalent microbiota found in these innovative products. Sulphite-reducing Clostridium spores, Escherichia coli, Listeria monocytogenes, Salmonella spp. or Staphylococcus aureus were not detected in any innovative samples. No differences were observed between traditional and innovative alheiras concerning pH and water activity, while nitrites, nitrates and biogenic amines were within accepted limits for these products. Regarding organic acids, lactic acid was found in all samples analyzed, while malic and succinic acid seemed predominant only in the innovative alheiras. In addition, and to find some similarities between different types of alheiras and/or different producers, a study was carried out using next-generation sequencing technology to characterize the microbial communities associated with these products. The bacterial and fungal communities associated with each alheira were obtained by sequencing 16S rRNA gene V3- V4 and Internal Transcribed Spacer 2 (ITS2) regions of rRNA gene amplicons. Significant differences in the microbiota composition were found between samples, which were reflected by large differences in the profiles of the dominant species. More than 500 taxa were identified, particularly belonging to the families Lactobacillaceae and Xanthomonadaceae, which were found in all samples. In the analysis of the bacterial communities, genera belonging to lactic acid bacteria and Xanthomonas were predominant. Concerning fungi, the yeast Pichia was found in almost all the samples, followed by the filamentous fungus Alternaria. A challenge test was conducted in order to investigate the behaviour of foodborne pathogens (E. coli, L. monocytogenes, Salmonella Enteritidis and Staph. aureus) in traditional and innovative alheiras (codfish and vegetarian) along the product shelf-life at 4 ºC. Each target pathogen showed a different behaviour on the alheira matrices, but most pathogens were not detected in vegetarian alheira before the expiration date. As part of the risk mitigation, 491 LAB were isolated from traditional and innovative alheiras, and their antimicrobial activity against several foodborne pathogens was investigated. Six strains revealed antimicrobial activity by possible bacteriocin production against L. monocytogenes, Enterococcus faecalis, Clostridium sporogenes and Clostridium perfringens. These strains were identified as Lactiplantibacillus plantarum (2), Leuconostoc mesenteroides (1) and Pediococcus acidilactici (3). Additionally, orthologues of several class II bacteriocins genes were detected, namely Plantaricin E, Plantaricin F, Pediocin PA, Enterocin X, Leucocin A, and Coagulin A. None of these strains produced biogenic amines, gelatinase or DNase, as well as no hemolytic activity or lipase enzyme production was observed. However, only Lpb. plantarum 9A3 was sensitive to all the antibiotics tested and was therefore selected for further testing. Bacteriocins produced by Lpb. plantarum (9A3) demonstrated a bacteriostatic mode of action and stability across a wide range of conditions (temperature, pH, surfactants, detergents, and proteases). In conclusion, unlike traditional alheiras, which often contain pathogens, neither harmful organisms nor chemical hazards were found in these new products, even though they were produced by the same companies. Characterization of microbial communities by WGS revealed distinct microbial diversity patterns in traditional and innovative alheiras, even when produced in the same facilities and conditions. While this study offers initial insights into microbial diversity in these products, it sheds light on the behaviour of foodborne pathogens in the alheira matrix. Lactic acid bacteria increased throughout the shelf-life, particularly in traditional alheiras. Although each target pathogen showed a different behaviour on alheira matrices in general, vegetarian alheira proved to have particular characteristics that influence the viability of foodborne pathogens, since all pathogens tested were below the detection limit of the enumeration technique at the end of shelf-life in this matrix. Lactiplantibacillus plantarum 9A3 emerged as a promising candidate for industrial use due to the production of stable bacteriocins targeting pathogens like L. monocytogenes and C. perfringens and absence of virulence factors.