ESB - Teses de Doutoramento / Doctoral Theses
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- Development and characterization of a novel probiotic germinated quinoa ice cream : nutritional, functional and biological validationPublication . García-Torres, Silvia Melissa; Gomes, Ana Maria; Silva, Cristina Luísa Miranda; Teixeira, José António CoutoIn the field of food research, new ingredients, compounds, and properties in raw materials are constantly being identified, opening knowledge gaps and generating opportunities for the development of innovative products. At the same time, global dietary habits have evolved, driving a growing demand for healthier, more sustainable alternatives aligned with ethical, environmental, and functional principles. Among the main trends are the preference for plant-based foods, the valorization of traditional crops, and the increasing interest in functional foods—those that offer benefits beyond basic nutritional value. The formulation of plant-based foods can be adapted for consumers with lactose intolerance, individuals following vegan diets, or the general public seeking more sustainable and responsible options, which significantly broadens the scope of functional foods. This research aimed to develop a functional ice cream using optimized germinated quinoa flour, incorporating the emerging probiotic Akkermansia muciniphila. Quinoa was selected for its balanced nutritional profile, the presence of bioactive compounds, and its technological adaptability, following a comparative evaluation with other Andean grains (kiwicha and cañihua). Two Peruvian varieties of different colors were used: Rosa de Huancayo (RH, white grain) and Pasankalla (PK, dark grain). Before germination, disinfection treatments were evaluated on the grain, including combinations of chemical agents, heat, and ultrasound. The combination of hot water (50 °C), hydrogen peroxide (2%), and acetic acid (0.1%) proved to be the most effective, significantly reducing microbial load (in aerobic mesophilic bacteria: 1.29–4.08 Log CFU/g) without affecting germination capacity. Enterobacteria showed greater resistance, especially in PK, but the combined treatment achieved the intended objective. Germination was optimized using a factorial design (24, 48, 72 h × 15, 20, 25 °C), evaluating its effect on nutritional content, bioactive compounds, antinutrients, and techno-functional properties. Improvements were identified in calcium (from 97.1 to 140.1 mg/100 g DW in PK; from 87.5 to 135.2 mg/100 g DW in RH), vitamin C (up to 70.22 µg/g DW in PK; 59.23 µg/g DW in RH), and protein content (from 17.09 % to 18.04 % in PK), as well as a reduction of tannins by up to 63% in RH. Optimal conditions for maximizing polyphenols, vitamin C, and protein content, and reducing tannins, were 25 °C for 24 h in PK, and 22.7 °C for 47.3 h in RH, although in the latter only the first two compounds were optimized. With the optimized germinated quinoa flour, a plant-based ice cream formulation was developed using a D-optimal mixture design. The final formulations—RH (8.86% flour, 14.8% fat, 21.3% sugar) and PK (6.00% flour, 13.0% fat, 23.0% sugar)—achieved physical properties comparable to conventional ice cream in terms of viscosity, hardness, and freezing point. The overrun was lower, representing an area for improvement. Nonetheless, the bioactive compounds derived from germinated quinoa were preserved after processing, highlighting antioxidant activity (110.7 ± 8.39 g Trolox equivalents/g DW) in the quinoa RH ice cream. The product’s functionality was evaluated through in vitro simulated gastrointestinal digestion. The ice cream made with RH flour showed sustained release of phenolic compounds, with high antioxidant activity during the gastric phase (up to 1672.57 µg TE/g DW). Regarding A. muciniphila, high viability was maintained during frozen storage (~10⁸ CFU/g), although a progressive decrease was observed during simulated digestion, reaching 10⁵ CFU/g on day 1 and 10³ CFU/g on day 60. Additionally, the impact of the quinoa-based ice cream formulations on gut microbial activity was evaluated by measuring the production of short-chain fatty acids (SCFAs), ammoniacal nitrogen and lactate during in vitro fecal fermentation. Fecal fermentation revealed that quinoa-based ice cream (QIC), with or without A. muciniphila (Akk), stimulated SCFA production, with butyrate (46.95 mM) predominating in QIC and acetate (33.78 mM) in QIC-Akk, while maintaining safe levels of ammoniacal nitrogen. Lactate dynamics indicated balanced microbial fermentation processes. This study presents an innovative strategy for the development of functional ice cream based on germinated quinoa and emerging probiotics. Despite limitations such as reduced probiotic viability during digestion, the results demonstrate the technological feasibility and nutritional potential of Andean grains in plant-based formulations. These findings contribute to the dialogue on sustainable food innovation and the valorization of traditional crops in the design of functional foods.
- Fate of micropollutants and recovery of EPS in aerobic granular sludge technology in events of salt intrusionPublication . Miranda, Catarina da Costa; Amorim, Catarina Raquel Leite; Castro, Paula Maria Lima e; Tiritan, Maria ElizabethWastewater treatment plants (WWTPs) face growing operational challenges, driven by evolving environmental, social, and regulatory demands. There is increasing pressure to enhance the quality of treated effluents to comply with newly imposed directives, as WWTPs must now contend with a wider spectrum of challenges, including the presence of contaminants of emerging concern (e.g., pharmaceuticals), and the fluctuating wastewater composition driven by intensive urbanization and climate change. Currently, WWTPs are transitioning into resource recovery facilities, aligned with circular economy principles. The aerobic granular sludge (AGS) technology stands out for its resilience to diverse stressors and is well-recognized for its resource recovery potential. Despite these capabilities, the combined effects of multiple stressors on AGS remain underexplored. The objective of this thesis was to understand how oscillating wastewater salinity content and pharmaceuticals simultaneously affect AGS performance and microbial dynamics, while also exploring the potential of waste derived materials for a specific environmental application. A lab-scale AGS reactor was operated for over 9 months under variable salinity conditions to evaluate treatment performance and AGS microbiome evolution. The operational stages included a stepwise increase in salinity (from 1.5 to 15 g/L) followed by daily salinity oscillations (between 7.5 and 22.5 g/L). Chemical oxygen demand (COO) was effectively removed from the wastewater, while ammonium and nitrite levels in the effluent reduced progressively, with nitrate persistently detected in the effluent. Phosphate removal initially declined but completely recovered during the daily salinity oscillations. A versatile core microbiome in the AGS was found, mainly composed of nitrifiers, denitrifiers, and extracellular polymeric substances (EPS)-producing bacteria, which most likely contributed to preventing system collapse under salinity stress. Additionally, over 4 months, the effect of daily salinity oscillations and intermittent dosing of the chiral pharmaceuticals venlafaxine (VNF), tramadol (TRA), and their metabolites in wastewater on treatment performance and EPS production was evaluated and linked to shifts in the AGS bacteriome. The liquid chromatography-mass spectrometry technique was used to monitor the concentration of pharmaceuticals and metabolites. Although the removal of pharmaceuticals improved over time, complete removal was not achieved. Even during pharmaceuticals' dosing, COO and ammonium removal remained effective. Nevertheless, EPS content in AGS increased markedly (up to 196 mg/g TSS) with pharmaceuticals' dosing, compared to 132 mg/g TSS reported in their absence. The AGS bacteriome demonstrated high adaptability, characterized by diverse bacterial niches and a core microbiome composed of nutrient-removing and EPS-producing genera (e.g., Paracoccus). The wide microbial diversity, coupled with functional redundancy, may have contributed to stable nutrient removal and system resilience under stressful conditions, providing practical knowledge of the effects of multiple stressors' presence in wastewater, further demonstrating the robustness of AGS under different environmental challenges. Due to the incomplete pharmaceutical removal by the AGS system, a complementary study was conducted to assess the adsorption potential of tuna bone char (TBC), obtained through the pyrolysis of bone waste-derived material, for VNF and TRA. Pharmaceuticals were supplied individually or in combination, in synthetic wastewater matrices with varying salinity content. F0< pharmaceuticals supplied individually, TBC preferentially adsorbed TRA, with experimental q, values varying from 0.77-1.70 for VNF and 0.72-2.14 mg/g for TRA. The adsorption capacity was affected by the salinity content and the co-existence of pharmaceuticals in wastewater, but TBC remained effective in adsorbing both pharmaceuticals from distinct wastewaters. TBC is, indeed, a promising candidate for the removal of pharmaceuticals from wastewater, addressing key targets outlined in updated discharge regulations while aligning with circular economy strategies for water. In line with the growing emphasis on resource recovery in WWTPs, the final study explores the reuse potential of EPS extracted from AGS as soil amendments for agricultural applications. EPS were recovered from different AGS sources, namely from the lab-scale reactor and biomass sourced from two full-scale urban WWTPs. In a 49-day greenhouse experiment, the effect of incorporating 0.5 and 1% EPS doses on maize growth, nutrient uptake, and soil enzymatic activity was investigated against vermicompost and a non amended control. The EPS source and applied dose greatly influenced the amendment performance, with lower doses showing a marginal influence on plant growth and nutritional parameters. Nevertheless, incorporating 1% of EPS offers agronomic benefits, such as increased chlorophyll content, enhanced magnesium and potassium uptake in maize shoots, and stimulated soil enzymatic activities, often outperforming vermicompost. These findings support the use of EPS in agriculture and underscore the value of recycling materials from WWTPs for sustainable agriculture practices.
- 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.
- 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.
- 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.
- 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.
- Design of paper-based analytical devices for chemical and biochemical assays of biomarkers in biological fluids of non-invasive collectionPublication . Ferreira, Francisca Teixeira Soares da Mota; Mesquita, Raquel Beatriz Ribeiro de; Rangel, Antonio Osmaro Santos SilvaEnsuring basic healthcare access around the world remains a challenge, particularly in lowincome regions. In response, international initiatives such as the Sustainable Development Goals (SDGs) and the World Health Organization (WHO) guidelines emphasize the need to develop innovative, accessible, and cost-effective diagnostic tools. Point-of-care testing has emerged as an ideal solution, enabling rapid and decentralized analysis. Among these type of devices, microfluidic paper-based analytical devices (µPADs) have gained attention due to their affordability, portability, and ease of use, making them a particularly valuable tool in resource-limited locations. The purpose of the work presented throughout this thesis was to design and develop innovative point-of-care methodologies based on the µPAD approach for the determination of several healthrelated parameters, that could serve as an adding tool in the diagnosis and monitoring of several health conditions. The use of biological samples of non-invasive collection, such as saliva and urine, enhances diagnostic accessibility, particularly in point-of-care settings where traditional sample collection may be impractical or even impossible. Additionally, this thesis also explores the use of colorimetric detection methods, a straightforward approach to quantifying the targeted analytes by producing visible colour changes. To further improve the specificity and accuracy of these diagnostic tools, enzymatic reactions were incorporated into some of the developed µPADs. The first developed device was dedicated to the quantification of total iron in urine samples by using the colorimetric reaction of bathophenanthroline with iron (II) coupled with hydroxylamine, a well-known reducing agent capable of converting iron (III) in iron (II). To handle the potential colour of the urine samples, a sample blank was included in the device. This feature was vital to ensure the applicability of the developed μPAD, as urine may present a wide variability of colour range, from light yellow to brownish. The determination of nitrate was also accomplished in urine samples with a newly developed µPAD. This device included the enzymatic reaction of nitrate reductase to perform the conversion of nitrate to nitrite and the Griess reagent which provided the colorimetric detection of the resulting nitrite. In order to delay the vertical flow and increase the extent of the enzymatic reaction, a hydrophilic membrane layer was also incorporated into the device. The small porosity of this membrane also led to the retention of the compound responsible for the colour of urine, enabling a direct analysis of the samples. The next devices developed performed the quantification of NHX and urea in saliva samples but with a more complex approach. Not only were the devices composed of four layers, but the detection relies on the diffusion of NH3 (g) through a gas-diffusion membrane to produce a colorimetric change of a pH indicator. The hydrophobicity of this membrane also helped eliminate possible interferences of the saliva sample in the colorimetric reaction, since it did not allow the sample to pass through the membrane and reach the colour reagent. The urea determination was accomplished by including urease in the device, since it selectively catalyzes the dissociation of urea in ammonia and carbon dioxide. The urease activity determination was accomplished using a similar structure and reactions to the one used for the urea µPAD. However, the determination itself was achieved using the kinetic capabilities of the enzyme by correlating to the urease activity with the variation of the signal obtained between two different enzymatic reaction times. This strategy not only allowed a more accurate quantification but also suppressed the influence of NHX already present in the samples. The last device developed using the µPAD structure approach was to quantify glucose in saliva samples. With only two layers in its composition, this device uses a combination of two enzymatic reactions. First, glucose oxidase converts glucose into gluconic acid and hydrogen peroxide, then followed by the release of oxygen from hydrogen peroxide performed by peroxidase. The colorimetric detection is accomplished with the oxidation of o-dianisidine. In this work, a correlation between the concentration of glucose in saliva and blood was also successfully established, using #5 saliva samples of diabetic patients and the correspondent glucometer measurements provided by the patients. A rather different approach was used to perform the semi-quantitative analysis of E. coli DNA. Instead of developing a µPAD, an option was made to design a barcode-style lateral flow strip that would allow the semi-quantitative detection of E. coli. The amplification of the DNA in the sample was performed using the Loop-Mediated Isothermal Amplification (LAMP) technique since it can be performed at a constant temperature and provides high sensitivity and specificity results. The developed methodology, although requiring further optimization, showed reliable performance and offers a rapid, cost-effective method for E. coli detection. Overall, this thesis demonstrates the potential of microfluidic paper-based analytical devices and lateral flow assays as innovative, cost-effective diagnostic tools for point-of-care applications. By addressing critical challenges such as sample complexity, reagent stability, and measurement accuracy, the developed devices enhance accessibility to reliable testing, particularly in resourcelimited settings. Their affordability and ease of use further emphasize their role in expanding diagnostic capabilities outside traditional laboratory environment.
- Effect of polyphenols on saccharomyces cerevisiae fed-batch fermentationPublication . Carvalho, Luís Carlos Rocha; Oliveira, Carla Cristina Marques de; Oliveira, Ana Lúcia da Silva; Çarsanba, ErdemThe industrial production of β-farnesene is traditionally achieved through Saccharomyces cerevisiae fed-batch fermentation, often employing sugarcane syrup as the feedstock. However, the presence of phenolic compounds in sugarcane syrup potentially impacts yeast performance. This thesis aimed to study the evolution of phenolic compounds over β-farnesene production by S. cerevisiae in bioreactor fermentations with sugarcane syrup, assess the phenolic compounds’ impact on this process, and explore the potential of beet syrup, a lower phenolic content alternative feedstock, for β-farnesene production via a similar fermentation process. The phenolic compounds present within both the sugarcane syrup and the fermentation broth were systematically identified and quantified. Sugarcane syrup presented 50.7 mg/L of total phenolic content (TPC), comprising hydroxybenzoic acids, hydroxycinnamic acids and flavonoids. Among two kinetic models assessed, the Weibull model exhibited superior fitting capacity (R2 ≥ 0.85) and accurately depicted phenolic compounds’ accumulation during the 13-day fed-batch fermentation in 2 L bioreactors. The concentration of most phenolic compounds ascended until day three, stabilizing until the end of the fermentation process. Nevertheless, hydroxybenzaldehyde and protocatechuic, caffeic, ferulic and p-coumaric acids declined after the second day, indicating yeast metabolism. This work provides a model that can be applied to describe the phenolic accumulation during similar fed-batch processes. To investigate the phenolic influence on the fermentation, sugarcane syrup phenolic compounds were effectively removed with activated charcoal. The syrup purification process was optimized with a central composite design, varying the type of charcoal, concentration of charcoal and contact time. Optimization yielded purification conditions with charcoal pellets at 115 g/L and 12.5 h of contact time, while removing 96.7 % of phenolic compounds and recovering 43.7% of syrup mass. Purified syrup introduction into fermentations displayed divergent scale-dependent outcomes. Although in shake-flasks there was an 11 % enhancement in β-farnesene productivity, no significant increase was detected in bioreactors. In contrast, an increment in biomass productivity was exclusive to bioreactors, registering an increase of 12 %. Therefore, for the conditions tested, the phenolic compounds did not influence the β-farnesene production at large-scale. Moreover, β-farnesene production using an alternative feedstock, sugarbeet syrup, was successful. Despite presenting lower TPC (21.3 mg/L) compared to sugarcane syrup (50.7 mg/L), beet syrup contained high glucose (193 g/L) and hydroxymethylfurfural (HMF; 1.4 g/L) concentrations. Inoculum production in the second bioreactor step required optimization due to slow growth and the desired cell density was achieved with a fed-batch mode with initial 40 g/L of sugars from beet syrup and 10 g/L of sugar pulses. Over 8 days, fed-batch fermentation yielded 127 g/L β-farnesene concentration, with 19.44 % cumulative yield and 1.70 g/L/h cumulative productivity, affirming the viability of using beet syrup as the fermentation feedstock. In conclusion, this thesis showcased the dynamic relationship between phenolic content and β-farnesene fed-batch process highlighting an increase in the fermentation broth's phenolic content. Notably, specific phenolic compounds were metabolized by the yeast. The effects of sugarcane syrup phenolic compounds on S. cerevisiae were dependent on the fermentation scale. While valuable for mitigating yeast oxidative stress, phenolic compounds’ removal did not enhance bioreactor fermentation productivity. On the other hand, the utilization of beet syrup as feedstock may enable β-farnesene production in mild climates without extensive sugarcane cultivation, fostering sustainable production across diverse regions.
- The bioactive effects of cannabinoids on skin : from daily care to disease management applicationsPublication . Veiga, Mariana da Luz Cabral; Fernandes, João Carlos Azevedo Cruz Gonçalves; João Pedro Azevedo, Silva; Sara Nunes da Costa e, SilvaCannabinoids were first isolated in the 1960s from the Cannabis sativa L. plant. The study into the pharmacological and therapeutic potential of cannabinoids has gained traction in the past decades. This comes as a result of their recognized bioactivity, including antimicrobial, anti-inflammatory, and immunomodulatory activity, and due to the decriminalization of Cannabis-derived products continuing to expand worldwide. Cannabidiol (CBD) is one of the most researched cannabinoids. In particular, its anti-inflammatory activity has been vastly investigated, with a consequent unravelling of direct and indirect interactions with a myriad of receptors, broadening the potential therapeutic applications of CBD. More recently, cannabigerol (CBG), the main precursor of all phytocannabinoids, has also been in focus, although reports on this cannabinoid are still scarce in comparison with its counterpart CBD. Studies concerning the use of CBD and CBG for different therapies have increased, although the current role of cannabinoids in treating dermatological conditions has yet to be defined. Therefore, this thesis aims to explore the use of CBD and CBG for topical applications and to enlighten their potential as promotors of skin health and skin care. To this end, we first assessed their safety through a range of chemical and biological assays, including sensitization and mutagenicity tests. Although CBD extracted from the plant has solidified its use for topical applications, the CBG used in this work is produced through fermentation technology, and its safety profile has yet to be elucidated. After concluding both molecules were safe for topical application, we evaluated their potential as cosmetic ingredients. Several assays were performed to determine if CBD and CBG could be used for skin care and hair care, with both compounds demonstrating their ability to prevent inflammation in keratinocytes after exposure to pollution, and CBG presenting the capacity to induce hair growth in an ex vivo hair follicle model. Next, and considering the antimicrobial activity reported for cannabinoids, we focused on evaluating the antimicrobial and antibiofilm activity of CBD and CBG against bacteria and fungi belonging to the skin microbiota or associated with skin disorders and assessed their anti-adhesion effect against Staphylococci in keratinocytes. For the first time, we reported the MIC and MBC for Cutibacterium acnes, Pseudomonas aeruginosa and Escherichia coli. Thus, this study cemented the use of cannabinoids as antimicrobial agents, besides reporting on their potential as preservative ingredients and their positive lack of impact on the normal skin microbiota. We then evaluated the capacity of CBD and CBG to ameliorate the profile of inflammatory skin conditions, including psoriasis, irritant contact dermatitis (ICD) and atopic dermatitis (AD). Firstly, we developed, optimized, and validated ex vivo skin models with these conditions, and then assessed the impact of cannabinoids on them. Regarding the psoriatic model, CBD and CBG significantly reduced the inflammation and the hyperproliferation of keratinocytes, two of the main characteristics of this disease. Finally, the treatment of CBD and CBG for ICD and AD was also successful, with a reestablishment of the epidermal barrier and a reduction in inflammatory markers. These results are important as inflammatory skin conditions are associated with a low quality of life, and although corticosteroids are the go-to medications to alleviate symptoms, it has been demonstrated these medicines lose efficiency over time and have several side effects. We also attested that neither CBD nor CBG have a side effect common to betamethasone, hypertrichosis. Overall, the works herein reported provide valuable contributions in the field of dermatology, providing a step towards developing new applications for cannabinoids and potentially uncovering new topical treatments for skin disorders.