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The effect of typical domestic microwave heating (015min, at 800W) on the thermal degradation of unflavored and flavored olive oils' minor bioactive compounds and related antioxidant activity was studied. Olive oils from cv. Arbequina were flavored with lemon verbena essential oil (0\%, 0.2\% and 0.4\%, w/w) leading to a linear increase of total phenols (112160 mg gallic acid kg1 oil, R-Pearson=+0.9870), total carotenoids (2.192.56 mg lutein kg1 oil, R-Pearson=+0.9611), and, to a less extent, of chlorophyll (2.323.19 mg pheophytin kg1 oil, R-Pearson=+0.8238). However, no such linear trend was observed for the oxidative stability (6.57.8 h) or the radical scavenging activity (inhibition rates: 40\%43\%). The contents of total phenols, total carotenoids, and chlorophyll decreased with the rise of the microwave heating time, following their thermal degradation, a second-order kinetic model (0.8784R-Pearson0.9926). The essential oil addition did not influence the estimated second-order rate reaction constants of total phenols (0.000700.00072kg oil min1 mg1 gallic acid)and total carotenoids (0.140.17kg oil min1 mg1 lutein), with a broader variation observed for chlorophyll (0.0140.022kg oil min1 mg1 pheophytin). Globally, total carotenoids degraded faster than total phenols and chlorophyll (half-life of 2.33.4, 8.812.8, and 14.530.8 min, respectively). Moreover, except for chlorophyll, the half-life of total phenols and carotenoids linearly decreased with the essential oil addition (R-Pearson: 0.9999 and 0.9421, respectively), showing that flavoring did not have a protective effect against degradation when subjected to a microwave heating., Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020), to CEB (UIDB/04469/2020) and to BioTecNorte operation (NORTE-01-0145-FEDER-000004) and Project “GreenHealth—Digital strategies in biological assets to improve well-being and promote green health” (Norte-01-0145-FEDER-000042) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Nuno Rodrigues thanks the National funding by FCT—Foundation for Science and Technology, P.I., through the institutional scientific employment program–contract., info:eu-repo/semantics/publishedVersion

New compounds with potential insecticide activity were synthesized by structural modifications performed in the monoterpenoids phenolic moieties of carvacrol and thymol, resulting in a set of derivatives with the ether function containing the propyl, chloropropyl or hydroxypropyl chains, as well as a bicyclic ether with an unsaturated chain containing a carboxylic acid terminal. In addition, an analogue of carvacrol and thymol isomers bearing methoxyl, 1-hydroxyethyl and (3-chlorobenzoyl)oxy, instead of the three original methyl groups, was also synthesized. Several structural changes that resulted in diminished insecticide activity have been identified, but two significantly active molecules have been synthesized, one of them being less toxic to human cells than the naturally-derived starting materials. Structured-based inverted virtual screening and molecular dynamics simulations demonstrate that these active molecules likely target the insect odorant binding proteins and/or acetylcholinesterase and are able to form stable complexes. For the most promising compounds, nanoencapsulation assays were carried out in liposomes of egg phosphatidylcholine/cholesterol (7:3) prepared by both thin film hydration and ethanolic injection methods. The compound-loaded liposomes were generally monodisperse and with sizes smaller or around 200 nm. The thin film hydration method allowed high encapsulation efficiencies (above 85%) for both compounds and a delayed release, while for the systems prepared by ethanolic injection the encapsulation efficiency is lower than 50%, but the release is almost complete in two days., This study was funded by project PTDC/ASP-AGR/30154/2017 (POCI-01-0145-FEDER-030154) of the COMPETE 2020 program, co financed by the FEDER and the European Union. The authors acknowledge also to Foundation for Science and Technology (FCT, Portugal), and FEDER-COMPETE-QREN-EU for financial support to the research centres CQ-UM (UID/QUI/00686/2020), CF-UM-UP (UIDB/04650/2020) and REQUIMTE (UIDB/50006/2020). The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network and was purchased within the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds from POCI 2010 (FEDER) and FCT.

Microalgae are a promising and sustainable ingredient for functional food development, due to their rich nutritional and health-promoting effects. Moreover, microalgae generally contain large amounts of structural biopolymers such as proteins and polysaccharides, which might display interesting technological features and rheological properties. This study aims at unravelling Phaeodactylum tricornutum extracts (PTE) potential role as structuring agents for food applications. Distilled water and a mixture of water/ethanol (75/25, v v1) were used as solvents. Extracts physicochemical properties were analysed regarding protein folding, molecular weight distribution, polymer interactions, and their main functional groups. PTE thermal properties, rheological behaviour, and emulsifying capacities were also evaluated. PTE showed different gelling mechanisms, with 40°C aqueous purified extracts reporting the strongest gel character. Moreover, 80°C ethanolic purified extracts showed the greatest emulsifying potential, with an initial emulsification index (EI) of around 90%. This EI decreased to 60% after 10 days, but remained constant for the following 15 days, showing an interesting emulsion stability. Overall, these findings suggest that PTE have a great potential for further application as thickening or emulsifier food ingredients. This diatom revealed to be a valuable source of structurally diverse, technologically active compounds., Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of UID/BIO/04469/2020 unit and OH2O (grant agreement nº POCI-01-0145-FEDER-029145). This study has also received funding from the European Fund for Regional Development (FEDER), COMPETE 2020 – Competitiveness and Internationalization Operational Program (Portugal 2020) and the European Regional Development Fund (Algarve 2020 and Lisboa 2020), under the scope of the project AlgaValor (grant agreement nº POCI-01-0247-FEDER-035234; LISBOA-01-0247-FEDER-035234; ALG-01-0247-FEDER-035234) and OH2O. Ricardo N. Pereira acknowledges FCT for its Assistant Research contract obtained under CEEC Individual 2017, info:eu-repo/semantics/publishedVersion

Tese de doutoramento em Chemical and Biological Engineering, Bacteriófagos (fagos) são uma potencial ferramenta no controlo de Escherichia coli produtora de toxina Shiga (STEC), com vários estudos a demonstrar a sua eficácia. O uso generalizado de produtos à base de fagos na União Europeia tem sido adiado devido a preocupações com a sua segurança. Para o controlo de STEC, isto torna-se ainda mais relevante devido ao impacto que os agentes de desinfeção terão na expressão do gene de virulência mais relevante (toxina Shiga), ou na microbiota comensal do intestino humano. O principal objetivo deste trabalho é a definição de um conjunto de ensaios que permita avaliar a segurança do uso de fagos no controlo de STEC. Três importantes preocupações foram identificadas: (1) a patogenicidade e virulência de Mutantes Insensíveis aos Bacteriófagos (BIMs); (2) indução dos fagos que contem o gene da toxina Shiga (fagos Stx) e da expressão da toxina; (3) efeito dos fagos na microbiota de indivíduos saudáveis. Inicialmente, foi realizado uma análise ao genoma completo dos fagos Stx para caracterizar a prevalência dos genes da toxina Shiga nos isolados clínicos e alimentares. A análise revelou uma grande diversidade de fagos Stx, capazes de infetar um amplo conjunto de serotipos, potenciando assim a capacidade de converter diferentes estirpes de E. coli em STEC. Foi também demonstrado que os genes stx encontram-se perto da cassete lítica, indicando que a expressão da toxina está ligada à indução e libertação dos fagos Stx. Em seguida, para avaliar as questões de segurança mencionados anteriormente, o fago Ace, isolado e caracterizado neste estudo, foi usado como fago modelo para os ensaios de segurança. O contacto recorrente do fago Ace com uma estirpe STEC, resultou em BIMs com perfis de persistência similares à estirpe original. No entanto, estes BIMs demonstraram ser mais sensíveis à atividade do complemento do soro humano. A ausência do antigene O157 foi demonstrado para algumas colónias, o que pode justificar a maior sensibilidade à atividade antimicrobiana do soro. Por outro lado, a infeção de STEC com fago Ace não induziu a libertação de fagos Stx, nem a produção da toxina Shiga. Para determinar o efeito do fago Ace na microbiota do intestino, foi proposto um modelo de fermentação in vitro, usando como inóculo o conteúdo fecal de indivíduos saudáveis. A estabilidade da microbiota foi avaliada por monitorização dos metabolitos da fermentação e validada por análise metagenómica. Este estudo demonstrou que o fago Ace é seguro quando introduzido em diferentes microbiotas. Em suma, este trabalho focou-se nos aspetos mais relevantes relacionados com a segurança do uso de fagos no controlo de patógenos alimentares. O trabalho centrou-se na caracterização da segurança de um fago no controlo de STEC e permitiu concluir que este fago é seguro. Uma vez que esta conclusão não pode ser generalizada para outros fagos, este estudo é sobretudo relevante como um documento orientador para estudos de segurança de fagos usados no biocontrolo de STEC., Bacteriophages (phages) are a promising tool for the biocontrol of Shiga toxin-producing Escherichia coli (STEC), with several studies demonstrating their efficacy. Safety concerns still impair the generalized usage of phage-based products in food biocontrol inside the European Union. Safety aspects are particularly relevant in STEC since these biocontrol agents may have impact on the expression of the major virulence genes (Shiga toxins), or on the commensal microbiota of the human gut. In this work, it was aimed at establishing an analytic pipeline to evaluate the safety of using lytic phages in STEC biocontrol. Three important safety concerns were identified: (1) Bacteriophage Insensitive Mutants (BIMs) virulence and pathogenicity; (2) Shiga toxin-encoding phages (Stx phages) induction and Shiga toxin expression; (3) lytic phage outcome when introduced in a healthy human colon microbiota. To address the aim of this work, firstly, it was performed a whole-genome analysis of Stx phages to characterize the prevalence of Shiga toxin genes among food and clinical isolates. The analysis revealed a great diversity of Stx phages, able to infect a wide range of STEC serotypes. In fact, it was shown that similar Stx phages can infect different serotypes, rendering them the ability to convert different E. coli strains into STEC pathotype. Importantly, without exception, it was shown that stx genes are near the lytic cassette, which indicates that toxin expression is linked to prophages induction and release. Secondly, to assess the three important safety concerns mentioned above, a newly isolated and characterized phage, the lytic phage Ace, was used as a model phage in the subsequent safety assays. The recurrent contact of phage Ace and STEC strain resulted on the appearance of BIMs, which were shown to have similar persistence profiles as the wild type (WT) strain. Moreover, BIMs tend to be less resistant to the serum complement activity. In fact, some colonies were lacking the O157 antigen, which might render the colonies more susceptible to serum killing. Phage Ace did not induce Stx phage or Shiga toxin production upon STEC infection. By controlling the growth of STEC strain, less prophage was being induced, and, subsequently, less production of Shiga toxin was observed. To assess the impact of phage Ace in the colon microbiota, an in vitro fermentation model using faecal content of healthy subjects, was proposed. This model has proved phage Ace safe when introduced in different microbiotas. Microbiota stability was assessed by monitorization of fermentation metabolites and validated by metagenomics analysis. Overall, this work focussed on the most relevant aspects of phage safety that are still impairing their widespread application as biocontrol agents. Considering STEC control, this work shows that the use of phage Ace is safe; however, due to the diversity of phages that might be used in biocontrol, this safety outcome cannot be generalized to other phages. Nevertheless, this study might be useful as a guiding strategy for future safety assessments of phages used to control STEC in the food industry.

Recently, plant derived proteins have increased in interest and use due to a combination of interesting properties and industry-wide trends to replace animal derived proteins. Electrohydrodynamic processing (EHDP), can be used to develop micro- and nanostructures of plant proteins in a facile manner and, thus, increase their opportunities in the food, pharmaceutical, and biomedical industries. One of the most currently studied and promising plant proteins is zein. This review covers the most studied strategies to produce electrospun and electrosprayed zein-based structures. The most relevant properties, such as size, morphology, and surface area, are discussed according to the potential areas of interest, for instance in the food, biomedical, and pharmaceutical industries. In addition, applications of other electrospun/electrosprayed plant derived proteins are also presented, confirming the increasing interest of different industries in alternative proteins which can help promote sustainability., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UIDB/04469/2020 unit and by the I&D&I AgriFood XXI project, operation number NORTE-01-0145-FEDER-000041, cofinanced by the European Regional Development Fund (FEDER) under the scope of NORTE 2020 (Programa Operacional Regional do Norte 2014/2020). This study was supported by the project cLabel+ (POCI-01-0247-FEDER-046080) cofinanced by Compete 2020, Lisbon 2020, Portugal 2020 and the European Union, through the European Regional Development Fund (ERDF). Pedro Silva is the recipient of a fellowship (SFRD/BD/130247/2017) supported by Fundação para a Ciência e a Tecnologia, (FCT, Portugal). S. Torres-Giner acknowledges Spanish Ministry of Science and Innovation (MICI) for the funding received during his Ramón y Cajal contract (RYC2019-027784-I), info:eu-repo/semantics/publishedVersion

Agro-food industries face the challenge of re-using their wastes following the circular economy concept. The co-bioprocessing of wastes produced by different agro-food industries located in the same region can be a suitable strategy for the valorization of these wastes. This work evaluated the use of mixtures of wastes from wineries, olive mill and breweries to produce lignocellulolytic enzymes and antioxidant compounds by Aspergillus niger under solid-state fermentation (SSF). The SSF of a mixture of brewery spent grain with crude and exhausted olive pomace led to the highest values of enzymatic activity (xylanase, 837 U/g, cellulase, 87 U/g; -glucosidase, 254 U/g), being higher than enzymes activities per mass of total substrates. The highest antioxidant activity was achieved after 1 day of fermentation, being 2.3-fold higher in comparison to the unfermented wastes. SSF allowed the co-management of agro-industrial wastes from different industries., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTec-Norte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, info:eu-repo/semantics/publishedVersion

The by-products/wastes from agro-food and in particular the fruit industry represents from one side an issue since they cannot be disposed as such for their impact on the environment but they need to be treated as a waste. However, on the other side, they are a source of bioactive healthy useful compounds which can be recovered and be the starting material for other products in the view of sustainability and a circular economy addressing the global goal of zero waste in the environment. An updated view of the state of art of the research on fruit wastes is here given under this perspective. The topic is defined as follows: (i) literature quantitative analysis of fruit waste/by-products, with particular regards to linkage with health; (ii) an updated view of conventional and innovative extraction procedures; (iii) high-value added compounds obtained from fruit waste and associated biological properties; (iv) fruit wastes presence and relevance in updated databases. Nowadays, the investigation of the main components and related bioactivities of fruit wastes is being continuously explored throughout integrated and multidisciplinary approaches towards the exploitation of emerging fields of application which may allow to create economic, environmental, and social value in the design of an eco-friendly approach of the fruit wastes., info:eu-repo/semantics/publishedVersion

Biodiversity strengthens the productivity of any ecosystem (agricultural land, forest, lake, etc.). The loss of biodiversity contributes to food and energy insecurity; increases vulnerability to natural disasters, such as floods or tropical storms; and decreases the quality of both life and health. Wild and managed bees play a key role in maintaining the biodiversity and in the recovery and restoration of degraded habitats. The novelty character of this perspective is to give an updated representation of bee products biodiversity, sustainability, and health relationship. The role of bees as bioindicators, their importance in the conservation of biodiversity, their ecosystem services, and the variety of the bee products are described herein. An overview of the main components of bee products, their biological potentials, and health is highlighted and detailed as follows: (i) nutritional value of bee products, (ii) bioactive profile of bee products and the related beneficial properties; (iii) focus on honey and health through a literature quantitative analysis, and (iv) bee products explored through databases. Moreover, as an example of the interconnection between health, biodiversity, and sustainability, a case study, namely the Cellulose Park, realized in Rome (Italy), is presented here. This case study highlights how bee activities can be used to assess and track changes in the quality of agricultural ecosystemshive products could be valid indicators of the quality and health of the surrounding environment, as well as the changes induced by the biotic and abiotic factors that impact the sustainability of agricultural production and biodiversity conservation in peri-urban areas., (undefined), info:eu-repo/semantics/publishedVersion

Tese de doutoramento em Food Science and Technology and Nutrition, The production of chestnuts has increased in the last years. This fruit has interesting nutritional properties, i.e., low in fat, but rich in starch, and vitamin C. Nonetheless, chestnuts tend to spoil rapidly, which leads to a limited shelf-life. For this reason, it is important to highlight the need of studying post-harvest processing technologies capable of mitigating the decay of chestnuts and extend their shelf-life while maintaining their quality as close as possible to fresh fruits. In this context, ohmic heating (OH) and high-pressure processing (HPP) were evaluated as a post-harvest technology to improve chestnuts' shelf-life (Castanea sativa Mill.) by controlling molds and insect larvae proliferation. Moreover, the properties of HPP-treated chestnut flour and starch were evaluated. Thus, chestnuts were processed by OH at temperatures of 35, 45, and 55 °C and HPP at pressures of 400, 500, 600 MPa, and compared with control samples that were not treated and samples treated hydrothermally (HT, 50 °C for 45 min), as it is currently used by the chestnut industry. Shelf-life studies were carried out at 25 °C and 40% of relative humidity (RH), and 5 °C and 70% RH for 60 days. The results show that the processing times necessary to disinfect chestnuts were reduced by around 91% when compared to the HT. OH conducted at 55 ºC (OH-55ºC) and HPP at 600 MPa (HPP-600MPa), in combination with storage at 5 °C and 70% RH, can extend up to 60 days the chestnut shelf-life, and suppress the sprout and growth of fungi and insect larvae. The OH and HPP treatments did not affect chestnuts' nutritional quality since samples suffered minimum changes in their proximate composition. After HPP-600MPa, the relative crystallinity, amylopectin, size granules, phenols, antioxidants activity, and the pasting parameters of starch increased contrary to the amylose content, which decreased. OH and HPP are emergent technologies in food processing, demonstrates for the first time that has the potential to be used by the chestnut industry for the post-harvest disinfestation of the fruits. Moreover, HPP-600MPa is a promissory processing alternative that induces some starch modifications., A produção de castanha tem aumentado nos últimos anos. Esta fruta tem propriedades nutricionais interessantes, nomeadamente, baixa em gordura, mas rica em amido e vitamina C. No entanto, as castanhas tendem a estragar-se rapidamente, o que leva a uma vida útil limitada. Por esta razão, é importante destacar a necessidade de estudar tecnologias de processamento pós-colheita capazes de mitigar o deterioro da castanha e, ao mesmo tempo, prolongar sua vida útil mantendo a sua qualidade o mais próxima possível das frutas frescas. Nesse contexto, o aquecimento ôhmico (OH) e o processamento em alta pressão (HPP) foram avaliados como tecnologias pós-colheita para melhorar a vida útil da castanha (Castanea sativa Mill.) por meio do controle da proliferação de bolores e larvas de insetos. Além disso, foram avaliadas as propriedades da farinha e do amido de castanha tratada com HPP. Assim, as castanhas foram processadas por OH em temperaturas de 35, 45 e 55 °C e HPP em pressões de 400, 500, 600 MPa, e comparadas com amostras de controle que não foram tratadas e amostras tratadas hidrotermicamente (HT, 50 °C por 45 min), uma vez que HT é utilizado atualmente pela indústria da castanha. O estudo de vida útil foi realizado a 25 °C e 40% de umidade relativa (UR), e 5 °C e 70% UR por 60 dias. Os resultados mostram que os tempos de processamento necessários para desinfetar as castanhas foram reduzidos em cerca de 91% quando comparado com HT. OH conduzido a 55 °C (OH-55ºC) e HPP a 600 MPa (HPP-600MPa), em combinação com armazenamento a 5 °C e 70% UR, pode prolongar a vida útil da castanha em até 60 dias, e suprime o brotamento e o crescimento de fungos e larvas de insetos. Os tratamentos OH e HPP não afetaram a qualidade nutricional das castanhas, uma vez que as amostras sofreram alterações mínimas em sua composição centesimal. Após HPP-600MPa, a cristalinidade relativa, amilopectina, tamanho dos grânulos, fenóis, atividade antioxidante e os parâmetros de pasta do amido aumentaram, enquanto o conteúdo de amilose diminuiu. OH e HPP são tecnologias emergentes no processamento de alimentos, demostrando pela primeira vez que tem potencial para serem utilizadas na indústria da castanha para a desinfestação pós-colheita dos frutos. Além disso, o HPP-600MPa é uma alternativa promissora de processamento que induz algumas modificações no amido., This thesis was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE 01 0145 FEDER 000004) funded by the European Regional Development Fund under the scope of Norte2020 Programa Operacional Regional do Norte.

Carboxymethylcellulose (CMC)-based films can act as a protective barrier in food surfaces and a carrier of bioactive compounds, such as curcumin. However, incorporating curcumin in hydrophilic matrixes can be a challenge, and new strategies need to be explored. In this work, CMC-based films containing free curcumin and curcumin-loaded nanohydrogels (composed of lactoferrin and glycomacropeptide) were produced and characterized. The incorporation of curcumin-loaded nanohydrogels showed a significant decrease in films thickness (from 0.0791 to 0.029 mm). Furthermore, the water vapor permeability of CMC-based films was significantly decreased (62%) by incorporating curcumin-loaded nanohydrogels in the films. The water affinitys properties (moisture, solubility, and contact angle) of films were also affected by incorporating encapsulated curcumin. The addition of nanohydrogels to CMC-based films reduced the tensile strength values from 16.46 to 9.87 MPa. Chemical interactions were analyzed using Fourier transform infrared spectroscopy. The release profile of curcumin from CMC-based films was evaluated at 25 °C using a hydrophilic food simulant and suggests that the release mechanism of the curcumin happens by Ficks diffusion and Case II transport. Results showed that protein-based nanohydrogels can be a good strategy for incorporating curcumin in edible films, highlighting their potential for use in food applications., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Maria José Costa is recipient of a fellowship supported by a doctoral program (SFRH/BD/122897/2016) funded by the Portuguese Foundation for Science and Technology (FCT, POPH-QREN and FSE Portugal), info:eu-repo/semantics/publishedVersion

Acetic acid has long been considered a molecule of great interest in the yeast research field. It is mostly recognized as a by-product of alcoholic fermentation or as a product of the metabolism of acetic and lactic acid bacteria, as well as of lignocellulosic biomass pretreatment. High acetic acid levels are commonly associated with arrested fermentations or with utilization as vinegar in the food industry. Due to its obvious interest to industrial processes, research on the mechanisms underlying the impact of acetic acid in yeast cells has been increasing. In the past twenty years, a plethora of studies have addressed the intricate cascade of molecular events involved in cell death induced by acetic acid, which is now considered a model in the yeast regulated cell death field. As such, understanding how acetic acid modulates cellular functions brought about important knowledge on modulable targets not only in biotechnology but also in biomedicine. Here, we performed a comprehensive literature review to compile information from published studies performed with lethal concentrations of acetic acid, which shed light on regulated cell death mechanisms. We present an historical retrospective of research on this topic, first providing an overview of the cell death process induced by acetic acid, including functional and structural alterations, followed by an in-depth description of its pharmacological and genetic regulation. As the mechanistic understanding of regulated cell death is crucial both to design improved biomedical strategies and to develop more robust and resilient yeast strains for industrial applications, acetic acid-induced cell death remains a fruitful and open field of study. © 2021 Chaves, Rego, Martins, Santos-Pereira, Sousa and Côrte-Real., This work was supported by the "Contrato-Programa" UIDB/04050/2020 funded by national funds through the FCT I.P., info:eu-repo/semantics/publishedVersion

Usually, wine-making by-products are discarded, presenting a significant environmental impact. However, they can be used as a source of bioactive compounds. Moreover, consumers increasing demand for naturally nutritious and healthy products requires new formulations and food product improvement, together with sustainable, environmentally friendly extraction methods. Thus, this work aimed to compare ohmic heating (OH) with conventional methodology (CONV), using food-grade solvents, mainly water, compared to standard methanol extraction of anthocyanins. No significant differences were found between the CONV and OH for total phenolic compounds, which were 2.84 ± 0.037 and 3.28 ± 0.46 mg/g DW gallic acid equivalent, respectively. The same tendency was found for antioxidant capacity, where CONV and OH presented values of 2.02 ± 0.007 g/100 g and 2.34 ± 0.066 g/100 g ascorbic acid equivalent, respectively. The major anthocyanins identified were malvidin-3-O-acetylglucoside, delphinidin-3-O-glucoside, petunidine-3-O-glucoside, cyanidin-3-O-glucoside, and peonidine-3-O-glucoside. These extracts displayed antimicrobial potential against microorganisms such as Yersinia enterocolitica, Pseudomonas aeruginosa, Salmonella enteritidis, methicillin-sensitive Staphylococcus aureus, a methicillin-resistant Staph. aureus (MRSA), and Bacillus cereus. In conclusion, OH provides similar recovery yields with reduced treatment times, less energy consumption, and no need for organic solvents (green extraction routes). Thus, OH combined with water and citric acid allows a safe anthocyanin extraction from grape by-products, thus avoiding the use of toxic solvents such as methanol, and with high biological potential, including antimicrobial and antioxidant activity., “MultiBiorefinery: Estratégias multiuso para a valorização de uma gama alargada de subprodutos agroflorestais e das pescas: Um passo em frente na criação de uma biorrefinaria” financiado pelo Programa Operacional Competitividade e Internacionalização (POCI-01-0145-FEDER-016403) e pelo Programa Operacional Regional de Lisboa (LISBOA-01-0145-FEDER-016403), na sua componente FEDER e pela Fundação para a Ciência e Tecnologia, I.P. na componente nacional (SAICTPAC/0040/2015). The authors would also like to thank the scientific collaboration under the FCT project UID/Multi/50016/2019, UID-BIM-00009- 2020, and UID/BIO/04469/2020. The author Marta Coelho would like to acknowledge FCT for your PhD grant with the reference [grant number SFRH/BD/111884/2015], info:eu-repo/semantics/publishedVersion

Pineapple is consumed on a large scale around the world due to its appreciated sensorial characteristics. The industry of minimally processed pineapple produces enormous quantities of by-products (3050%) which are generally undervalued. The end-of-life of pineapple by-products (PBP) can be replaced by reuse and renewal flows in an integrated process to promote economic growth by reducing consumption of natural resources and diminishing food waste. In our study, pineapple shell (PS) and pineapple core (PC), vacuum-packed separately, were subjected to moderate hydrostatic pressure (225 MPa, 8.5 min) (MHP) as abiotic stress to increase bromelain activity and antioxidant capacity. Pressurized and raw PBP were lyophilized to produce a stable powder. The dehydrated samples were characterized by the following methodologies: chemical and physical characterization, total phenolic compounds (TPC), antioxidant capacity, bromelain activity, microbiology, and mycotoxins. Results demonstrated that PBP are naturally rich in carbohydrates (6688%), insoluble (1628%) and soluble (24%) fiber, and minerals (45%). MHP was demonstrated to be beneficial in improving TPC (24%), antioxidant activity (26%), and bromelain activity (632%) without affecting the nutritional value. Furthermore, microbial and mycotoxical analysis demonstrated that powdered PC is a safe by-product. PS application is possible but requires previous decontamination to reduce the microbiological load., Fundação para a Ciência e a Tecnologia (FCT), Portugal, through a PhD grant (SFRH/BD/109124/2015) and for financing the PhD grant of Renata A. Amaral (SFRH/BD/146009/2019). This work was supported by the national funding of FCT, under the scope of the strategic funding to the research units LEAF (UIDB/04129/2020), LAQV-REQUIMTE Associate Laboratory (UIDB/50006/2020) and CEB (UIDP/04469/2020) through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement, and PDR2020-101-031856 ProEnergy—New food products and bioenergy from fruits of low commercial value and agro industrial wastes, info:eu-repo/semantics/publishedVersion

Tese de Doutoramento em Ciência e Tecnologia Alimentar e Nutrição, Atualmente, o esgotamento de recursos, o acúmulo de resíduos e as mudanças climáticas são os principais fatores que impulsionam a necessidade de práticas sustentáveis. Algumas das estratégias para a redução do impacto ambiental gerado são o reaproveitamento de biorresíduos industriais, bem como o aumento do uso de recursos subexplorados por processos efetivos/intensificados para obtenção de produtos sustentáveis, ecológicos, seguros e de alta qualidade. A presente tese teve como objetivo desenvolver metodologias eficientes e ecologicamente corretas envolvendo a aplicação de campos elétricos e enzimas, além de solventes de baixo custo e não tóxicos para a valorização de biorecursos subvalorizados, como casca de Pinus pinaster e microalga Spirulina platensis. Os extratos de casca de pinheiro (PBE) apresentaram alta atividade antioxidante, antibacteriana e anti-hiperglicêmica, com baixa citotoxicidade e atividade seletiva contra células cancerosas, pois foram afetados negativamente. Essas propriedades foram atribuídas ao seu perfil fenólico e flavonóide. Além disso, o Aquecimento Óhmico (OH) evidenciou um aumento na extração de compostos fenólicos antioxidantes sem degradação. Os resultados também indicam extração preferencial sob o solvente apropriado, presumivelmente devido à ação do campo elétrico agindo em sinergia com o calor e solventes, promovendo efeitos de eletroporação da membrana. A microencapsulação por spray-drying de PBE utilizando maltodextrina foi eficaz na proteção de compostos fenólicos durante as condições gastrointestinais, controlando sua liberação e aumentando os seus benefícios para a saúde, diminuindo a produção de espécies reativas de oxigênio implicadas no processo de estresse oxidativo associado a algumas patologias. A Spirulina platensis é interessante para a indústria alimentar devido à sua composição geral e alto teor em C-ficocianina (corante azul natural). A presente tese mostrou que campos elétricos moderados associados à temperatura promovem efeitos de desintegração celular e eletroporação melhorando a extração e estabilidade de compostos intracelulares da Spirulina, tendo também um efeito positivo no rendimento de extração de C-ficocianina com tempos de tratamento mais curtos. Uma extração sequencial em múltiplos estágios avaliada por extração térmica convencional, OH, tratamento enzimático (EAE), e suas combinações, evidenciou que a extração combinada de OH e EAE permitiu a recuperação seletiva de ficobiliproteínas na etapa de extração aquosa. Pigmentos, como clorofila e carotenóides, foram extraídos seletivamente com etanol (95%), bem como a fração lipídica da microalga. De modo geral, a tecnologia OH tem potencial para ser uma alternativa "verde", melhorando a extração de compostos intracelulares, além de reduzir o tempo de processo, solventes e consumo de energia, oferecendo a possibilidade de ajustar a seletividade e estabilidade dos compostos extraídos. Além disso, processos integrados que abordem simultaneamente diferentes frações, em direção a uma economia de “desperdício zero”, são desejáveis e podem até ser benéficos para a eficácia de cada fração individual. Os biocompostos obtidos podem ser usados como aditivo tecnológico natural e/ou ingredientes funcionais para aplicações alimentares, cosméticas e farmacêuticas., Nowadays, resources depletion, waste accumulation and climate change are key factors driving the need for sustainable practices. Some of the strategies for the reduction of generated environmental impact are the reuse of industrial biowastes, as well as increasing the use of underexploited resources by effective/intensified processes to obtain sustainable, ecological, safe and high quality products. The present thesis aimed to develop efficient and environmentally friendly methodologies involving the application of electric fields and enzymes, as well as low cost and non-toxic solvents for the valorization of undervalued bioresources, as Pinus pinaster bark and Spirulina platensis microalgae. Pine bark extracts (PBE) presented high antioxidant, antibacterial and antihyperglycemic activities, with low cytotoxicity and a selective activity against cancer cells as these were negatively affected. These properties were ascribed to their phenolic and flavonoid profile. Moreover, Ohmic Heating (OH) evidenced an increased in the extraction of antioxidant phenolic compounds with no degradation. Results also indicate preferential extraction under the appropriate solvent, presumably due to the action of electric field effects acting in synergy with heat and solvents, promoting membrane electroporation effects. Spray-drying microencapsulation of PBE using maltodextrin was effective in protecting phenolic compounds during gastrointestinal conditions, controlling their delivery and enhancing its health benefits, decreasing the production of reactive oxygen species (ROS) implicated in the process of oxidative stress associated with some pathologies. Spirulina platensis is interesting for the food industry due to its overall composition and high content in C-phycocyanin (natural blue colorant). The present thesis showed that moderated electric fields associated with temperature promotes cell structure disintegration and electroporation effects improving the extraction and stability of intracellular compounds from Spirulina, having also a positive effect on the extraction yield of C-phycocyanin at shorter treatment times. A sequential multi-stage extraction evaluated by conventional thermal extraction, OH, enzymatic treatment (EAE), and their combinations evidenced that the combined OH and EAE extraction allowed selective recovery of phycobiliproteins in the aqueous extraction step. Pigments, such as chlorophyll and carotenoids were selectively extracted with 95% ethanol, as well as the lipid fraction of the microalgae. Overall, OH technology holds the potential to be a "green" alternative, improving intracellular compounds extraction, in addition to reducing the process time, solvents and energy consumption, offering the possibility of tuning the selectivity and stability of the extracted compounds. Moreover, integrated processes simultaneously addressing different fractions, towards a “zero waste” economy, are desirable and may be even beneficial to the effectiveness of each individual fraction. The obtained biocompounds can be used as natural technological additive and/or functional ingredient for food, cosmetic and pharmaceutical applications.

7 pages, 3 figures, 3 tables, Fish are largely dependent on dietary phosphorus for skeletal development and mineralization. In aquaculture, commercial diets commonly have higher phosphorus concentration than the basal requirements in most fish species to ensure growth and prevent bone mineral disorders. Excessive phosphorus in feeds is harmful for metabolism and results in an increase of wastes in farm effluents, which impact aquatic ecosystems. Previous studies have shown that depletion/excess of dietary phosphorus cause skeletal malformations and reduced/enhanced mineralization in fish. There is scarce information on dietary phosphorus requirements for optimal bone mineralization in species with different types of bone (cellular vs. acellular bone), which is particularly relevant for sustainable aquaculture. Thus, the aim of our study was to analyse the effect of dietary phosphorus concentrations on bone mineralization of turbot, a demersal acellular-boned fish and valuable aquaculture species. Our results show that the dietary phosphorus concentration did not cause changes to the bone mineral density and the phosphate/calcium concentrations. No apparent skeletal malformations were detected. Additionally, we did not find an altered expression of genes involved in bone mineral metabolism. Taken together, our data show that the phosphorus requirements for optimum growth and bone mineralization in turbot are below those currently used commercially at least for the time period examined: 55–195 days postfertilization (dpf), This work was funded by the Spanish Economy and Competitiveness Ministry project AGL2014-52473R and AGL2017-89648P to JR. PS-B was supported by AGL2014-52473R and AGL2017-89648P project contracts

Our previous research was focused on the effects of hydrophobicity on the antioxidant (AO) efficiency of series of homologous antioxidants with the same reactive moieties. In this work we evaluate the antioxidant efficiency of hydrophobic phenolipids in 4:6 olive oil-in-water emulsions, with different phenolic moieties (derived from caffeic, 4-hydroxycinnamic, dihydrocaffeic acids, tyrosol and hydroxytyrosol), with alkyl chains of 8 and 16 carbons, and compare the antioxidant efficiency with that of the parent compounds. All catecholic phenolipids, in particular the C8 derivatives, have proven to be better antioxidants for the oxidative protection of emulsions than their parental compounds with octyl dihydrocafffeate being the most efficient (16-fold increase in relation to the control). To understand the importance of some factors on the antioxidant efficiency of compounds in emulsions, Pearson’s correlation analysis was carried out between antioxidant activity and the first anodic potential (Epa), reducing capacity (FRAP value), DPPH radical scavenging activity (EC50) and the concentration of antioxidants in each region of the emulsified system. Results confirm the importance of the effective concentration of AOs in the interfacial region (AOI) (ρ = 0.820) and of the Epa (ρ = −0.677) in predicting their antioxidant efficiency in olive oil-in-water emulsions., Research was funded by FCT/MCTES (UIDB/QUI/50006/2020; PTDC/OCE-ETA/32492/ 2017–POCI-01-0145-FEDER-032492; doctoral grant SFRH/BD/100889/2014), FCT and REQUIMTELAQV for a post-doc fellowship (PTDC/OCE- ETA/32492/2017), Xunta de Galicia (10TAL314003PR), the University of Vigo (postdoctoral grant-Talent Recruitment Program 2018) and the IACOBUS 2019 program. The manuscript was prepared during a sabbatical leave of C. B.-D., supported by the University of Vigo.

New or used barrels can be applied in ageing of alcoholic beverages. Compounds adsorbed in wood migrate between beverages along with wood extractives. As barrel ageing is costly and time-consuming, processes using wood fragments have been gaining interest. These generate wood residues for which the reuse is still not well established. This work aims at the reuse of oak fragments for the additive ageing of alcoholic beverages. Oak chips, previously immersed in fortified wine, were applied to beer, wine and grape marc spirit. Wood compounds and adsorbed wine volatiles were extracted, with more impact and satisfactory yields on beer composition. Also, wood adsorbed beverages compounds in a subtractive ageing phenomena. Beer formulations using different binomial wood concentration/temperature combinations were generated and presented to trained tasters. Higher temperatures and wood concentrations led to prominence of wood descriptors and lower perception of fruity and floral aromas, reflecting the changes in chemical composition., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. Fermentum – Engenharia das Fermentações Lda. also participated in co-funding and provided beer samples used in this work, which authors would like to acknowledge. Lastly, authors would like to thank Mr. Benoît Verdier and Seguin Moreau for supplying the woods and Mr. Paulo Coutinho and Quinta do Portal for supplying the fortified wine, info:eu-repo/semantics/publishedVersion

Dissertação de mestrado em Tecnologia e Ciência Alimentar, A qualidade do vinho depende de fatores como o processo de vinificação, solo, condições climáticas e casta. Na sua produção podem ser usadas uma grande variedade de castas de Vitis vinífera, contudo apenas um pequeno número destas possuem importância comercial. O desenvolvimento de métodos confiáveis para rastrear videiras e vinhos têm-se tornado um forte objetivo devido à crescente procura pela proteção de origem de produtos. Os métodos baseados em ADN têm-se demonstrado objetivos na diferenciação de castas. A extração e purificação eficaz de ADN é condição essencial para que estes métodos possam ser aplicados, sendo necessário obter ADN em quantidade suficiente e puro. Este estudo teve o intuito de desenvolver um protocolo eficiente de extração e purificação de ADN de amostras vegetais de videira num novo protótipo de dispositivo microfluídico, comparando-o ao kit comercial DNeasy® PowerSoil® Pro Kit (Qiagen). A performance fluídica e de diversos protocolos foram avaliados no protótipo para o otimizar. A extração e purificação de ADN foi realizada para 10 castas da região do Douro e 3 do Minho, seguindo o procedimento do kit selecionado e no dispositivo desenvolvido com o protocolo selecionado na otimização. O protótipo de 15 mm usa menos volumes de soluções e é mais rápido relativamente ao de 21 mm. Ambos os protocolos 2.1 e 3 apresentam rendimentos aproximados de 50 %, contudo o protocolo P2.1 é preferível a este por possibilitar a integração e possuir menor variabilidade de resultados. Assim, o protótipo de 15 mm e o protocolo P2.1 foram usados no seguimento do estudo. O dispositivo miniaturizado demonstrou extração de ADN eficiente em folhas de videira e uvas em comparação ao kit comercial DNeasy® PowerSoil® Pro Kit (Qiagen). Futuramente, são necessários estudos com matrizes mais complexas e melhorar a pureza das amostras obtidas, avaliar a amplificação do ADN extraído pelo sistema e, por fim, a sua integração com os módulos de amplificação e deteção para um sistema completo de análise de ADN (μTAS)., Wine quality depends on several factors such as grape variety, the winemaking process, soil, and climatic conditions. Despite the wide range of Vitis vinífera varieties used in winemaking, only a small number of these have commercial importance. The demands of protection to the product provenience lead to the development of reliable methods to track grapevines and wines becoming a growing objective. DNA-based methods are objective in differentiating grapevine varieties. Effective DNA extraction and purification is an essential condition for these methods to be applied. It is needed to obtain enough and pure DNA. This study aimed to develop an efficient DNA extraction and purification protocol from vegetable grapevine samples in a new microfluidic device prototype, comparing it to the commercial kit DNeasy® PowerSoil® Pro-Kit (Qiagen). Fluidics performance was evaluated in the prototype, as well, four protocols to optimize the extraction. DNA extraction and purification were performed in ten varieties from Douro region, and three from Minho. DNA extraction and purification were carried by the protocol from the kit selected and to the device developed. The 15 mm prototype uses fewer volumes of solutions and it is faster than the 21 mm. Both protocols 2.1 and 3 show approximate yields of 50 %. However, the P2.1 protocol is preferable since it allows integration and has less variability in results. Thus, the 15 mm prototype and the P2.1 protocol were selected to carry on the study. The miniaturized device demonstrated efficient DNA extraction from grape leaves and grapes compared to the commercial kit DNeasy® PowerSoil® Pro-Kit (Qiagen). In the future, it is needed studies with more complex matrices, to improve the purity of the samples obtained; evaluate the amplification of the DNA extracted by the system, and finally, its integration with the amplification and detection modules for a complete DNA analysis system (μTAS).

Dissertação de mestrado em Biotecnologia, The growing consumers’ interest in healthier foods has led the food industry to develop functional foods specifically designed to improve human health, well-being, and performance. Curcumin is a natural polyphenol that presents numerous biological activities. However, curcumin has low water solubility, poor chemical stability, and low bioavailability. Lipid-based nanostructured delivery systems can be used to encapsulate curcumin. In this study, solid lipid nanoparticles (SLNs) encapsulating curcumin were developed and incorporated into a gelatine food matrix. SLNs (before and after incorporation in the gelatine) were characterized through mean diameter (Z-average diameter), polydispersity index (PDI) and zeta potential (ζ-potential) by Dynamic light scattering (DLS). Gelatine and gelatine-SLNs were evaluated through colour, texture, and rheology during 21 days of storage. In vitro digestion of the SLNs and gelatine-SLNs was performed and curcumin bioaccessibility, stability, and bioavailability were measured. The degree of hydrolysis (DH) of proteins and the production of free fatty acids (FFA) of gelatine and gelatine-SLNs were evaluated at gastric and intestinal phases, respectively. During 21 days, the Z-average diameter of the SLNs was maintained and the ζ-potential slightly decreased. The incorporation of the SLNs into gelatine did not significantly change neither the initial particle size nor PDI. ζ-potential of SLNs and gelatine-SLNs is different and this is due to the fact that gelatine has a slightly positive surface charge. The total colour difference (TCD) of the gelatine and gelatine-SLNs remained constant during 21 and 14 days of storage, respectively. The addition of SLNs did not lead to changes in the textural parameters of the gelatine, however, they promoted more solid behaviour to this food matrix. The value of DH of proteins in the gelatine and gelatine-SLNs were 47.8 % and 52.2 %, respectively. The total production of FFA of gelatine-SLNs was almost double of that of SLNs. Curcumin bioaccessibility and bioavailability were not statistically different between SLNs and gelatine-SLNs. However, the stability was higher in the gelatine-SLNs sample, suggesting that this food matrix provides a protective effect to curcumin. Finally, these results suggest that SLNs encapsulating curcumin and their incorporation in the gelatine food matrix is a promising application for the food industry.

This work aims to provide the first study on the mycobiota present in Chilean pepper Capsicum annuum L. cv. Cacho de Cabra throughout the early production stages. Two hundred and forty berry fruits were sampled: 1) at the ripe fruits harvest day; 2) during drying; and 3) smoking processes. A total of 192 strains, encompassing 11 genera and 44 species, were identified through analysis of -tubulin (benA) gene and internal transcribed spacer of ribosomal DNA (ITS) region. All collection points showed samples with high fungal contamination, but the mycobiota composition varied as a result of different environmental conditions. Alternaria spp. and Fusarium spp. were predominantly isolated from fresh fruits of C. annuum. Penicillium spp. was the most frequent genus in all analysed points. Penicillium brevicompactum and P. crustosum were the most abundant species. Among Aspergillus, A. niger and A. flavus were dominant after the drying phase. In our study, none of the analysed strains of Penicillium (113) and Aspergillus (35) produced Ochratoxin A at detectable levels. The broad characterization of the fungal community of C. annuum carried out in this study, could be a guideline for future mycotoxin analyses performed directly on the pod. Understanding the role and dynamics of mycobiota and its relationship with the toxins present in this substrate, will be useful to establish and improve control measures considering the specificities of each point in the C. annuum production chain., CONICYT/Chile for her Ph.D. grant no 21181445. R.R thanks to CONICYT/Chile for his MSc. grant Folio N° 7317076, Application N° 73170764. The present work was partially funded by the Universidad de La Frontera (Temuco, Chile) through the Project DIUFRO PIA19-0001. Moreover, this study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

The potential of plant extracts as bioinsecticides has been described as a promising field of agricultural development. In this work, the extracts of Punica granatum (pomegranate), Phytolacca americana (American pokeweed), Glandora prostrata (shrubby gromwell), Ulex europaeus (gorce), Tagetes patula (French marigold), Camellia japonica red (camellia), Ruta graveolens (rue or herb-of-grace) were obtained, purified, and their activity against Spodoptera frugiperda (Sf9) insect cells was investigated. From the pool of over twenty extracts obtained, comprising different polarities and vegetable materials, less polar samples were shown to be more toxic towards the insect cell line Sf9. Among these, a dichloromethane extract of R. graveolens was capable of causing a loss of viability of over 50%, exceeding the effect of the commercial insecticide chlorpyrifos. This extract elicited chromatin condensation and the fragmentation in treated cells. Nanoencapsulation assays of the cytotoxic plant extracts in soybean liposomes and chitosan nanostructures were carried out. The nanosystems exhibited sizes lower or around 200 nm, low polydispersity, and generally high encapsulation efficiencies. Release assays showed that chitosan nanoemulsions provide a fast and total extract release, while liposome-based systems are suitable for a more delayed release. These results represent a proof-of-concept for the future development of bioinsecticide nanoformulations based on the cytotoxic plant extracts., This research was funded by COMPETE 2020 program, co-financed by the FEDER and the European Union, PTDC/ASP-AGR/30154/2017 (POCI-01-0145-FEDER-030154). Foundation for Science and Technology (FCT, Portugal), and FEDER-COMPETE-QREN-EU funded research centers CQ-UM(UIDB/00686/2020), CF-UM-UP (UIDB/04650/2020) and REQUIMTE (UIDB/50006/2020).

Personalizing the nutrition and sensorial attributes of 3D printed foods primarily requires various multiscale properties to be individually tailored. Herein, multiscale inks are produced by segregative phase separation, a candidate for further 3D inks texture control, of gellan gum (GG), and whey protein isolate (WPI). The inks microstructure, rheological properties, flow dynamics, their impact on printability, and properties-variables interactions are analyzed using experimental design and clustering. The gels are a GG matrix structured with WPI beads or fibers ranging from 100??m in diameter. A straightforward, six-step printability test determines that high-quality prints require increasing viscosity, which is obtained by reducing the size and length of the WPI beads. Also, flow dynamics and rheology models predict the shear stress and extrusion force, according to the print settings and food-inks fluid properties. The phase-separated inks enable printing at high speed (>25/50?mm/s) upon low extrusion forces (, Nanotechnology-based functional solutions project, funded by ERDF and CCDR-N, under the call Norte2020 (Ref. NORTE-01-0145-FEDER-000019) and Enhance Microalgae (High added-value industrial opportunities for microalgae in the Atlantic Area), funded by ERDF, under the Call Interreg Atlantic Area 2014–2020 (Ref. EAPA_338/2016), info:eu-repo/semantics/publishedVersion

Since Listeria monocytogenes has a high case-fatality rate, substantial research has been devoted to estimate its growth rate under different conditions of temperature, pH and water activity (aw). In this study, published findings on L. monocytogenes growth in broth were extracted and unified by constructing meta-regression models based on cardinal models for (i) temperature (CM[T]), (ii) temperature and pH (CM[T][pH]), and (iii) temperature, pH and aw (CM[T][pH][aw]). After assessing all the sources retrieved between 1988 until 2017, forty-nine primary studies were considered appropriate for inclusion. Apart from the modelling variables, study characteristics such as: type of broth (BHI, TSB, TPB), reading method (colony-forming-units, CFU; or binary-dilution optical density methods, OD), inoculum concentration and strain serotype, were also extracted. Meta-regressions based on CM[T] and CM[T][pH] were fitted on subsets of the 2009 growth rate measures and revealed that type of broth and reading method significantly modulated the cardinal parameter estimates. In the most parsimonious CM[T][pH][aw] meta-regression model, whereby the variability due to type of broth was extracted in a nested random-effects structure, the optimum growth rate ?opt of L. monocytogenes was found to be lower when measured as CFU (0.947 h-1; SE=0.094 h-1) than when measured as OD (1.289 h-1; SE=0.092 h-1). Such a model produced the following cardinal estimates: Tmin=-1.273°C (SE=0.179 °C), Topt=37.26°C (SE=0.688 °C), Tmax=45.12°C (SE=0.013 °C), pHmin=4.303 (SE=0.014), pHopt=7.085 (SE=0.080), pHmax=9.483 (SE=0.080), aw min=0.894 (SE=0.002) and aw opt=0.995 (SE=0.001). Integrating the outcomes from numerous L. monocytogenes growth experiments, this meta-analysis has estimated pooled cardinal parameters that can be used as reference values in quantitative risk assessment studies., Foundation for Food Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020). U. Gonzales-Barron acknowledges the national funding by FCT, P.I., through the Institutional Scientific Employment Program contract, info:eu-repo/semantics/publishedVersion

Whey protein gelation and final gel properties are dependent of the gel forming solution characteristics (e.g. protein concentration, pH, ionic strength), and physical variables involved in the method used for gel preparation. Ohmic heating (OH) is an emerging technology in food processing and its application in heat-induced gelation of whey proteins has demonstrated its capacity to influence the physicochemical properties of protein gels. In this work, we studied the OH process and its inherent moderate electric field (MEF) variables - i.e. electric field (EF) strength and frequency - in order to establish their influence in protein aggregation and gelation during WPI cold-set gels formation. The presence of the EF during OH, particularly at higher EF strengths conjugated with lower frequencies, contributed to the formation of smaller aggregates with lower content of reactive thiol groups and lower viscosity. The cold-set gels produced from the aggregates suspension presented distinctive properties, influenced by the EF variables. i.e. higher EF strength and lower frequency. EF treatments give rise to more fine-stranded gels with lower disulphide crosslinking but higher number of hydrophobic interactions and hydrogen bonds. The EF effects during the treatments resulted in weaker, more elastic gels with higher water retention and swelling capacity. These results open novel perspectives for the use of OH as a tool for fine-tuning protein gel networks aiming at enhanced functionality for various applications (e.g. use as texturizer or encapsulating agents)., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020, Programa Operacional Regional do Norte. Rui M. Rodrigues gratefully acknowledge FCT for their financial grants with references SFRH/BD/110723/2015, info:eu-repo/semantics/publishedVersion

lactoglobulin (-Lg) has the ability to form three-dimensional networks when heated above denaturation temperature (ca. 76°C), since it undergoes conformational changes followed by subsequent protein-protein interactions, which allows designing stable micro- and nanostructures with affinity to bind to a wide range of molecules. In this sense, -Lg micro (with particle size from 200 to 300nm) and nano (with particle size100nm) structures were developed as a delivery system for the controlled release of hydrophilic and hydrophobic model compounds. Several concentrations of bioactive compounds were incorporated into -Lg micro- and nanostructures and their association efficiency (AE) and loading capacity (LC) were determined. -Lg structures were characterized in terms of structural properties, morphology, binding mechanisms, conformational changes and secondary structure. The impact of several conditions (e.g., pH, thermal processing, ionic strength and storage temperature) on the stability of -Lg structures was also investigated. The release profile of bioactive compounds from -Lg structures was determined in vitro using two food simulants with different hydrophobicities under different temperature conditions (at 4°C and 25°C). Data recorded showed that -Lg nanostructures had the highest AE and LC comparing with -Lg microstructures, for both bioactive compounds tested. -Lg micro- and nanostructures with or without association of bioactive compounds showed to be stable under acidic (pH 2 to 3), neutral (pH 6) or alkaline (pH 10) conditions, thermal treatments up to 70°C and during storage for 50 and 90 days at 25°C and 4°C, maintaining their particle size, PDI and surface charge (p>0.05). The release kinetics of bioactive compounds from micro- and nanostructures fitted well the Linear Superimposition Model, being the relaxation the main release mechanism. Both compounds showed an initial burst effect followed by a slow release. All these findings provide new insights on which conditions the -Lg micro- and nanostructures are more stable, and therefore more suitable to act as potential delivery systems for hydrophilic and hydrophobic bioactive compounds., FCT under the scope of the strategic funding of UID/Multi/50016/2019 and UID/BIO/04469/2013 units and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This study was also supported by FCT under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), info:eu-repo/semantics/publishedVersion

This paper proposes a design methodology for a synthesizable, fully digital TDC architecture. The TDC was implemented using a hardware description language (HDL), which improves portability between platforms and technologies and significantly reduces design time. The proposed design flow is fully automated using TCL scripting and standard CAD tools configuration files. The TDC is based on a Tapped Delay Line architecture and explores the use of Structured Data Path (SDP) as a way to improve the TDL linearity by homogenizing the routing and parasitic capacitances across the multiple TDL’s steps. The studied approach also secures a stable, temperature independent measurement operation. The proposed TDC architecture was fabricated using TSMC 180nm CMOS process technology, with a 50MHz reference clock and a supply voltage of 1.8V. The fabricated TDC achieved an 111ps RMS resolution and a single-shot precision of 54ps (0.48 LSB) and 279ps (2.51 LSB), with and without post-measurement software calibration, respectively. The DNL across the channel is mostly under 0.3 LSB and a maximum of 8 LSB peak-to-peak INL was achieved, when no calibration is applied., (037902)

Bovine lactoferrin (bLF) has been extensively described as a wide spectrum antimicrobial protein. bLF bactericidal activity has been mainly attributed to two different mechanisms: environmental iron depletion and cell membrane destabilization. Due to its antimicrobial properties, bLF has been included in the formulation nutraceutical food products and edible active packages. This work comprises the experimental evidence of the requirement of bLF unrestricted mobility (free bLF) to effectively perform its bactericidal action. To assess the unrestricted and restricted bLF activity, a nontoxic matrix of bacterial nanocellulose (BNC) was used as carrier, and as an anchoring scaffold, respectively. Therefore, BNC was functionalized with bLF through two different methodologies: (i) bLF was embedded within the three-dimensional structure of BNC and; (ii) bLF was covalently bounded to the nanofibrils of BNC. bLF efficiency was tested against two bacteria isolated from clinical specimens, Escherichia coli and Staphylococcus aureus. bLF concentration after covalent binding to BNC was two-fold higher in comparison to the embedding method. Nevertheless, only the embedded bLF exhibited a significant bactericidal activity, due to bLF ability to permeate the BNC matrix and execute its bactericidal action., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/ 04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER- 000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of UID/FIS/04650/2020 and projects PTDC/BTM-MAT/28237/ 2017 and PTDC/EMDEMD/28159/2017, Portuguese Foundation for Science and Technology (FCT) SFRH/BD/64901/2009 and SFRH/BD/111478/2015, Spanish State Research Agency (AEI) and the European Regional Development Fund through the project PID2019-106099RBC43/ AEI/10.13039/501100011033 and from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively, info:eu-repo/semantics/publishedVersion

Mycotoxins are toxic compounds mainly produced by fungi of the genera Aspergillus, Penicillium and Fusarium. They are present, often as mixtures, in many feed and food commodities including cereals, fruits and vegetables. Their ubiquitous presence represents a major challenge to the health and well being of humans and animals. Hundreds of compounds are listed as possible mycotoxins occurring in raw and processed materials destined for human food and animal feed. In this study, mycotoxins of major toxicological relevance to humans and target animal species were investigated in a range of crops of interest (and their derived products). Extensive Literature Searches (ELSs) were undertaken for data collection on: (i) ecology and interaction with host plants of mycotoxin producing fungi, mycotoxin production, recent developments in mitigation actions of mycotoxins in crop chains (maize, small grains, rice, sorghum, grapes, spices and nuts), (ii) analytical methods for native, modified and co-occurring mycotoxins (iii) toxicity, toxicokinetics, toxicodynamics and biomarkers relevant to humans and animals (poultry, suidae (pig, wild boar), bovidae (sheep, goat, cow, buffalo), rodents (rats, mice) and others (horses, dogs), (iv) modelling approaches and key reference values for exposure, hazard and risk modelling. Comprehensive databases were created using EFSA templates and were stored in the MYCHIF platform. A range of approaches were implemented to explore the modelling of external and internal exposure as well as dose-response of mycotoxins in chicken and pigs. In vitro toxicokinetic and in vivo toxicity databases were exploited, both for single compounds and mixtures. However, large data gaps were identified particularly with regards to absence of common statistical and study designs within the literature and constitute an obstacle for the harmonisation of internal exposure and dose-response modelling. Finally, risk characterisation was also performed for humans as well as for two animal species (i.e. pigs and chicken) using available tools for the modelling of internal dose and a component-based approach for selected mycotoxins mixtures.

Atlantic cod is processed industrially for food purposes, with several by-products being directed to animal feed and other ends. Looking particularly into swim bladders, the extraction of collagen can be a valuable strategy for by-product valorization, explored in the present work for the first time. Collagen was extracted using acetic acid (ASCsb) and pepsin (PSCsb) with yields of 5.72% (w/w) and 11.14% (w/w), respectively. SDS-PAGE profile showed that the extracts were compatible with type I collagen. FTIR, CD and XRD results suggest that the PSCsb structure underwent partial denaturation, with microDSC showing a band at 54 ºC probably corresponding to a melting process, while ASCsb structure remained intact, with preserved triple helix and a denaturation temperature of 29.6 ºC. Amino acid composition indicates that the total content of proline-like amino acids was 148/1000 residues for ASCsb and 141/1000 residues for PSCsb, with a hydroxylation degree of about 37%. The extracts exhibited a typical shear thinning behavior, interesting property regarding their further processing toward the development of biomaterials. In this regard, assessment of metabolic activity of human fibroblast cells cultured in the presence of collagen extracts with concentrations up to 3mg/mL revealed the absence of cytotoxic behavior. Collagen extracts obtained from Atlantic cod swim bladders shown attractive properties regarding their use in cosmetic or biomedical applications., The authors would like to acknowledge to European Union for the financial support under the scope of European Regional Development Fund (ERDF) through the Structured Project NORTE-01-0145-FEDER-000021 (Norte2020) and under the scope of the European Union Seventh Framework Programme (FP7/2007-2013) through grant agreement ERC-2012-ADG 20120216-321266 (ERC Advanced Grant ComplexiTE). The Portuguese Foundation for Science and Technology is also acknowledged for the PhD grant of A. L. A under Doctoral Programme Do ~ Mar (PD/BD/127995/2016), as well as Norte2020 Regional Programme for the PhD grant of C. O. (Norte-08-5369-000037).

Reports on the effect of droplet size on the oxidative stability of emulsions and nanoemulsions are scarce in the literature and frequently contradictory. Here, we have employed a set of hydroxytyrosol (HT) esters of different hydrophobicity and fish oil-in-water emulsified systems containing droplets of different sizes to evaluate the effect of the droplet size, surfactant, (&Phi, I) and oil (&Phi, O) volume fractions on their oxidative stability. To quantitatively unravel the observed findings, we employed a well-established pseudophase kinetic model to determine the distribution and interfacial concentrations of the antioxidants (AOs) in the intact emulsions and nanoemulsions. Results show that there is a direct correlation between antioxidant efficiency and the concentration of the AOs in the interfacial region, which is much higher (20&ndash, 200 fold) than the stoichiometric one. In both emulsified systems, the highest interfacial concentration and the highest antioxidant efficiency was found for hydroxytyrosol octanoate. Results clearly show that the principal parameter controlling the partitioning of antioxidants is the surfactant volume fraction, &Phi, I, followed by the O/W ratio, meanwhile, the droplet size has no influence on their interfacial concentrations and, therefore, on their antioxidant efficiency. Moreover, no correlation was seen between droplet size and oxidative stability of both emulsions and nanoemulsions.

Edible coatings incorporated with bacteriocin-producing lactic acid bacteria (LAB) can provide an alternative natural preservation method as they can offer an additional microbiological protection to food products. The bacteriocin-producing strains Lactococcus lactis L3A21M1 and Lc. garvieae SJM17 isolated from artisanal Azorean cheeses were incorporated into an edible fresh-cheese coating composed of alginate, maltodextrin and glycerol. The immobilization of Lactococcus cells into the coating had no negative effect on their viability and antibacterial activity throughout 10 days storage at 4 °C and 10 °C. The application of coating with immobilized Lactococcus cells on cheeses reduced significantly (p, Portuguese Foundation for Science and Technology (FCT), Project UID/CVT/00153/2019. Sofia P.M. Silva received support through grant SFRH/BD/139525/2018 from the Portuguese Foundation for Science and Technology (FCT), info:eu-repo/semantics/publishedVersion

Commonly used as biological chemosensors in toxicity assays, Vibrio fischeri bacteria were systematically characterized using complementary physicochemical and biological techniques to elucidate the evolution of their properties under varying environmental conditions. Changing the pH above or below the optimal pH 7 was used to model the long-term stress that would be experienced by V. fischeri in environmental toxicology assays. The spectral shape of bioluminescence and cell-surface charge during the exponential growth phase were largely unaffected by pH changes. The pH-induced modulation of V. fischeri growth, monitored via the optical density (OD), was moderate. In contrast, the concomitant changes in the time-profiles of their bioluminescence, which is used as the readout in assays, were more significant. Imaging at discrete timepoints by scanning electron microscopy (SEM) and helium-ion microscopy (HIM) revealed that mature V. fischeri cells maintained a rod-shaped morphology with the average length of 2.2 ± 1 µm and diameter of 0.6 ± 0.1 µm. Detailed morphological analysis revealed subpopulations of rods having aspect ratios significantly larger than those of average individuals, suggesting the use of such elongated rods as an indicator of the multigenerational environmental stress. The observed modulation of bioluminescence and morphology supports the suitability of V. fischeri as biological chemosensors for both rapid and long-term assays, including under environmental conditions that can modify the physicochemical properties of novel anthropogenic pollutants, such as nanomaterials and especially stimulus-responsive nanomaterials., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and COMPETE 2020 (POCI‐ 01‐0145‐FEDER‐006684) and BioTecNorte operation (NORTE‐01‐0145‐FEDER‐000004) funded by European Regional Development Fund under the scope of Norte2020 Programa Operacional Re‐ gional do Norte. A.R.S. holds an FCT fellowship SFRH/BD/131905/2017. Measurements using the helium‐ion microscope were supported by the Karlsruhe Nano Micro Facility (KNMF) of the Karls‐ ruhe Institute of Technology (KIT) under projects 2016‐015‐010689 and 2018‐019‐021475, info:eu-repo/semantics/publishedVersion

The viability of 2nd generation bioethanol processes is dependent on achieving high ethanol titers, which requires the use of high solid loadings that will negatively affect the fermentative microorganism besides increasing enzyme-associated costs. To solve this, and also problems of feedstock availability, lignocellulosic biomass can be mixed with dairy by-products to increase carbon content. In this study, industrial strains of Saccharomyces cerevisiae, with improved thermotolerance and stress resistance, were engineered for the cell surface display of cellulolytic enzymes and were evaluated in consolidated bioprocessing of cellulose. Additionally, -galactosidase was also displayed to enable lactose consumption, resulting in high ethanol titers (>50 g/L) from the simultaneous use of cheese whey and pretreated corn cob as substrate. The multi-feedstock valorization approach together with this lactose-consuming cellulolytic yeast allowed the reduction on materials costs by 60% with a 2.5-fold increase in the annual ethanol production, therefore contributing to the establishment of economic viable ethanol processes., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020, the MIT-Portugal Program (Ph.D. Grant PD/BD/128247/2016 to Joana T. Cunha), and Biomass and Bioenergy Research Infrastructure (BBRI)-LISBOA-01-0145-FEDER-022059, supported by Operational Programme for Competitiveness and Internationalization (PORTUGAL2020), by Lisbon Portugal Regional Operational Programme (Lisboa 2020) and by North Portugal Regional Operational Programme (Norte 2020) under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors acknowledge Professor Emile van Zyl and Stellenbosch University for providing the sequences encoding cellobiohydrolase 1 from Talaromyces emersonii (CBH1) and cellobiohydrolase 2 from Chrysosporium lucknowense (CBH2)., info:eu-repo/semantics/publishedVersion

Microorganism assessment plays a key role in food quality and safety control but conventional techniques are costly and/or time consuming. Alternatively, electronic tongues (E-tongues) can fulfill this critical task. Thus, a potentiometric lab-made E-tongue (40 lipid sensor membranes) was used to differentiate four common food contamination bacteria, including two Gram positive (Enterococcus faecalis, Staphylococcus aureus) and two Gram negative (Escherichia coli, Pseudomonas aeruginosa). Principal component analysis and a linear discriminant analysis-simulated annealing algorithm (LDA-SA) showed that the potentiometric signal profiles acquired during the analysis of aqueous solutions containing known amounts of each studied bacteria allowed a satisfactory differentiation of the four bacterial strains. An E-tongue-LDA-SA model (12 non-redundant sensors) correctly classified 98 ± 5% of the samples (repeated K-fold-CV), the satisfactory performance of which can be attributed to the capability of the lipid membranes to establish electrostatic interactions/hydrogen bonds with hydroxyl, amine and/or carbonyl groups, which are comprised in the bacteria outer membranes. Furthermore, multiple linear regression models, based on selected subsets of E-tongue sensors (1215 sensors), also allowed quantifying the bacteria contents in aqueous solutions (0.993 ± 0.011 R2 0.998 ± 0.005, for repeated K-fold-CV). In conclusion, the E-tongue could be of great value as a preliminary food quality and safety diagnosis tool., Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020) and to CEB (UIDB/04469/2020), as well as to the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Ítala M.G. Marx also acknowledges the Ph.D. research grant (SFRH/BD/137283/2018) provided by FCT., info:eu-repo/semantics/publishedVersion

Mead is a traditional beverage that results from the alcoholic fermentation of diluted honey performed by yeasts. Although the process of mead production has been optimized in recent years, studies focused on its sensory properties are still scarce. Therefore, the aim of this work was to analyse the sensory ttributes of mead produced with free or immobilized cells of the Saccharomyces cerevisiae strains QA23 and ICV D47, and to establish potential correlations with its olatile composition. In the volatile composition of mead, the effect of yeast condition was more important than the strain. In respect to sensory analysis, the most pleasant aroma descriptors were correlated with mead obtained with free yeast cells, independently of the strain. Both sensory analysis and volatile composition indicates that the most pleasant mead was produced by free yeast cells. Although this study has provided a significant contribution, further research on the sensory quality of mead is still needed., Portuguese Foundation for Science and Technology (FCT) through a PhD grant (SFRH/BD/45820/2008). This research was partially supported through the PTDC project (PTDC/AGR-ALI/68284/2006) awarded by the FCT. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte, info:eu-repo/semantics/publishedVersion

In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genome-scale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described., Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES) – Finance code 001. The authors also acknowledge the support of CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil); International cooperation project CAPES-FCT (Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior/Brazil - Fundação para a Ciência e Tecnologia/Portugal - Process 315/11); CAPES, Brazil (Atração de Jovens Talentos - Process 064922/2014-01); Fundação para a Ciência e Tecnologia, Portugal (strategic funding of UID/BIO/04469 unit) and COMPETE 2020, Portugal (POCI-01-0145-FEDER-006684), in addition to the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, info:eu-repo/semantics/publishedVersion

Nanoparticles are commonly used to improve the bioavailability of several bioactive compounds. In this work, nanostructured lipid carriers (NLC) have been optimized by response-surface methodology to protect phenolic compounds from Hibiscus sabdariffa. The influence of lipid and surfactant concentration on the mean particle size, polydispersity index and zeta potential of NLC was analysed. The results showed that the effect of both independent variables was statistically significantly on the selected responses. The optimized formulation obtained from the predicted model was composed of 2.21% (w/w) of lipid phase and 1.93% (w/w) of surfactant, with a desirability function of 0.77. The loading of polyphenol-enriched Hibiscus sabdariffa extract - obtained by microwave assisted extraction or by pressurized liquid extraction - within the NLC resulted in particles with a mean size of 470±8nm (encapsulation efficiency of 52.9±0.9% for quercetin and 60±2% for anthocyanins) and 344±12nm (encapsulation efficiency of 93±3% for quercetin and 84±4% for anthocyanins), respectively. Transmission electron microscopy images and Fourier transformed infra-red analysis of extracts-loaded NLC confirmed that interaction between lipids and polyphenols improved the physicochemical stability of the particles. Our findings highlight the added value of NLC as a delivery system for phenolic compounds extracted from Hibiscus sabdariffa., Research group AGR274 “Bioactive ingredients” from the Analytical Chemistry Department of the University of Granada, and the Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra. Also, the authors would like to thank the financial support received from the Spanish Ministry of Economy and Competitiveness (MINECO) (project AGL2015-67995-C3-2), from Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) under the project M-ERA-NET/0004/2015-PAIRED, co-financed by FEDER, under the Partnership Agreement PT2020. We would like to thank the Ministry of Education, Culture and Sport (MECD) for supporting the grant FPU15/01125 of SPM and the stay in the University of Coimbra supported by FPU short – term stay. MCT wishes to acknowledge FCT and Dendropharma - Investigação E Serviços De Intervenção Farmacêutica, Sociedade Unipessoal Lda. for the individual fellowship (PD/BDE/135086/2017), info:eu-repo/semantics/publishedVersion

Biofilms are responsible for most of the interference caused by microorganisms in food processing. The aim of this study was to evaluate the cinnamon (Cinnamomum zeylanicum) essential oil and eugenol sanitizer and anti-biofilm activity against biofilms. Concentrations used of essential oil were 0.0% (control) 0.12%; 0.48%; 0.96% and 1.92%; the amount of eugenol was 0.76%. Concentrations were determined from other published studies. Number of viable cells and quantification the bacterial biomass were determined. Anti-biofilm treatment was effective in preventing the formation of biofilms. The 1.92% concentration was the most satisfactorily with Escherichia coli reduction of 5.91log CFUcm-2 and Staphylococcus aureus reduction of 5.17log CFUcm-2 (P, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Foundation for Science and Technology and European Community fund European Regional Development Fund (FEDER), in the ambit of the Project PTDC/SAUESA/6460912006/FCOMP-01-0124-FEDER-007480, info:eu-repo/semantics/publishedVersion

Dissertação de mestrado em Biotecnologia, No tempo corrente há um incremental desejo por parte da população global de obter o mais variadíssimo tipo de alimentos, sem que estes acarretem qualquer risco. Esta tarefa vê-se dificultada, uma vez que, ainda que haja uma exigência por alimentos seguros, há também uma pressão contra vários tipos de tratamentos e métodos de conservação. Um dos métodos de conservação usados com enorme frequência hoje em dia é o tratamento térmico, que tem vindo a ser usado desde que Louis Pasteur o criou a meio do século XIX. Enquanto este tipo de tratamento é eficaz, é necessário otimizá-lo para os organismos e matriz alimentar em causa, permitindo a destruição microbiana sem comprometer as propriedades organoléticas dos alimentos. Uma fonte externa descobriu, após tratamento térmico, uma contaminação por parte do fungo Cadophora fastigiata nos seus produtos. Devido à falta de informação sobre este fungo filamentoso, tomase em causa este caso prático como ímpeto para levar a cabo um estudo térmico sobre este fungo, determinando os seus valores de D e z e apontando se estes parâmetros se mostram de tal forma insólitos que justifiquem a contaminação. No final do estudo, obtiveram-se os valores de 0,69 minutos para a inativação de 90% dos esporos a 70 ºC para uma amostra de água destilada a pH 6,5. Assim como, valores de D de 9,19 e 9,60 minutos, a 55 ºC, respetivamente para amostras a pH 4 de solução de sacarose a 20% e de água acidificada. Adicionalmente os valores de z obtidos para todas as amostras variaram entre 7,8 ºC e 10,6 ºC, tornando desta forma improvável uma contaminação fúngica por parte deste fungo após o devido tratamento térmico., In current times there is a growing desire by the global population to obtain the most varied sorts of foods, without any associated risks. This task sees itself hardened by the fact that, although there is a demand for safe products, there is also a pressure of sorts against a lot of the treatments and methods used for food preservation. One of the preservation methods used with vast frequency nowadays is the thermal processing, this method has been used since Louis Pasteur created it in the middle of the 19th century. While this type of treatment is very effective, it is necessary to optimize it depending on the organisms and food matrix at hand, in order to allow for microbial destruction without jeopardizing the products’ organoleptic proprieties. An external source has discovered, after thermal processing of their products a contamination by Cadophora fastigiata. Due to the lack of information regarding this filamentous fungus, this practical case is used as a throttle to start off a series of thermal studies on this fungus, in order to determine its D and z values and highlighting whether these parameters are so outlandish that they are able to justify the contaminations found. By the end of this study, we obtained the value of 0.69 minutes for the inactivation of 90% of the spores at 70 ºC, for a distilled water sample at pH 6.5. Aside from these, we also found D values of 9.19 and 9.60 minutes, at 55 ºC, respectively for the samples of 20% sucrose and acidified water both at a pH of 4. Additionally, the z values obtained for all samples varied between 7.8 ºC and 10,6 ºC, making it unlikely for a fungal contamination by this species to take place with the proper thermal treatments.

Olive oil is highly appreciated due to its nutritional and organoleptic characteristics. However, a huge compositional variation is observed between olive oils, requiring the use of diverse analytical techniques for its classification including titration, spectrophotometry and chromatography, as well as sensory analysis. Chemical analysis is usually time-consuming, expensive and require skilled technicians, while the sensorial ones are dependent upon individual subjective evaluations, even if performed by trained panellists. This work evaluated and demonstrated the feasibility of using a potentiometric electronic tongue, comprising non-specific lipid polymeric and cross-sensitive sensor membranes, coupled with chemometric tools based on different sub-sets of sensors (from 11 to 14 sensors), to predict key quality parameters of olive oils based on single-run assays. The multivariate linear models established for 23 centenarian olive trees from different cultivars allowed predicting peroxide value, oxidative stability, total phenols and tocopherols contents, CIELAB scale parameters (L*, a* and b* values), as well as 11 gustatory-retronasal positive attributes (green, sweet, bitter, pungent, tomato and tomato leaves, apple, banana, cabbage, fresh herbs and dry fruits) with satisfactory accuracy (0.90±0.07 R2 0.98±0.02 for the repeated K-fold-CV procedure, which ensured that 25% of the data was used for internal-validation purposes). The electronic tongue device had an accuracy statistically similar to that achieved with standard analytical techniques, pointing out the versatility of the device for the fast and simultaneous chemical and sensory analysis of olive oil., Project POCI-01-0145-FEDER-006984 – Associate Laboratory LSRE-LCM, Project UID/BIO/04469/2013 – CEB, Project UID/QUI/50006/2013 - REQUIMTE-LAQV and strategic project PEst-OE/AGR/UI0690/2014 – CIMO all funded by European Regional Development Fund (ERDF) through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT – Fundação para a Ciência e a Tecnologia I.P., info:eu-repo/semantics/publishedVersion

Dissertação de mestrado em Biotecnologia, Os processos fermentativos realizados por microrganismos são amplamente utilizados na produção industrial de vários produtos e compostos. Existem vários processos nos quais a atividade simultânea de duas ou mais espécies de microrganismos (co-culturas) num ambiente controlado é de elevada importância para o produto final. Nestes casos, os compostos produzidos em co-cultura são diferentes daqueles produzidos em culturas puras de todos os microrganismos presentes, o que tem impacto nas propriedades sensoriais do produto final. O presente trabalho visa o estudo de co-culturas de Saccharomyces cerevisiae e Dekkera bruxellensis aplicadas no envelhecimento e acidificação acelerados de cerveja. Pretende-se conhecer a dinâmica de co-culturas, tanto a nível do crescimento microbiano das estirpes envolvidas, como ao nível da produção de metabolitos, descrevendo as interações entre as diferentes estirpes em co-cultura, sejam elas neutras, sinérgicas ou competitivas. Para tal, realizaram-se 8 ensaios fermentativos diferentes em condições de cultura pura e co-cultura, 5 dos quais para analisar o impacto no processo fermentativo da alteração da taxa de inóculo, elevada concentração de glicose, temperatura, e de etanol, e os restantes 3 foram aplicados a matrizes alimentares. Os ensaios foram acompanhados de análise dos perfis de crescimento por citometria de fluxo, perfis de produção de metabolitos maioritários por HPLC e análise de metabolitos secundários por GC-MS. Observou-se que S. cerevisiae e D. bruxellensis apresentam uma relação competitiva, sendo que em co-cultura em meio ideal, S. cerevisiae consome glicose mais rapidamente e apresenta um crescimento bastante superior a D. bruxellensis, sendo o mesmo verificado para elevadas concentrações de açúcar. Já D. bruxellensis consegue um crescimento exponencialmente superior em situação de aumento da taxa de inóculo relativamente a S. cerevisiae, sendo que na situação contrária há um equilíbrio das populações. Verificou-se ainda uma morte celular precoce por parte de ambas as leveduras com a diminuição da temperatura para 21 ℃. D. bruxellensis apresenta maior resistência ao etanol e aos compostos extraídos da madeira de barrica, demonstrando uma tolerância ao etanol até concentrações de 8-10 %. Relativamente aos compostos secundários, verifica-se essencialmente a produção de ésteres, álcoois e ácidos orgânicos pelas leveduras, mas apenas D. bruxellensis é responsável pela produção dos etilfenóis 4-etilguaicol e 4-etilfenol, apresentado as maiores concentrações destes metabolitos nos ensaios de refermentação de produtos cervejeiros acabados., Fermentative processes performed by microorganisms are widely used in several compound’s industrial production. There are currently several food processes in which the simultaneous activity of two or more microorganisms (co-cultures), in a controlled manner, is crucial for the end product. In these cases, the compounds produced in co-culture are different from those produced in pure cultures of different microorganism populations, which will have an impact on the sensory characteristics of the final product. This project aims to study co-cultures of Saccharomyces cerevisiae and Dekkera bruxellensis applied to accelerated ageing and souring of beer. It is intended to understand the dynamic of co-cultures, both in the growth rate of all the species involved as well as the profile of metabolites produced, and to describe the existent interactions between them, whether neutral, synergic or competitive. This knowledge is to be accompanied by a compilation of analysis’ methodologies usually used in the study of co-culture fermentations´ dynamics. With this purpose in mind, 8 fermentation tests were assayed, 5 of which were conducted in synthetic medium intended to show the impact of different pitching rates, high glucose concentration, temperature and ethanol in the fermentation process, and the last 3 were applied to real matrices. The different assays were analyzed by flow cytometry to determine the growth profile, HPLC to determine the profile of production of primary metabolites and GC-MS to determine the secondary metabolite profile. It was concluded that S. cerevisiae and D. bruxellensis compete for glucose, being that in synthetic co-culture medium, S. cerevisiae consumes glucose and grows much faster than D. bruxellensis, even in high glucose media. D. bruxellensis outcompetes S. cerevisiae when in superior cell concentration at the start of the fermentation. On the other hand, when a bigger pitching rate benefits S. cerevisiae an equilibrium is reached between both populations. It was also verified early cell death by both yeast when there was a decrease in temperature to 21℃. D. bruxellensis shows greater tolerance to wood compounds and ethanol, demonstrating growth up to 8-10 % ethanol in extraction medium. Regarding secondary compounds, it was verified the production of mainly esters, alcohols and organic acids by both yeasts, but only D. bruxellensis shows production capability of ehtylphenol compounds such as 4-ethylguaicol and 4-ethylphenol, showing higher production in the refermentation of beer products.

Dissertação de mestrado em Biotecnologia, Society growing awareness of excessive sugar consumption impact in health has led to an increased attention towards several sugar substitutes. Despite the existing alternatives in the market, D-tagatose possesses unique features. The enzymatic isomerization of D-galactose has achieved more projection as it is a more environmentally friendly compared to chemical isomerization. On the other hand, the search of renewable sources of D-galactose is necessary to attain a sustainable growth. Nevertheless, D-galactose production from renewable resources requires a pre-treatment that includes a hydrolysis of polysaccharides or disaccharides to obtain monomer sugars. Thus, the main objective of this thesis was the production of D-tagatose from several renewable resources by enzymatic isomerization of D-galactose using a L-arabinose isomerase from Bacillus subtilis (BSAI). For that, production and purification of BSAI enzyme was performed. Moreover, Gelidium amansii, κ-carrageenan and cheese whey were evaluated as sources of D-galactose by acid and enzymatic hydrolysis of complex sugars. BSAI was produced and purified with a yield of 34 mg/g cell. Moreover, isomerase activity for D-galactose substrate was verified by HPLC (High performance liquid chromatography). The BSAI enzyme was used for D-tagatose production from D-galactose obtained from several raw materials. Regards, red seaweed pre-treatment for D-galactose production, 12% (w/w) of biomass using a ratio of 0.125 g of sulphuric acid/g at 150 ºC for 10 min was suitable for the extraction of galactan as galactose achieving a concentration of 22.28 g/L. On other hand, optimization of hydrolysis of κ-carrageenan was performed by an experimental design. Under selected conditions (sulphuric acid concentration of 2.25% (w/w) and a treatment time of 45 minutes), percentage of hydrocolloid was increased to 8% (w/w) in order to obtain a liquor with approximately 20 g/L of D-galactose. Finally, cheese whey powder was enzymatically hydrolysed using a lactase (5UI/g of lactose) achieving 26.67 g/L of D-galactose. For liquors from Gelidium amansii there are no significant differences in the conversion between the use of detoxified and non-detoxified liquors. So, HMF did not inhibited the action of the BSAI enzyme. For the same BSAI concentration (7 mg/mL) conversions of 50.9, 52.0, 55.6 and 27.8% were obtained in the isomerization assays with Gelidium amansii detoxified and non-detoxified liquor, cheese whey hydrolysate and κ-carrageenan hydrolysate, respectively. The results obtained are very similar to those obtained in the group previously with pure galactose solutions. Thus, the use of liquors instead of pure solutions does not affect BSAI enzyme activity., crescente consciencialização da sociedade sobre o impacto do consumo excessivo de açúcar na saúde levou a uma atenção crescente para vários substitutos do açúcar. Apesar das alternativas existentes no mercado, D-tagatose possui características únicas. A isomerização enzimática de D-galactose alcançou mais projeção, uma vez que é menos prejudicial para o ambiente quando comparada com a isomerização química. Por outro lado, a busca de fontes renováveis de D-galactose é necessária para alcançar um crescimento sustentável. No entanto, a produção de D-galactose a partir de recursos renováveis requer um pré-tratamento que resulte na hidrólise de polissacarídeos ou dissacarídeos para obter monómeros de açúcares. Assim, o principal objetivo desta tese foi a produção de D-tagatose a partir de vários recursos renováveis através da isomerização enzimática de D-galactose, utilizando a L-arabinose isomerase de Bacillus subtilis (BSAI). Para isso, foi realizada a produção e purificação da enzima BSAI. Além disso, Gelidium amansii, κ-carragenano e o soro de queijo foram avaliados como fontes de D-galactose por hidrólise ácida e enzimática de açúcares complexos. A enzima BSAI foi produzida e purificada com um rendimento de 34 mg/g de células e a sua atividade para o substrato D-galactose foi verificada por HPLC (High performance liquid chromatography). A enzima BSAI foi utilizada para a produção de D-tagatose a partir de D-galactose obtida a partir de várias matérias primas. O pré-tratamento de Gelidium amansii foi adequado atingindo uma concentração de 22,28 g/L de D-galactose, com 12% (p/p) de biomassa utilizando uma proporção de 0,125 g de ácido sulfúrico/g a 150 ºC durante 10 min. Por outro lado, a otimização da hidrólise do κ-carragenano foi realizada por um desenho experimental. Sob condições selecionadas (concentração de ácido sulfúrico de 2,25% (p/p) e tempo de tratamento de 45 minutos), a percentagem do sólido foi aumentada para 8% (p/p), a fim de obter um licor com aproximadamente 20 g/L de D-galactose. Finalmente, o soro de queijo foi hidrolisado enzimaticamente usando uma lactase (5UI/g de lactose) atingindo 26,67 g/L de D-galactose. Utilizando os licores de Gelidium amansii, destoxificado e o não destoxificado, não se verificaram diferenças significativas na conversão. Assim, o HMF não inibiu a ação da enzima BSAI. Para a mesma concentração de BSAI (7 mg/mL), conversões de 50,9; 52,0; 55,6 e 27,8% foram obtidas nos ensaios de isomerização com o licor destoxificado e não destoxificado (Gelidium amansii), o hidrolisado do soro de queijo e o hidrolisado de κ-carragenano, respetivamente. Os resultados obtidos são muito semelhantes aos obtidos no grupo anteriormente com soluções puras de D-galactose. Assim, o uso de licores em vez de soluções puras não afeta a atividade da enzima BSAI.

Polymyxin B (PMB), a member of polypeptide antibiotics, is widely used for the treatment of infection in animals such as cattle, sheep, pigs, and chickens. However, it is toxic on the kidneys and nervous system, and polymyxin resistance is increasingly reported, which leaves a serious threat to human health. Therefore, it is essential to establish rapid methods for detecting PMB with high sensitivity and specificity. In this study, an anti-PMB polyclonal antibody (pAb) was obtained by immunizing New Zealand white rabbits with PMB conjugated with glycosylated bovine serum albumin (GBSA). Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) were developed. Under the optimal conditions, inhibitory concentrations (IC50) of PMB were 257.1 ng/mL (ic-ELISA) and 250.8 ng/mL (ic-CLEIA); the limits of detection (LOD) were 17.4 ng/mL (ic-ELISA) and 14.5 ng/mL (ic-CLEIA), respectively. Cross-reactivity of the pAb toward polymyxin E (PME) was 257.1%, and no response was found with other antibiotics. The recovery rates in spiked meat samples were 77.4 similar to 106.1% (ic-ELISA) and 84.1 similar to 107.1% (ic-CLEIA), respectively.

Dissertação de mestrado em Biotecnologia, The increased consumer awareness of the importance of a healthy lifestyle to prevent diseases and maintain the well-being led to the advance of the functional foods and beverages market, that exhibits an expected revenue of €252.079 million worldwide by 2025. Functional food is described as food consumed as part of the daily diet but has demonstrated physiological benefits beyond basic nutrition. Health and well-being improvement via gut microbiota modulation is an attractive approach to shift composition and metabolic signatures of the microbiota, namely through the ingestion of prebiotics. Prebiotics include non-digestible carbohydrates and non-carbohydrates with proven ability to modulate microbial populations, such as the gut microbiota. Fructooligosaccharides (FOS) and galactooligosaccharides (GOS) are very well-studied prebiotic oligosaccharides with proven health benefits in humans. These compounds can be added to foods and beverages due to their nutritional value and technological properties, being used for instance to improve the mouthfeel, to replace sugar and fat content and to increase the fibre content. In this work, the prebiotic potential of commercial FOS and GOS, individually and in mixtures (GF), was studied using an in vitro model of static batch culture fermentations with human faecal inoculum (48h at 37oC). The production of lactate and short-chain fatty acids (SCFAs), ammonia and gases was evaluated, as well as the pH variation. The fermentation of GOS led to the highest production of SCFAs, namely acetate and propionate, 125.10±19.62 mM and 18.09±5.08 mM, respectively. PH values and ammonia concentrations decreased with the supplementation of prebiotics, with the mixture GF 3:1 yielding the major ammonia reduction, namely 9- fold. Overall, the production of CO2 increased with the addition of all prebiotics, while the CH4 production decreased with GOS supplementation. In addition, a healthier traditional Portuguese rice cake containing GOS and GF 3:1, and a 25% sugar reduction, was herein developed. The sensory test results revealed no significant differences in appearance, aroma, taste, texture and overall impression between the original recipe and the 3 modified recipes tested. However, based on market acceptability, recipes 4 (25% sugar reduction) and 5 (25% sugar reduction and 1% GOS addition) were selected as the most accepted ones., O aumento da consciência dos consumidores sobre a importância de um estilo de vida saudável na prevenção de doenças e na manutenção do bem-estar, levou à expansão do mercado de alimentos e bebidas funcionais, que apresenta uma receita mundial expectável de €252.079 milhões até 2025. Alimentos funcionais são alimentos consumidos numa dieta diária normal, mas que apresentam benefícios fisiológicos comprovados além da nutrição convencional. Melhoria da saúde e do bem-estar através da modulação da microbiota intestinal é uma estratégia em crescimento para alterar a composição e assinatura metabólica da mesma, que pode ser obtida pela ingestão de prebióticos. Os prebióticos incluem hidratos de carbono não digeríveis e outros compostos que não são carboidratos. Fruto-oligossacáridos (FOS) e galato-oligossacáridos (GOS), são oligossacáridos prebióticos muito bem estudados com benefícios provados para a saúde humana. Estes substratos podem ser adicionados a alimentos e bebidas funcionais pelo seu valor nutricional e pelas suas propriedades tecnológicas para, por exemplo, melhorar a textura de produtos, substituir o teor de açúcar e gordura e aumentar o teor de fibra. Neste trabalho foi estudado o potencial prebiótico de FOS e GOS, individualmente e em misturas (GF), através de um modelo in vitro de fermentações descontínuas inoculadas com microbiota fecal humana (48h a 37oC). Foi avaliada a produção de lactato e ácidos gordos de cadeia curta (SCFAs), amónia e gases, assim como a variação de pH. A fermentação com GOS resultou na maior produção de SCFAs, nomeadamente de acetato e propionato com 125.10±19.62 mM e 18.09±5.08 mM, respetivamente. A concentração de amónia e os valores de pH diminuíram como resultado da adição de prebióticos, com a mistura GF na proporção 3:1 a resultar na maior redução de amónia, nomeadamente 9 vezes. De um modo geral, a suplementação com todos os prebióticos causou um aumento na produção de CO2, enquanto que a produção de CH4 diminui como resultado da suplementação com GOS. Adicionalmente, desenvolveu-se a receita de um bolo de arroz tradicional mais saudável com a incorporação de GOS e GF 3:1 e com uma redução de açúcar de 25%. Os testes sensoriais não revelaram diferenças significativas entre a receita tradicional e as 3 receitas modificadas nos parâmetros de aparência aroma, sabor, textura e impressão global. No entanto, a aceitabilidade do mercado sugere que as receitas 4 (redução de 25% de açúcar) e 5 (redução de 25% de açúcar e adição de 1% de GOS) são as mais aceites., Este estudo recebeu financiamento do projeto NewFood (NORTE-01-0246-FEDER-000043)

Dissertação de mestrado em Biotecnologia, Atualmente, há um crescente interesse no uso de recursos naturais como fonte de compostos bioativos, com potenciais benefícios para a saúde. Desde a antiguidade que é recorrente o uso plantas ao nível da medicina tradicional, nomeadamente a flor de sabugueiro (Sambucus nigra L.). Recentemente, esta planta tem sido a base de vários produtos nutracêuticos, contudo, ainda não existem evidências científicas claras que fundamentem a sua bioatividade, bem como os mecanismos celulares associados. De acordo com a literatura, os extratos de sabugueiro são constituídos sobretudo por compostos terpénicos voláteis, triterpénicos, esteróis, e compostos fenólicos. É, contudo, importante salientar que a composição de cada uma destas famílias é dependente do método de extração (solvente, temperatura e tempo). O principal objetivo deste estudo foi a caracterização química de um extrato aquoso de flor de sabugueiro e a sua validação como agente anticancerígeno. A extração aquosa da flor de Sabugueiro foi realizada a diferentes temperaturas (50, 70 e 90 ºC). O extrato aquoso obtido a 90 ºC apresentou a atividade anti-radicalar mais elevada (0.157 ± 0.001 mmol TE g-1) e uma concentração de compostos fenólicos mais elevada (45.32 ± 2.20 mg CAE g-1). As análises qualitativas e quantitativas de GC-MS e HPLC-MS do extrato aquoso de flor de sabugueiro permitiram identificar 46 compostos, sendo a quercetina e o ácido clorogénico representativos de 86 % da totalidade dos compostos fenólicos identificados na fração hidrofílica, e a naringenina (27.16 %) o composto maioritário na fração lipofílica. A atividade epigenética do extrato da flor de sabugueiro na desmetilação do MLH1 foi testada em combinação com o 5-fluorouracil em células do cancro colorretal (RKO), usando a 5-azacitidina como controlo positivo. Os resultados sugerem que a combinação do extrato de sabugueiro com o 5-FU não resulta num efeito sinergético, o que indica que o extrato não tem influência na desmetilação do MLH1. Apesar do extrato da flor sabugueiro ter diversas moléculas com capacidade antioxidante, o extrato não demonstrou proteção ou indução de reparação de danos do DNA após um insulto oxidativo (H2O2). Ao nível da atividade antimicrobiana, verificou-se que bactérias gram-positivas são mais suscetíveis à presença deste extrato, nomeadamente, Staphylococcus aureus clinicamente isolado e Staphylococcus epidermidis. Em conclusão, verificou-se que o extrato de flor de sabugueiro não possui capacidade de modular atividade epigénica e estimular a proteção/reparação de danos oxidativos. Ao invés, verificou-se que o extrato possui atividade microbiana, nomeadamente contra bactérias gram-positivas., Nowadays, there is a growing interest on the use of natural resources as sources of bioactive compounds with potential health benefits. Since the antiquity, that is recurrent the use of plants in folk medicine, such as the elderflower (Sambucus nigra L.). Recently, this plant has been the basis of several nutraceutical products. Thus, there is not a clear scientific explanation for its bioactivity, as well as the responsible cellular mechanisms. According to the literature, elderflower extracts are mainly composed by volatile terpenic, triterpenic, sterols and phenolic compounds. However, the composition of each one of this families will be dependent of extraction method (solvent, temperature, time). The main gold of this study was the chemical characterization of an aqueous elderflower extract and its validation as an anticarcinogenic agent. The aqueous extraction of elderflower was performed at different temperatures (50,70 and 90 ºC). The aqueous extract obtained at 90 ºC exhibited the highest antiradical activity (0.157 ± 0.001 mmol TE g-1) and the highest concentration of phenolic compounds (45.32 ± 2.20 mg CAE g-1). The qualitative and quantitative analysis of GC-MS and HPLC-MS of elderflower aqueous extract allowed the identification of 46 compounds, being quercetin and chlorogenic acid representative of 86 % of the total of phenolic compounds identified in hydrophilic fraction, and naringenin (27.16 %), the major compound in lipophilic fraction. The epigenetic activity of elderflower aqueous extract on demethylation of MLH1 was tested in combination with 5-fluorouracil (5-FU) in colorectal cancer cells (RKO), using 5-azacytidine (5-azaC) as a positive control. The results suggest that the combination of elderflower extract with 5-FU did not result in a synergetic effect, which indicates that elderflower extract did not have an influence on MLH1 demethylation. Even though, the elderflower extract has several molecules with antioxidant potential, the extract did not exhibit protection or induction of repair of DNA damage after exposure to oxidative stress (H2O2). The antimicrobial activity of elderflower extract was evaluated, and it was verified that gram-positive bacteria were the most susceptible to the presence of this extract, namely, Staphylococcus aureus clinically isolated and Staphylococcus epidermidis. In conclusion, it was verified that S. nigra extract does not have the ability to modulate epigenetic activity and stimulate DNA protection/repair against oxidative stress. Although, this extract exhibits antimicrobial activity, particularly on gram-positive bacteria.

Viscosity-modified diet through thickeners is used as a strategy to circumvent swallowing problems by oropharyngeal dysphagia patients. Most commercial products present xanthan and starch in their formulations, but flaxseed gum (FG) is a potential thickener for liquid food that provides additional health benefits. FG was mixed either with modified starch (MS) and/or xanthan gum (XG), varying biopolymersâ concentration according to a central composite rotational design in which rheological and colour properties in water were the evaluated responses. All formulations showed a shear time-independent and shear-thinning behaviour, mainly influenced by XG and MS concentrations. In oscillatory measurements, the formulations presented a prevailing elastic character attributed to MS and mainly to XG, which despite the lower concentration in which it was incorporated, exerted a similar influence on this rheological property. However, the increase of FG concentration was the most significant factor influencing viscosity, but also favoured an increase of both viscoelastic moduli mainly G'. Analysis of the microstructure disclosed different network structures as a result of biopolymers interactions, which was related to rheological behaviour giving insights to design new thickeners for dysphagia management. In addition, the amount of glucose released after in vitro digestion was evaluated and compared to a commercial MS-based thickener. Interestingly, the commercial formulation showed a glucose release significantly higher than the proposed FG/MS/XG-based formulations. These results open the opportunity to tailor the rheological characteristics of food systems by adding and combining natural ingredients, improving technological and nutritional properties., Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (Finance code 001); São Paulo Research Foundation - FAPESP (Process numbers 2016/05448–8; 2011/51707–1; EMU 2009/54137–1; 2007/58017–5; 2006/03263–9; 2004/08517–3) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Process 307168/2016–6). Okuro and Santos thanks São Paulo Research Foundation (FAPESP) (Grant numbers 2018/20308–3 and 2017/18109–0), info:eu-repo/semantics/publishedVersion

The effect of thermal sterilization processes on the physicochemical parameters and sensory characteristics of Californian-style black olives, from Hojiblanca and Manzanilla Cacereña brine solutions, were evaluated. Two-way ANOVA showed that Hojiblanca olives had a lower phenols content and defect intensity and that increasing the sterilization period resulted in a decrease in total phenols and an increase of the cooked defect. The impact of thermal sterilization was further evaluated using a potentiometric electronic tongue, which was capable of discriminating the different sterilization treatments (repeated K-fold cross-validation sensitivity: 89.0?±?15.0% to 97.0?±?6.0%). Moreover, multiple regressions allowed the prediction of phenols (R2???0.95?±?0.03) and the intensity of the defect (R2???0.95?±?0.04). These results point out the feasibility of the electronic tongue as an analytical tool for monitoring the effects of thermal sterilization treatments. Furthermore, the satisfactory results obtained for the brine solutions may foresee a practical non-destructive method for indirect quality assessment of table olives., Consejería de Educación y Empleo of the Junta de Extremadura to work with a scholarship at the Polytechnic Institute of Bragança for 4 months (Resolución de 18 de julio de 2019, DOE de 25 de julio de 2019, no 143) (Expedient number A28). Nuno Rodrigues thanks to National funding by FCT- Foundation for Science and Technology, P.I., through the institutional scientific employment program-contract. The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER, under Programme PT2020, for the financial support of CIMO (UID/AGR/00690/2019) and of CEB (UID/BIO/04469/2019) and to BioTecNorte operation (NORTE-01-0145-FEDER-000004), info:eu-repo/semantics/publishedVersion

Lactosucrose is a synthetic trisaccharide composed of galactose, glucose and fructose. This compound is obtained through enzymatic synthesis using lactose and sucrose as substrates. The enzymes involved in the process are able to catalyse both hydrolysis and transfer reactions. The yield and productivity of the process are usually affected by the occurrence of parallel hydrolysis of the newly formed product (lactosucrose). Therefore, it is important to find efficient strategies to avoid or minimize product degradation. Furthermore, in the last decades the demand for lactosucrose has significantly increased. This compound is considered a potential prebiotic and several beneficial effects associated to its consumption have been described. As a result, it has been included in the formulations of functional foods. This review covers the most relevant information about lactosucrose, including its synthesis and purification, beneficial effects at physiological level, and also its potential applications., Fundação para a Ciência e a Tecnologia (FCT) Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the project “BioInd – Biotechnology and Bioengineering for im- proved Industrial and Agro-Food processes”, REF. NORTE-07- 0124-FEDER-000028 co-funded by the Programa Operacional Regional do Norte (ON. 2–ONovo Norte), QREN, FEDER.

A new hybrid bionanomaterial composed of zinc oxide nanoparticles (ZnO NPs) and chitosan was constructed after enzymatic immobilization of papain for biomedical applications. In this work, we report the preparation and characterization steps of this bionanomaterial and its biocompatibility in vitro. The properties of the immobilized papain system were investigated by transmission electron microscopy, zeta potential, DLS, UV-vis absorption spectroscopy, FTIR spectroscopy, and X-ray diffraction. The prepared bionanomaterial exhibited a nanotriangular structure with a size of 150 nm and maintained the proteolytic activity of papain. In vitro analyses demonstrated that the immobilized papain system decreased the activation of phagocytic cells but did not induce toxicity. Based on the results obtained, we suggest that the novel bionanomaterial has great potential in biomedical applications in diseases such as psoriasis and wounds., Financial support from CNPq (310678/2014-5), FAPEPI and CAPES (Rede nBioNet) is gratefully acknowledged

This work reports on a large-scale potential neuropeptide activity screening in human gut microbiomes deposited in public databases. In our experimental approach, the sequences of the bioactive peptides collected in the MAHMI database, mainly predicted as immunomodulatory or antitumoral, were crossed with those of the neuroactive/digestive peptides. From 91,325,790 potential bioactive peptides, only 581 returned a match when crossed against the 5949 neuroactive peptides from the NeuroPep database and the 15 digestive hormones. Relevant bacterial taxa, such as Ruminococcus sp., Clostridium sp. were found among the main producers of the matching sequences, and many of the matches corresponded to adiponectin and the hormone produced by adipocites, which is involved in glucose homeostasis. These results show, for the first time, the presence of potentially bioactive peptides produced by gut microbiota members over the nervous cells, most notably, peptides with already predicted immunomodulatory or anti-inflammatory activity. Classical (Lactobacillus sp.) and next-generation (Faecalibacterium sp.) probiotics are shown to produce these peptides, which are proposed as a potential mechanism of action of psychobiotics. Our previous experimental results showed that many of these peptides were active when incubated with immune cells, such as dendritic cells, so their effect over the nervous system innervating the gut mucosa holds significant potential and should be explored., This work was supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (Grant AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales”, grant PS-2016). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). SING group thanks CITI (Centro de Investigación, Transferencia e Innovación) from University of Vigo for hosting its IT infrastructure. This work was partially supported by the Asturias Regional Plan I+D+i for research groups (FYCYT-IDI/2018/000236) and by the Consellería de Educación, Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group.