Microencapsulation of probiotic bacteria

This manuscript describes some general reflections on the research around the microencapsulation and its application in the preparation of stable consortia of probiotic bacteria. Its predominant application focuses on the agro-food, nutraceutical and pharmaceutical industry but despite the highest industrial interest, the wide diversity of applications and the existence of a broad and diversified consumer market yet to be explored, there is still a quite long way to go to improve the bioactivity, functionality and applicability of these innovative (bio) technological products

Optimization of the production of fucoxanthin-rich fractions from the microalga Isochrysis galbana

As microalgas são organismos microscópicos uni- ou multicelulares com a habilidade de converterem energia solar em energia química num processo conhecido por fotossíntese. Estes seres vivos autotróficos sintetizam ácidos gordos polinsaturados (PUFA) como os acidos eicosapentoenoico (EPA) ou docosahexaenoico (DHA) conhecidos pelas suas aplicações terapêuticas em ensaios clínicos como agentes antioxidantes, anticancerígenos ou no combate à obesidade. Apesar dos benefícios conhecidos do EPA e DHA os seres humanos e outros vertebrados como os peixes não têm capacidade para os sintetizarem e por isso existe uma grande necessidade de encontrar novas fontes sustentáveis destes compostos de maneira a aliviar a pressão sobre os stocks de pesca continuando a dar resposta a um mercado em crescimento. As microalgas utilizam a sua pigmentação não só como forma de capturarem a luz solar, mas também, como uma ferramenta no combate ao stress oxidativo. O pigmento em destaque nesta tese é a fucoxantina, uma xantofila com capacidade para desempenhar as duas funções acima descritas dependendo da espécie de microalga. As bioactividades associadas à fucoxantina fazem com que seja alvo de inúmeros ensaios clínicos demonstrando ser não só um poderoso agente antioxidante bem como um promissor agente no combate a inflamações, diabetes e obesidade, tendo por isso tem um valor de mercado acrescido uma vez que pode ser utilizado em diversas áreas com implicações muito revelantes na saúde humana. A variabilidade da biomassa de microalgas atraiu nos últimos anos inúmeras indústrias que vêm nestes seres vivos a solução para diversos problemas como: a redução de CO2 atmosférico; uma fonte sustentável de biodiesel devido ao alto nível lipídico de algumas espécies; uma nova fonte promissora de proteína ou o potencial para isolar novas moléculas com relevância nas áreas das ciências biomédicas, nas indústrias cosméticas ou farmacêuticas. Na última década a União Europeia tem reunido esforços e financiado projectos nas mais diversas áreas de caracterização de microalgas sendo promotora da aprovação das espécies Chlorella e Tetraselmis para consumo humano. O desafio prende-se agora com o estabelecimento de protocolos universais que permitam aos diversos países da UE comparar resultados e criar novos consensos levando à regulamentação de cada sector e de um mercado global em crescimento. Assim sendo, a microalga haptofita Isochrysis galbana foi escolhida com base no seu perfil bioquímico como uma fonte promissora para fucoxantina, um carotenoide com elevada actividade antioxidante e DHA. Nas instalações da Necton, os ALGEM® environmental labscale photobioreactors foram usados para averiguar quais as melhores condições de crescimento celular da cultura bem como de acumulação de fucoxantina e DHA. Estes reactores permitem submeter as culturas às condições abióticas desejáveis tendo a capacidade de simular: fotoperíodo, intensidade luminosa (LED), temperatura e agitação. Os ensaios nos reactores ALGEM® revelaram que a estação do ano com maior acumulação de fucoxantina foi o Outono (6.4 ± 0.1 mg/g peso seco) e que o pH mais propicio para a bioacumulação deste carotenoide é pH 7.5 (7.88 ± 0.05 mg/g peso seco). O perfil de ácidos gordos realizado no GC-MS identificou a estação do ano Outono e o pH 8.5 como aqueles que produziram maiores níveis de DHA com 11.9 % e 16.7 % do total de ácidos gordos, respectivamente. A biomassa produzida nos reactores tubulares da Necton continha 17.69 ± 1.06 mg/g peso seco de fucoxantina e foi usada para identificar o melhor método de extracção testando as condições: rácio de biomassa/solvente e tempo de extracção. Os resultados identificaram a proporção 1/2 (10g biomassa humida/ 20 mL etanol) com 1 hora de agitação como a melhor para extrair fucoxantina. Com base neste resultado, vários extractos foram preparados para testar a extracção líquido-líquido (LTPS) na recolha de fucoxantina e DHA. Esta nova técnica permite o fracionamento de um extracto de microalgas separando compostos de elevado valor como o DHA, a fucoxantina e outros ácidos gordos, por isso a eficácia da técnica foi averiguada testando diferentes proporções de hexano e água (H/W: 1/2, 1/1, 2/1) e quantificando a quantidade de fucoxantina e DHA em cada sistema. O LTPS demonstrou ser uma técnica apropriada para a extração destes compostos, com o ratio 2/1 (160/80, H/W) a extrair mais fucoxantina, enquanto a maior quantidade de DHA foi recolhida no sistema 1/1 (120/120, H/W). Por estas razões, conclui-se que o sistema de solventes mais promissor para o LTPS foi o ratio 1/1 (H/W) assumindo a combinação de critérios: quantidade de fucoxantina; quantidade de DHA; peso da fração coloidal. Idealmente, o melhor sistema de solventes será aquele que conseguirá extrair a maior quantidade de fucoxantina sem comprometer significativamente a quantidade de DHA e de fase coloidal. Novos ensaios serão preparados para testar esta e outras condições capazes de influenciar a dinâmica das emulsões.Microalgae are microscopic unicellular and multicellular organisms capable of harvesting sunlight and convert it to chemical energy in a process known as photosynthesis. These photoautotrophs synthesize PUFA, including eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids known for their therapeutic applications such as antioxidant, anti-cancer or anti-obesity effects. Despite the benefits of these molecules, humans and other vertebrates like fish lack the metabolic pathways to be able to biosynthesize them, therefore, there is an urgent need to find new sustainable sources for antioxidant agents, DHA and other essential PUFAs. The haptophyte Isochrysis galbana was chosen based on its biochemical profile, as a promising new feedstock for fucoxanthin and DHA. In Necton facilities, the ALGEM® environmental labscale photobioreactors were used to assess the ideal abiotic conditions for the cultivation of I.galbana and its intracellular accumulation of fuoxanthin and DHA. The ALGEM® annual variation trial revealed a significant higher bioaccumulation of fucoxanthin during Autumn season while the pH trial identified the highest value of fucoxanthin at pH 7.5 (7.88 mg/g DW). The fatty acid profile analysis of each Algem® trial identified Autumn season and pH 8.5 as the ones accumulating more DHA, 11.9 % TFA and 16.7 % TFA, respectively. The biomass from the tubular photobioreactors contained 17.69 ± 1.06 mg/g DW of fucoxanthin and it was used to identify the best extraction conditions focusing on: biomass/solvent ratio and time of extraction. The results identified the proportion 1/2 (10g WB/ 20 mL ethanol) at 1 hour mixing as the best for the extraction of fucoxanthin. Several extracts were prepared in this way to test the efficiency of the LTPS in extracting fucoxanthin as well as DHA. This novel liquid-liquid extraction system allows to fractionate a microalgae extract effectively separating high-value compounds such as fucoxanthin, DHA and other PUFA, therefore, the efficiency of LTPS was assessed by testing different solvent systems using hexane and water (H/W: 1/2, 1/1, 2/1) and quantifying the amount of DHA and fucoxanthin in each fraction of all solvent systems. The LTPS revealed to be a suitable technique with the solvent system 2/1 (160,80 H/W) being the one that extracted more fucoxanthin, while the majority of DHA was collected in the ratio 1/1 (120/120, H/W). For these reasons, the best LTPS solvent system was selected based on a combination of the following criteria: quantity of fucoxanthin; amount of DHA and yield of colloidal fraction. The most promising solvent system was considered to be the ratio 1/1 (H/W). Despite that, new trials are being study aiming to determine a solvent proportion that extracts the highest possible amount of fucoxanthin without significantly compromise the DHA content and the amount of colloidal fraction. Although more trials should be performed, this work revealed to be an important step towards the confirmation of the LTPS technique as a promising protocol for a biorefinery concept

Analysis of 140 published GSMs and identification of the most common representation problems

The number of publications related to GSMs is increasing exponentially, but as most of these models are scattered across the Internet there is a need to centralize these data in a way that users can easily access and load them into stoichiometric modelling tools. This work presents a web platform to collect scientific work related with the reconstruction of GSMs, providing links to the original publications and the available models (www.optflux.org/models). The platform also indicates which models are compatible with OptFlux, an open-source reference computational platform for the optimization of cellular factories by the application of in silico ME methods, designed for non-computational experts by providing a user-friendly interface. The compatible models can be automatically loaded into OptFlux via a repository manager. This work also presents a thorough analysis on more than 140 published GSMs available in the platform. This analysis highlights some common problems in published models, such as the lack of standards to represent them. The SBML format has been adopted as the main standard by the community, despite some limitations in representing all the information required for modelling purposes. As consequence, this format has been extended ad-hoc by several authors, thus making its automatic interpretation a non-trivial problem. This analysis provides some insight into the limitations of formats used and the recurrent problems in the representation of GSMs

Development of deep learning approaches to predict relationships between chemical structures and sweetness

The non-caloric sweeteners market is catching up with the market of conventionally used sugars due to the benefits of preventing obesity, tooth decay and other health problems. Developing strategies for designing easier-to-produce novel molecules with a sweet taste and less toxicity are up-to-date motivations for the food industry. In this sense, Machine Learning (ML) approaches have been reported as cutting-edge technologies to guide the design of new molecules towards specific objectives, including sweet taste. The largest known dataset of sweet molecules is here provided. The dataset contains fully integrated 9541 sweeteners and 1141 bitterants from FooDB, FlavorDB and literature. This robust dataset allowed the development of standard Machine and Deep Learning pipelines towards conceiving Structure-Activity Relationships (SAR) between molecules and sweetness. In this work, we showcase that Textual Convolutional Neural Networks (TextCNN), Graph Convolutional Networks (GCN), and Deep Neural Networks (DNNs) outperformed most of traditional shallow learning approaches. These Deep Learning (DL) models produced platforms to guide the design of new sweeteners and repurposing existing compounds. Sixty million compounds from PubChem were evaluated using these models. Herein, we deliver a dataset of 67724 compounds that present high probabilities of being sweet. Quick searches in literature allowed us to find 13 molecules reported as potent sweetening agents, revealing that our approach is suitable for finding new sweeteners, valuable to expand food chemistry databases, repurposing existing chemicals and designing novel molecules with a sweet taste.info:eu-repo/semantics/publishedVersio

Quality improvement of traditional dry fermented sausages based on innovative technological strategies

Dry fermented sausages are highly appreciated food specialties, mainly in Portugal and other southern European countries. Therefore, all research efforts aiming at improving the food quality and safety of traditional dry sausages are of interest, since they are likely to result in products with higher added value and quality standards most suited to the requirements and concerns of the modern consumers. Among those efforts, it may be highlighted the studies involving innovative processing parameters and technologies to overcome practical problems gathered in the meat industry, which are mostly associated with food quality and safety. Additionally, characterization of traditional dry sausages and rationalization of their processing are essential for further achievement of any official certification. Thus, this article attempts to point out some research lines of highest interest in meat science (and particularly to the broad variety of regional dry fermented sausages), towards to the valorisation of technological, nutritional and commercial features. In addition, it is here emphasized the importance for the continuous improvement of the quality and safety of meat products as a way to respond to the current concerns regarding its consumption and the general advices in reducing its daily intake

Combining evolutionary algorithms with reaction rules towards focused molecular design

Designing novel small molecules with desirable properties and feasible synthesis continues to pose a significant challenge in drug discovery, particularly in the realm of natural products. Reaction-based gradient-free methods are promising approaches for designing new molecules as they ensure synthetic feasibility and provide potential synthesis paths. However, it is important to note that the novelty and diversity of the generated molecules highly depend on the availability of comprehensive reaction templates. To address this challenge, we introduce ReactEA, a new open-source evolutionary framework for computer-aided drug discovery that solely utilizes biochemical reaction rules. ReactEA optimizes molecular properties using a comprehensive set of 22,949 reaction rules, ensuring chemical validity and synthetic feasibility. ReactEA is versatile, as it can virtually optimize any objective function and track potential synthetic routes during the optimization process. To demonstrate its effectiveness, we apply ReactEA to various case studies, including the design of novel drug-like molecules and the optimization of pre-existing ligands. The results show that ReactEA consistently generates novel molecules with improved properties and reasonable synthetic routes, even for complex tasks such as improving binding affinity against the PARP1 enzyme when compared to existing inhibitors.Centre of Biological Engineering (CEB, University of Minho) for financial and equipment support. Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and through a Ph.D. scholarship awarded to João Correia (SFRH/BD/144314/2019). European Commission through the project SHIKIFACTORY100 - Modular cell factories for the production of 100 compounds from the shikimate pathway (Reference 814408).info:eu-repo/semantics/publishedVersio

Behavior of the complex micro-ecology in maize and rye flour and mother-dough for broa throughout storage

Besides water, maize and rye flour are the main constituents of broa a unique sourdough bread, manufactured following traditional protocols at the farm level in Portugal. Mother-dough, i.e., a piece of leavened dough kept aside from batch to batch under refrigeration conditions, constitutes the only starter culture used throughout breadmaking. Maize and rye flour, as well as mother-dough, were accordingly assayed for their microbiological profiles throughout storage time, to characterize the evolution in viability of the adventitious microorganisms: total viable counts, as well as viable yeasts, molds, gram-negative rods, gram-positive rods (endospore-forming and nonsporing) and gram-positive cocci (catalase+ and catalase). In general, all microbial groups exhibited an outstanding resistance to storage, so use of mother-dough appears technologically effective in this form of breadmaking, and an appropriate storage of flour does not convey any important changes to their microbiological profile.Several members of the Regional Directorate of Agriculture of Entre-Douro-e-Minho (DRAEDM, Portugal) and several local farmers are hereby gratefully acknowledged for their cooperation, on a volunteer basis, in supplying samples for analysis. Financial support for author J. M. R. was provided by a Ph.D. fellowship within the program PRAXIS XXI – ref. PRAXIS XXI/BD/16060/98, administered by Fundação para a Ciência e a Tecnologia (Portugal) and supervised by author F. X. M. Partial financial support was received within program PAMAF – IED, through research grant “Pão de milho: caracterização do processo tradicional de produção e melhoramento tecnológico” – ref. PAMAF 1022, administered by Ministério da Agricultura, Desenvolvimento Rural e Pescas (Portugal) and coordinated by author F. X. M

Electrically stimulated neural stem cells encapsulated in gellan gum hydrogel for spinal cord injury repair

Dissertação de mestrado em Biomedical Engineering, Biomaterials, Rehabilitation and BiomechanicsAs lesões da medula espinal podem ter um impacto dramático na vida de um individuo, resultando em incapacidades motoras, sensoriais, psicológicas e sociais. A patofisiologia das lesões da medula espinal é extremamente complexa com vários eventos moleculares e celulares a ocorrerem após a lesão e contribuindo para o desenvolvimento de um microambiente inibitório à regeneração tecidular, o que dificulta o desenvolvimento de terapias eficazes. Neste contexto, terapias que combinam diferentes estratégias têm a vantagem de moderar diferentes eventos críticos após este tipo de lesões. Vários tipos de células têm sido transplantadas de forma a substituir o tecido perdido após lesão. As células mais promissoras são as Células Estaminais Neuronais induzidas (iNSCs), pois permitem transplantes autologos e têm a capacidade de se diferenciarem em neurónios, astrócitos e oligodendrócitos. Para promover a sobrevivência das células transplantadas, biomateriais têm sido utilizados para funcionar como uma matriz extracelular artificial. Adicionalmente, a modulação do ambiente inibitório e por consequente o sucesso do transplante pode ser melhorado através da suplementação com secretoma de células mesenquimais estaminais (MSCs). Por fim, a estimulação eléctrica epidural tem-se revelado como uma terapia muito promissora em pessoas com lesões medulares. Neste projecto propõem-se uma terapia mais holística, que tem em conta vários eventos patofisiológicos da lesão ao invés do foco em apenas um. Desta forma, pretende-se alcançar uma terapia eficaz que combina, simultaneamente, células estaminais, biomaterias, secretoma e estimulação epidural.Spinal Cord Injuries (SCI) can have a devastating impact on an individual’s life, leadind to motor, sensory, psychological and social impairments. The SCI pathophysiology associated is extremely complex, with numerous molecular and cellular events occurring after the initial trauma and that lead to the development of an inhibitory microenvironment in the injury site, which impairs the development of new therapies. Therefore, therapies that combine different strategies have been on the rise for focusing on different critical events. For instance, several types of cells have been implanted in order to replace lost tissue after these types of lesions. Induced Neural Stem Cells (iNSCs) have been the most promising for autologous transplant and differentiation in neurons, astrocytes and oligodendrocytes. To promote the survival of transplanted cells, biomaterials have been implemented to act as an artificial extracelullar matrix. Additionally, some pathological events and, as a consequence, the success of the transplanted cells, can be enhanced using the secretome of mesenchymal stem cells (MSCs). At last, epidural electrical stimulation has revealed to be a promising therapy for SCI, enabling physical rehabilitation and promoting neural plasticity. Therefore, this project aims to combine all this approaches in order to target several pathophysiolgical events of the spinal cord injury, instead of just one. As a result, we perform in vitro studies on the combination of stem cells, biomaterials, secretome and epidural electrical stimulation in order to obtain an effective therapy