Browsing by Author "Filipe, Alexandra"
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- Cellulose-stabilized oil-in-water emulsions: structural features, microrheology, and stabilityPublication . Costa, Carolina; Rosa, Pedro; Filipe, Alexandra; Medronho, Bruno; Romano, Anabela; Liberman, Lucy; Talmon, Yeshayahu; Norgren, MagnusCellulose-based oil-in-water (O/W) emulsions were studied by diffusing wave spectroscopy (DWS) regarding the effect of the cellulose concentration and mixing rate on the average droplet size, microrheological features and stability. Furthermore, the microstructure of these emulsions was imaged by cryo-scanning electron microscopy (cryo-SEM). The micrographs showed that cellulose was effectively adsorbed at the oil-water interface, resembling a film-like shell that protected the oil droplets from coalescing. The non-adsorbed cellulose that was observed in the continuous aqueous medium, contributed to the enhancement of the viscosity of the medium, leading to an improvement in the stability of the overall system. Generally, the higher the cellulose concentration and mixing rate, the smaller the emulsion droplets formed, and the higher was their stability. The combination of both techniques, DWS and cryo-SEM, revealed a very appealing and robust methodology for the characterization and design of novel emulsion-based formulations.
- Emulsion formation and stabilization by biomolecules: the leading role of cellulosePublication . Costa, C.; Medronho, Bruno; Filipe, Alexandra; Mira, I.; Lindman, B.; Edlund, H.; Norgren, M.Emulsion stabilization by native cellulose has been mainly hampered because of its insolubility in water. Chemical modification is normally needed to obtain water-soluble cellulose derivatives. These modified celluloses have been widely used for a range of applications by the food, cosmetic, pharmaceutic, paint and construction industries. In most cases, the modified celluloses are used as rheology modifiers (thickeners) or as emulsifying agents. In the last decade, the structural features of cellulose have been revisited, with particular focus on its structural anisotropy (amphiphilicity) and the molecular interactions leading to its resistance to dissolution. The amphiphilic behavior of native cellulose is evidenced by its capacity to adsorb at the interface between oil and aqueous solvent solutions, thus being capable of stabilizing emulsions. In this overview, the fundamentals of emulsion formation and stabilization by biomolecules are briefly revisited before different aspects around the emerging role of cellulose as emulsion stabilizer are addressed in detail. Particular focus is given to systems stabilized by native cellulose, either molecularly-dissolved or not (Pickering-like effect).
- Isolation and analysis of the terpene synthases codifying sequences from the aromatic plants: thymus albicans and Thymus mastichinaPublication . Filipe, Alexandra; Marques, Natália Tomás; Cardoso, JoãoA família Lamiaceae engloba um conjunto de espécies onde se inclui o género Thymus. Plantas do género Thymus são conhecidas pelas suas propriedades aromáticas de elevado potencial comercial e económico, com várias aplicações na medicina tradicional ou como plantas aromáticas de interiores e no uso de fragrâncias em sabonetes, perfumes e detergentes. Estas características são conferidas pelos óleos essenciais produzidos por estruturas especializadas denominadas de tricomas glandulares que se encontram nos seus órgãos aéreos. Esta família apresenta dois tipos de tricomas glandulares, os tricomas peltados e os capitados, sendo os primeiros os maiores contribuintes para a síntese do óleo essencial. O óleo essencial produzido é composto por uma mistura heterogénea de metabolitos maioritariamente secundários, como alcalóides, fenóis, aldeídos e terpenos, sendo esta última a classe de compostos mais abundante. Os terpenos são a maior família de produtos naturais, com aproximadamente 20 000 compostos que se dividem em várias classes de acordo com o número de átomos de carbono que os constituem, sendo denominados de monoterpenos (C10), sesquiterpenos (C15), diterpenos (C20) e triterpenos (C30) e são sintetizados respectivamente pelas monoterpeno, sesquiterpeno, diterpeno e triterpeno sintases. Os monoterpenos são sintetizados a partir do geranil difosfato (GDP) e requerem a presença de dois metais divalentes (Mn2+ ou Mg2+) para a sua ionização em 3R- ou 3Slinalil difosfato (LDP, dependendo da posição inicial do geranil difosfato). A partir deste intermediário universal a reacção pode adoptar vários fins, podendo desta forma resultar na síntese de diferentes terpenóides. As monoterpeno sintases são caracterizadas pela presença de várias regiões conservadas, entre as quais os motivos DDXXD e (M/L)L(S/Q/N)L(F/Y)EAS que estão envolvidos na interacção com os metais divalentes e na sua actividade enzimática. O objectivo deste trabalho consistiu em isolar e caracterizar as sequências codificantes de duas monoterpeno sintases presentes em plantas do género Thymus, respectivamente Thymus albicans Hoffmans & Link quimiótipo cineol e Thymus mastichina (L.) L. spp. mastichina quimiótipo linalol. Neste estudo a região codificante das enzimas 1,8-cineol e linalol sintases, que estão envolvidas na síntese dos monoterpenos 1,8-cineol e linalol, monoterpenos predominantes nos óleos essenciais das plantas em estudo, foram isoladas, totalmente ou parcialmente e os tricomas glandulares caracterizados a nível estrutural em T. albicans. Inicialmente avaliou-se a distribuição de tricomas peltados nos órgãos aéreos de T. albicans e constatou-se que os tricomas peltados se encontram em maior abundância nas folhas, brácteas e sépalas, em comparação com o ovário e as pétalas. Observações ao microscópio óptico das secções das folhas revelaram que a estrutura dos tricomas peltados de T. albicans é similar à maioria das espécies da família Lamiaceae, apresentando uma célula basal ligada à epiderme da folha seguindose uma célula pedícular e uma cabeça composta por células secretoras distribuídas por dois círculos concêntricos, com 8 células secretoras em torno de 4 células centrais. Em contrapartida, os tricomas capitados são compostos por duas células pediculares e uma cabeça alongada, maior que as células pediculares. A estrutura apresentada pelos tricomas capitados contrasta com a maioria dos tricomas observados na família Lamiaceae, que são caracterizados por apresentarem uma célula pedicular maior que metade do comprimento da cabeça. Com base nestas observações as folhas foram o órgão da planta seleccionado para isolar a sequência codificante destas enzimas a partir do seu RNA total. Este estudo não incluiu a caracterização dos tricomas dos órgãos aéreos de T. mastichina devido à difícil disponibilidade do material vegetal desta espécie e também porque os seus tricomas glandulares já foram analisados em outros estudos. A estratégia utilizada para isolar a região codificante das enzimas baseou-se na amplificação do DNA (PCR e PCR-RACE) utilizando primers degenerados em zonas conservadas da sequência nucleotídica, tais como as regiões DDXXD e (M/L)L(S/Q/N)L(F/Y)EAS ou em regiões especificas do transcripto amplificado em cDNA de folha de T. albicans e T. mastichina. Como resultado obteve-se a sequência completa correspondente à região codificante de uma possível 1,8-cineol sintase de T. albicans (TalbCyn), com 1857 pb e 70% de similaridade com a 1,8-cineol de Rosmarinus officinalis. Obtiveram-se também dois fragmentos de possíveis monoterpeno sintases em T. albicans com 972 pb (TalbLin) e T. mastichina com 795 pb (TmlCyn), que partilham 71% de similaridade com uma linalol sintase de Perilla setoyensis e com 57% de similaridade para com a 1,8-cineol sintase de Rosmarinus officinalis, respectivamente. Alinhamentos entre as sequências de aminoácidos de TalbCyn, TmlCyn, TalbLin e outras 1,8-cineol sintases e linalool sintases revelou motivos característicos dos membros desta família (RRX8W, DDXXD, (M/L)L(S/Q/N)L(F/Y)EAS e (N/D)DX2(S/T)X3E) e também três regiões (região 1, região 2 e região 3) menos conservados entre as 1,8-cineol e linalol sintases, que podem ser determinantes para a estrutura terciária das proteínas, conferindo-lhes especificidade na síntese do produto. viii TalbLin apresenta uma substituição no motivo DDXXD no último ácido aspártico (D) por uma serina (S), o que poderá justificar o baixo rendimento de extracção de linalol (0.4%) comparando com 1,8-cineol (67.9%) em T. albicans verificado em estudos anteriores. A Região1 menos conservada, presente em ambas as sequências isoladas, contém um resíduo de asparagina que não está presente em TalbLin e está envolvido na deprotonação da água necessária para a síntese do 1,8-cineol. A Região 3, sendo uma região característica das terpeno ciclases, está presente nas ciclases 1,8-cineol sintases mas não se encontra nas acíclicas linalol sintases. Estas diferenças encontradas entre as 1,8-cineol e linalol sintases reflectem diferenças por parte das enzimas na especificidade do produto, resultando na síntese de diferentes monoterpenos. Para caracterizar funcionalmente as enzimas isoladas, a região correspondente à proteína madura da 1,8-cineol sintase de T. albicans, respectivamente a partir da região RRX8W, foi clonada no vector de expressão pCR®T7/NT-TOPO® utilizando o hospedeiro E. coli BL21 (DE3) e incluindo uma cauda de histidinas na sua extremidade amino-terminal para possibilitar o isolamento e purificação da proteina recombinante produzida. Ensaios preliminares de indução da expressão na presença de 0.7 mM de IPTG a 20 ºC foram realizados para avaliar o padrão de expressão da proteina recombinada e o extracto proteico da fracção insolúvel de amostras da cultura retiradas no final de 4, 10, 24, 48 e 72 horas foram analisadas por SDS-PAGE. Após 10 horas de indução, obteve-se na fracção insolúvel uma proteína com 70 KDa, peso molecular aproximado ao estimado para a proteína recombinante, 72.92 KDa respectivamente. No entanto, a identidade desta proteina carece ainda de ser confirmada através de Western blot, com anticorpos específicos para a cauda de histidinas e por sequenciação. De uma forma geral, os resultados obtidos neste trabalho constituem os passos iniciais que permitirão o isolamento e caracterização das enzimas 1,8-cineol e linalol sintases de T. albicans e T. mastichina, com o objectivo de elucidar o mecanismo reaccional complexo das monoterpeno sintases.
- Legume beverages from chickpea and lupin, as new milk alternativesPublication . Lopes, Mariana; Pierrepont, Chloé; Duarte, Carla Margarida; Filipe, Alexandra; Medronho, Bruno; Sousa, IsabelRecently, milk consumption has been declining and there is a high demand for non-dairy beverages. However, market offers are mainly cereal and nut-based beverages, which are essentially poor in protein (typically, less than 1.5% against the 3.5% in milk) and are not true milk replacers in that sense. In this work, new beverages from different pulses (i.e., pea, chickpea and lupin) were developed using technologies that enable the incorporation of a high level of seed components, with low or no discharge of by-products. Different processing steps were sequentially tested and discussed for the optimization of the sensorial features and stability of the beverage, considering the current commercial non-dairy beverages trends. The lupin beverage protein contents ranged from 1.8% to 2.4% (w/v) and the chickpea beverage varied between 1.0% and 1.5% (w/v). The "milk" yield obtained for the optimized procedure B was 1221 g/100 g of dry seed and 1247 g/100 g of dry seed, for chickpea beverage and lupin beverage, respectively. Sensory results show that chickpea beverage with cooking water has the best taste. All pulses-based beverages are typical non-Newtonian fluids, similarly to current non-dairy alternative beverages. In this respect, the sprouted chickpea beverage, without the cooking water, presents the most pronounced shear-thinning behavior of all formulations.
- Levulinic acid: a novel sustainable solvent for lignin dissolutionPublication . Melro, Elodie; Filipe, Alexandra; Valente, Artur J. M.; Antunes, Filipe E.; Romano, Anabela; Norgren, Magnus; Medronho, BrunoLignin is a natural, renewable resource with potential to be used in biomaterials. Due to its complex structure, its efficient dissolution is still challenging, which hinders its applicability at large scale. This challenge become harder considering the current need of sustainable and environmentally friendly solvents. To the best of our knowledge, this work reports for the first time the dissolution of kraft lignin in levulinic acid, a "green" solvent, and compares its efficiency with common carboxylic acids and sulfuric acid. It has been found that levulinic acid has a high capacity to dissolve kraft lignin at room temperature (40 wt% solubility), and it efficiency is not compromised when diluting the acid with water (up to 40 wt% water content). the Kamlet-Taft pi* parameter of the different acidic solvents was estimated and found to correlate well with their solubility performance. Lignins previously dissolved in levulinic and formic acids were selected to be regenerated and minor differences were found in thermal stability and morphological structure, when compared to native kraft lignin. However, an increase in the content of the carbonyl groups in the regenerated lignin material was observed. (C) 2020 Elsevier B.V. All rights reserved.
- Lignin extraction from waste pine sawdust using a biomass derived binary solvent systemPublication . Magalhães, Solange; Filipe, Alexandra; Melro, Elodie; Fernandes, Catarina; Vitorino, Carla; Alves, Luís; Romano, Anabela; Rasteiro, Maria G.; Medronho, Bruno; MDPILignocellulosic biomass fractionation is typically performed using methods that are somehow harsh to the environment, such as in the case of kraft pulping. In recent years, the development of new sustainable and environmentally friendly alternatives has grown significantly. Among the developed systems, bio-based solvents emerge as promising alternatives for biomass processing. Therefore, in the present work, the bio-based and renewable chemicals, levulinic acid (LA) and formic acid (FA), were combined to fractionate lignocellulosic waste (i.e., maritime pine sawdust) and isolate lignin. Different parameters, such as LA:FA ratio, temperature, and extraction time, were optimized to boost the yield and purity of extracted lignin. The LA:FA ratio was found to be crucial regarding the superior lignin extraction from the waste biomass. Moreover, the increase in temperature and extraction time enhances the amount of extracted residue but compromises the lignin purity and reduces its molecular weight. The electron microscopy images revealed that biomass samples suffer significant structural and morphological changes, which further suggests the suitability of the newly developed bio-fractionation process. The same was concluded by the FTIR analysis, in which no remaining lignin was detected in the cellulose-rich fraction. Overall, the novel combination of bio-sourced FA and LA has shown to be a very promising system for lignin extraction with high purity from biomass waste, thus contributing to extend the opportunities of lignin manipulation and valorization into novel added-value biomaterials.
- Molecular cloning and functional characterization of a monoterpene synthase isolated from the aromatic wild shrub Thymus albicansPublication . Filipe, Alexandra; Cardoso, João; Miguel, Maria Graca; Anjos, Liliana; Trindade, Helena; Figueiredo, Ana Cristina; Barroso, Jose; Power, Deborah; Marques, N T.The essential oil of Thymus albicans Hoffmanns. & Link, a native shrub from the Iberian Peninsula, is mainly composed of monoterpenes. In this study, a 1,8-cineole synthase was isolated from the 1,8-cineole chemotype. A partial sequence that lacked the complete plastid transit peptide but contained an extended C-terminal when compared to other related terpene synthases was generated by PCR and Rapid Amplification of cDNA Ends (RACE). The predicted mature polypeptide was 593 amino acids in length and shared 78% and 77% sequence similarity with the homologue 1,8-cineole synthase from Rosmarinus officinalis and Salvia officinalis, respectively. The putative protein possessed the characteristic conserved motifs of plant monoterpene synthases including the RRx(8)W and DDxxD motifs and phylogenetic analysis indicated that the amplified 1,8-cineole synthase bears greater sequence similarity with other 1,8-cineole synthases from Lamiaceae family relative to the terpene synthases from the genus Thymus. Functional expression of the recombinant protein in Escherichia coli revealed that in the presence of geranyl diphosphate (GPP) 1,8-cineole was the major product but that its production was too low for robust quantification. Other minor conversion products included a-pinene, beta-pinene, sabinene and beta-myrcene suggesting the isolated 1,8-cineole synthase may be a multi-product enzyme. To our knowledge, this is the first report of a functionally characterized monoterpene synthase from Thymus albicans.
- New deep eutectic solvent assisted extraction of highly pure lignin from maritime pine sawdust (Pinus pinaster Ait.)Publication . Fernandes, Catarina; Melro, Elodie; Magalhaes, Solange; Alves, Luis; Craveiro, Rita; Filipe, Alexandra; Valente, Artur J. M.; Martins, Gabriela; Antunes, Filipe E.; Romano, Anabela; Medronho, BrunoLignocellulosic biomass is a renewable and sustainable feedstock, mainly composed of cellulose, hemicellulose, and lignin. Lignin, as the most abundant natural aromatic polymer occurring on Earth, has great potential to produce value-added products. However, the isolation of highly pure lignin from biomass requires the use of efficient methods during lignocellulose fractionation. Therefore, in this work, novel acidic deep eutectic solvents (DESs) were prepared, characterized and screened for lignin extraction from maritime pine wood (Pinus pinaster Ait.) sawdust. The use of cosolvents and the development of new DES were also evaluated regarding their extraction and selectivity performance. The results show that an 1 h extraction process at 175 ?C, using a novel DES composed of lactic acid, tartaric acid and choline chloride, named Lact:Tart:ChCl, in a molar ratio of 4:1:1, allows the recovery of 95 wt% of the total lignin present in pine biomass with a purity of 89 wt%. Such superior extraction of lignin with remarkable purity using a ?green? solvent system makes this process highly appealing for future large-scale applications. ? 2021 Elsevier B.V. All rights reserved.
- New insights on the role of urea on the dissolution and thermally-induced gelation of cellulose in aqueous alkaliPublication . Alves, Luis; Medronho, Bruno; Filipe, Alexandra; E. Antunes, Filipe; Lindman, Björn; Topgaard, Daniel; Davidovich, Irina; Talmon, YeshayahuThe gelation of cellulose in alkali solutions is quite relevant, but still a poorly understood process. Moreover, the role of certain additives, such as urea, is not consensual among the community. Therefore, in this work, an unusual set of characterization methods for cellulose solutions, such as cryo-transmission electronic microscopy (cryo-TEM), polarization transfer solid-state nuclear magnetic resonance (PTssNMR) and diffusion wave spectroscopy (DWS) were employed to study the role of urea on the dissolution and gelation processes of cellulose in aqueous alkali. Cryo-TEM reveals that the addition of urea generally reduces the presence of undissolved cellulose fibrils in solution. These results are consistent with PTssNMR data, which show the reduction and in some cases the absence of crystalline portions of cellulose in solution, suggesting a pronounced positive effect of the urea on the dissolution efficiency of cellulose. Both conventional mechanical macrorheology and microrheology (DWS) indicate a significant delay of gelation induced by urea, being absent until ca. 60 °C for a system containing 5 wt % cellulose, while a system without urea gels at a lower temperature. For higher cellulose concentrations, the samples containing urea form gels even at room temperature. It is argued that since urea facilitates cellulose dissolution, the high entanglement of the cellulose chains in solution (above the critical concentration, C*) results in a strong three-dimensional network.
- On the formation and stability of cellulose-based emulsions in alkaline systems: effect of the solvent qualityPublication . Costa, Carolina; Medronho, Bruno; Filipe, Alexandra; Romano, Anabela; Lindman, Björn; Edlund, Håkan; Norgren, MagnusWith amphiphilic properties, cellulose molecules are expected to adsorb at the O/W interface and be capable of stabilizing emulsions. The effect of solvent quality on the formation and stability of cellulose-based O/W emulsions was evaluated in different alkaline systems: NaOH, NaOH-urea and tetrabutylammonium hydroxide (TBAH). The optimal solvency conditions for cellulose adsorption at the O/W interface were found for the alkaline solvent with an intermediate polarity (NaOH-urea), which is in line with the favorable conditions for adsorption of an amphiphilic polymer. A very good solvency (in TBAH) and the interfacial activity of the cation lead to lack of stability because of low cellulose adsorption. However, to achieve long-term stability and prevent oil separation in NaOH-urea systems, further reduction in cellulose's solvency was needed, which was achieved by a change in the pH of the emulsions, inducing the regeneration of cellulose at the surface of the oil droplets (in-situ regeneration).