Fructo-oligosaccharides: production, characterization and purification

GLUPOR 12 - 12nd International Meeting of the Portuguese Carbohydrate Chemistry GroupThe consumers interest in healthy and high nutritional food has significantly increased in the recent years. This trend towards the adoption of healthier lifestyles has been the main driver for the great demand of functional ingredients, such as the prebiotics fructo-oligosaccharides (FOS). Industrially, FOS are produced from sucrose through purified enzymes, in two-step bioprocesses, with low theoretical yields (0.50-0.55 gFOS.gSucrose-1) and purities (50-55%). Downstream steps are therefore needed to remove the non-prebiotic sugars and enable the incorporation of these FOS mixtures in diabetic, dietetic and healthy foods. In the last ten years, we have been investigating new strategies to produce FOS with higher contents, purities and differentiated functionalities. We have been exploring Aureobasidium pullulans and Aspergillus ibericus as FOS producers, in one-step fermentation processes, using the whole cells of the microorganisms instead of the isolated enzymes. This strategy proved to be efficient, fast and economic, yielding 0.64 gFOS.gSucrose-1. The FOS mixtures produced were able to stimulate the growth of probiotic strains and were simultaneously resistant to hydrolysis along the gastrointestinal system confirming their health claims as prebiotics. The probiotic strains preferentially metabolized the FOS mixture synthesized by A. ibericus, followed by the one from A. pullulans and lastly the commercial FOS. The purification of FOS is not straightforward due to the physicochemical similarities between the different oligosaccharides and the smaller saccharides. To increase the FOS purity, we have been exploring different strategies including microbial treatments and downstream treatments as activated charcoal and ion-exchange chromatography. As microbial treatments, we studied the use of a Saccharomyces cerevisiae strain, able to metabolize the small saccharides without FOS hydrolyse, in co-culture with the FOS microorganism producer or in a two-step fermentation, in which FOS are firstly synthesized and then purified by the S. cerevisiae. Fermentations in two-steps were found to be more efficient than the co-culture ones and purities of 82% (w/w) in FOS were obtained [1]. To avoid competition by the subtract in the co-culture, we are now evaluating the use of a S. cerevisiae strain with the SUC2 gene for invertase expression repressed. Using this strategy, FOS are being produced with yields of 0.64 gFOS.gSucrose-1 and purities up to 93% (w/w). As downstream treatment we optimized an adsorption/desorption process of sugars using activated charcoal and ethanol as eluent. Mixtures containing 50.6% (w/w) of FOS were purified to 92.9% (w/w) with a FOS recovery of 74.5% (w/w) and some fractions were obtained with purities up to 97% (w/w) [2]. Acknowledgements: Clarisse Nobre acknowledges the Portuguese Foundation for Science and Technology (FCT) for her Post-Doc Grant [ref. SFRH/BPD/87498/2012] and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) and the project MultiBiorefinery (POCI-01-0145-FEDER-016403) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. References [1] Nobre, C, Castro, CC, Hantson, A-L, Teixeira, JA, Weireld, G, Rodrigues, LR, Strategies for the production of high-content fructo-oligosaccharides through the removal of small saccharides by co-culture or successive fermentation with yeast, Carbohydrate Polymers, 136, 274281, 2016. [2] Nobre, C, Teixeira, JA, Rodrigues, LR, Fructo-oligosaccharides purification from a fermentative broth using an activated charcoal column. New Biotechnology, 29(3), 395401, 2012.Clarisse Nobre acknowledges the Portuguese Foundation for Science and Technology (FCT) for her Post-Doc Grant [ref. SFRH/BPD/87498/2012] and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER027462), the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) and the project MultiBiorefinery (POCI-01-0145-FEDER016403) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Strategies for Fructo-oligosaccharides production with high-content and purity

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] The consumers’ interest in healthy and high nutritional food has significantly increased in the recent years. This trend towards the adoption of healthier lifestyles has been the main driver for the great demand of functional ingredients, such as the prebiotics fructo-oligosaccharides (FOS). Industrially, FOS are produced from sucrose through purified enzymes, in two-step bioprocesses, with low theoretical yields (0.50-0.55 gFOS.gSucrose-1) and purities (50-55%). Downstream steps are therefore needed to remove the non-prebiotic sugars and enable the incorporation of these FOS mixtures in diabetic, dietetic and healthy foods. In the last ten years, we have been investigating new strategies to produce FOS with higher contents, purities and differentiated functionalities. We have been exploring Aureobasidium pullulans and Aspergillus ibericus as FOS producers, in one-step fermentation processes, using the whole cells of the microorganisms instead of the isolated enzymes. This strategy proved to be efficient, fast and economic, yielding 0.64 gFOS.gSucrose-1. The FOS mixtures produced were able to stimulate the growth of probiotic strains and were simultaneously resistant to hydrolysis along the gastrointestinal system confirming their health claims as prebiotics. The probiotic strains preferentially metabolized the FOS mixture synthesized by A. ibericus, followed by the one from A. pullulans and lastly the commercial FOS. [...]info:eu-repo/semantics/publishedVersio

From antimicrobial to anticancer peptides : a review

Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms. Although AMPs have been essentially studied and developed as potential alternatives for fighting infectious diseases, their use as anticancer peptides (ACPs) in cancer therapy either alone or in combination with other conventional drugs has been regarded as a therapeutic strategy to explore. As human cancer remains a cause of high morbidity and mortality worldwide, an urgent need of new, selective, and more efficient drugs is evident. Even though ACPs are expected to be selective toward tumor cells without impairing the normal body physiological functions, the development of a selective ACP has been a challenge. It is not yet possible to predict antitumor activity based on ACPs structures. ACPs are unique molecules when compared to the actual chemotherapeutic arsenal available for cancer treatment and display a variety of modes of action which in some types of cancer seem to co-exist. Regardless the debate surrounding the definition of structure-activity relationships for ACPs, great effort has been invested in ACP design and the challenge of improving effective killing of tumor cells remains. As detailed studies on ACPs mechanisms of action are crucial for optimizing drug development, in this review we provide an overview of the literature concerning peptides' structure, modes of action, selectivity, and efficacy and also summarize some of the many ACPs studied and/or developed for targeting different solid and hematologic malignancies with special emphasis on the first group. Strategies described for drug development and for increasing peptide selectivity toward specific cells while reducing toxicity are also discussed.The authors thank Fundação para a Ciência e a Tecnologia (FCT- MEC, Portugal) for funding—PTDC/QUI-BIO/112929/2009. Diana Gaspar also acknowledges FCT for fellowship SFRH/BPD/ 73500/2010 and A. Salome Veiga for funding within the FCT Investigator Programme (IF/00803/2012

Global characterization of the Quercus suber ectomycorrhizal transcriptome using 454 pyrosequencing

In temperate forests, trees live in symbiosis with fungi and depend on them for their survival. Species from the Fagaceae family, including oaks, establish a symbiotic relationship in their roots with basidiomycetous fungi, called ectomycorrhizas (ECM). ECM symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with well developed ECM root tips are more tolerant to environmental stresses, such as drought, and iotic stresses such as root pathogens. There is a general agreement that ECM fungi increase plant survival and productivity. Cork oak is well adapted to water scarcity typical of the Mediterranean summer, due to a root system that can reach several metres in depth, and to the abundance of roots at the soil surface associated with ECM. The development of ECM symbiosis is characterized by the successive development of three structural components: a mantle of fungal tissue that encloses the root, the intraradical network of hyphae (Hartig net), where plant and fungus exchange metabolites, and the extraradical mycelium, which extends into the soil and is responsible for nutrient and water uptake. All these processes are highly regulated and are accompanied by alterations on gene expression in both partners. The current project is integrated in the effort for covering the transcriptome of Quercus suber and is focused on the identification of ESTs involved in the development of ECM symbiosis in cork oak. Since only 236 ESTs from Quercus suber are currently known, the use of genomics approaches for gene discovery or functional studies is far from being achieved for this plant species.This work was supported by the FCT project SOBREIRO/0034/200

Star Formation in the Outer Filaments of NGC 1275

We present photometry of the outer star clusters in NGC 1275, the brightest galaxy in the Perseus cluster. The observations were taken using the Hubble Space Telescope Advanced Camera for Surveys. We focus on two stellar regions in the south and south-east, far from the nucleus of the low velocity system (~22 kpc). These regions of extended star formation trace the H alpha filaments, drawn out by rising radio bubbles. In both regions bimodal distributions of colour (B-R)_0 against magnitude are apparent, suggesting two populations of star clusters with different ages; most of the H alpha filaments show no detectable star formation. The younger, bluer population is found to be concentrated along the filaments while the older population is dispersed evenly about the galaxy. We construct colour-magnitude diagrams and derive ages of at most 10^8 years for the younger population, a factor of 10 younger than the young population of star clusters in the inner regions of NGC 1275. We conclude that a formation mechanism or event different to that for the young inner population is needed to explain the outer star clusters and suggest that formation from the filaments, triggered by a buoyant radio bubble either rising above or below these filaments, is the most likely mechanism.Comment: Accepted for publication in MNRAS, 14 pages, 14 figures, 3 table

Shifting gear in antimicrobial and anticancer peptides biophysical studies : from vesicles to cells

© 2015 European Peptide Society and John Wiley & Sons, Ltd.Despite the intensive study on the mechanism of action of membrane-activemolecules such as antimicrobial and anticancer peptides, most of the biophysical work has been performed using artificial model systems, mainly lipid vesicles. The use of these systems allows full control of the experimental parameters, and to obtain molecular-level detail on the action of peptides, the correlation with biological action is intangible. Recently, several biophysicalmethodologies have been translated to studies using bacterial and cancer cells. Here, we review biophysical studies on the mechanism of action of antimicrobial and anticancer peptides performed directly on cells. The data in these studies allow to correlate vesicle-based and cell-based studies and fill the vesicle-cell interdisciplinary gap.Projects Ciência Sem Fronteiras PVE 171/2012 (CAPES, Brazil) and MSCA-RISE-2014-Nr644167 are acknowledged. D. Gaspar and J. Freire are recipients of fellowships SFRH/BPD/73500/2010 and SFRH/BD/70423/2010 from FCT, Portugal, respectively. A.S. Veiga acknowledges FCT, Portugal, for funding within the FCT Investigator Programme (IF/00803/2012)

Tailoring carbon nanotubes to enhance their efficiency as electron shuttle on the biological removal of acid orange 10 under anaerobic conditions

Anaerobic treatments have been described for the biodegradation of pollutants. However, the reactions proceed slowly due to the recalcitrant nature of these compounds. Carbon nanomaterials (CNM) intermediate in, and favor, the electron transfer, accelerating the anaerobic reduction of pollutants, which act as final electron acceptors. In the present work, different carbon nanotubes (CNT) with modified surface chemistry, namely CNT oxidized with HNO3 (CNT_HNO3) and CNT doped with nitrogen in a ball milling process (CNT_N_MB) were prepared using commercial CNT as a starting material. The new CNM were tested as redox mediators (RM), 0.1 g L−1, in the biological reduction of the azo dye, Acid Orange 10 (AO10), with an anaerobic granular sludge, over 48 h of reaction. Methane production was also assessed to verify the microorganism’s activity and the CNM’s effect on the methanogenic activity. An improvement in the biological removal of AO10 occurred with all CNM (above 90%), when compared with the control without CNM (only 32.4 ± 0.3%). The best results were obtained with CNT_N_MB, which achieved 98.2 ± 0.1% biological AO10 removal, and an 11-fold reduction rate increase. In order to confer magnetic properties to the CNM, tailored CNT were impregnated with 2% of iron-samples: CNTThis study was 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, as well as FCT/MCTES trough national funds (PIDDAC) and Base Funding – UIDB/50020/20 of the Associate Laboratory LSRE‐LCM – funded by national funds rough FCT/MCTES (PIDDAC). Ana Rita Silva holds a Grant from FCT, reference SFRH/BD/131905/2017. Salomé. G.P. Soares acknowledges FCT funding under the Scientific Employment Stimulus ‐ Institutional Call CEECINST/00049/2018.info:eu-repo/semantics/publishedVersio

Modulation of butyrate-degrading methanogenic communities by conductive materials

Butyrate is a volatile fatty acid commonly present in anaerobic bioreactors. Previous research showed that methane production (MP) rates from butyrate, by lake sediment microbiomes, doubled by addition of carbon nanotubes, which was accompanied by changes in the microbial community composition, with enrichment of typical fatty-acid degrading bacteria (Syntrophomonas spp.), well known to exchange electrons with methanogens via hydrogen or formate formation1. But the authors suggested that electrons exchange via conductive materials (CM) may take place instead. In our study, anaerobic butyrate-degrading enrichment cultures were developed with other CM, namely activated carbon (AC) and magnetite (Mag) at 0.5 g/L. MP started earlier in AC enrichment and complete degradation was achieved faster in Mag enrichment. Syntrophomonas spp. were enriched in all cultures (representing 60 to 80 % of the total bacterial community), but hydrogenotrophic methanogens were highly stimulated by AC (78 % of Methanomicrobiales), while the methanogenic community of Mag culture was more diverse in acetoclastic methanogens (43% of Methanosarcina and Methanosaeta). It is still unclear if the improvement on butyrate degradation is associated to the role of CM in interspecies electron transfer, but it is undoubtful that they differentially modulate the methanogenic communities towards faster MP.info:eu-repo/semantics/publishedVersio