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Background. Osteosarcoma is a highly malignant bone tumor, most frequently occurring in the rapid bone growth phase. Effective treatment of this disease is hindered by the lack of specific probes for early diagnosis and the fast cancer widespread. Methods. To find such probes, the cell-Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) methodology was implemented against the human osteosarcoma MG-63 cell line towards the selection of new specific aptamers. After 10 rounds of selection, the aptamer DNA pool was Sanger sequenced and the sequences were subjected to a bioinformatic analysis that included sequence alignment, phylogenetic relationship, and secondary structure prediction. Results. A DNA aptamer (OS-7.9), with a dissociation constant (Kd) value in the nanomolar range (12.8\thinspace\textpm\thinspace0.9 nM), revealed high affinity against the target cells at the physiological temperature. Furthermore, the selected aptamer also recognized lung carcinoma and colon colorectal adenocarcinoma cell lines, which are reported as common metastasis sites of osteosarcoma. Conclusions. These results suggest that OS-7.9 could recognize a common protein expressed in these cancer cells, possibly becoming a potential molecular probe for early diagnosis and targeted therapies for metastatic disease. Moreover, to the best of our knowledge, this was the first attempt to generate a DNA aptamer (OS-7.9 aptamer) against the MG-63-cell line by cell-SELEX., This work was supported by the Hacettepe University Scientifc Research Projects Coordination Unit (project no. FHD 2018–17245) and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020unit. Diana A. Sousa (DAS) acknowledge FCT for the grant PD/BD/139083/2018. Débora Ferreira (DF) is recipient of a fellowship supported by a doctoral advanced training (call NORTE 69–2015-15) funded by the European Social Fund under the scope of Norte2020—Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. The authors acknowledge FCT for funding MRC doctoral grant SFRH/BD/132998/2017., info:eu-repo/semantics/publishedVersion

Cellobiose 2-epimerases are interesting enzymes that can catalyze the production of lactose-based prebiotics, namely, epilactose and lactulose, through a single substrate reaction with noteworthy yields. In this work, the GRAS yeast Saccharomyces cerevisiae was for the first time used as a host for the heterologous production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. The enzyme showed an optimal temperature and an optimal pH of 80 °C and 7.5, respectively. Under these conditions, the time-course production of epilactose from lactose (50 g/L) was evaluated. The maximum yield and productivity obtained were 27\% and 9 g L1 h1, respectively. This epilactose yield represents the highest value obtained from the several approaches reported using cellobiose 2-epimerase from C. saccharolyticus. In conclusion, it was shown that the enzyme produced by S. cerevisiae is suitable for the production of epilactose-enriched prebiotic mixtures with potential applications in the food industry., FCT -Fundação para a Ciência e a Tecnologia(Lisboa2020), info:eu-repo/semantics/publishedVersion

Lactoferrin (Lf), a bioactive milk protein, exhibits strong anticancer and antifungal activities. The search for Lf targets and mechanisms of action is of utmost importance to enhance its effective applications. A common feature among Lf-treated cancer and fungal cells is the inhibition of a proton pump called V-ATPase. Lf-driven V-ATPase inhibition leads to cytosolic acidification, ultimately causing cell death of cancer and fungal cells. Given that a detailed elucidation of how Lf and V-ATPase interact is still missing, herein we aimed to fill this gap by employing a five-stage computational approach. Molecular dynamics simulations of both proteins were performed to obtain a robust sampling of their conformational landscape, followed by clustering, which allowed retrieving representative structures, to then perform protein-protein docking. Subsequently, molecular dynamics simulations of the docked complexes and free binding energy calculations were carried out to evaluate the dynamic binding process and build a final ranking based on the binding affinities. Detailed atomist analysis of the top ranked complexes clearly indicates that Lf binds to the V1 cytosolic domain of V-ATPase. Particularly, our data suggest that Lf binds to the interfaces between A/B subunits, where the ATP hydrolysis occurs, thus inhibiting this process. The free energy decomposition analysis further identified key binding residues that will certainly aid in the rational design of follow-up experimental studies, hence bridging computational and experimental biochemistry., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding UIDP/04378/2020, UIDB/04378/2020, UIDB/04469/2020 and UIDB/04050/2020; and by 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. Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral program in Applied and Environmental Microbiology (DP_AEM). Juliana F. Rocha is the recipient of the SFRH/BD/136746/2018 fellowship supported by FCT. Henrique S. Fernandes acknowledges FCT for his Ph.D. grant SFRH/BD/115396/2016. Some of the calculations were produced with the support of INCD funded by FCT and FEDER under the project 01/SAICT/2016 number 022153., info:eu-repo/semantics/publishedVersion

Most biosurfactants are obtained using costly culture media and purification processes, which limits their wider industrial use. Sustainability of their production processes can be achieved, in part, by using cheap substrates found among agricultural and food wastes or byproducts. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of surfactin production by Bacillus subtilis #309. The culture medium containing soap-derived waste glycerol led to the best surfactin production, reaching about 2.8 g/L. To the best of our knowledge, this is the first report describing surfactin production by B. subtilis using stearin and soap wastes as carbon sources. A complete chemical characterization of surfactin analogs produced from the different waste glycerol samples was performed by liquid chromatographymass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR). Furthermore, the surfactin produced in the study exhibited good stability in a wide range of pH, salinity and temperatures, suggesting its potential for several applications in biotechnology., National Science Centre, Poland, project 2020/37/B/NZ9/ 01519. The Article Processing Charge (APC) was financed under the Leading Research Groups support project from the subsidy increased for the period 2020–2025 in the amount of 2% of the subsidy referred to in Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained in 2019. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the 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, info:eu-repo/semantics/publishedVersion

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.seppur.2018.07.062., Esterases catalyze the cleavage and formation of ester bonds of a broad range of substrates presenting a widespread spectrum of industrial applications. This work aimed to partially purify and characterize an esterase from Aureobasidium pullulans LABIOTEC 01 produced in a culture medium containing olive mill wastewater. Esterase purification was evaluated using different strategies, namely the enzyme recovery by PEG-salt aqueous two-phase systems (ATPSs); and the enzyme precipitation with ammonium sulfate, acetone, and ethanol. The best purification factor (18±2) was obtained when the ATPS composed of 20% (w/w) polyethylene glycol (PEG) 6000 and 5.8% (w/w) potassium phosphate buffer (PPB) pH 8.0 was combined with acetone precipitation. The partially purified enzyme presented an optimum pH of 5.0, although it remained active in the pH range of 4.5 to 7.5 ( 50% relative activity). The optimum temperature was found to be 60°C. Furthermore, the addition of salts such as FeCl3, CuSO4 and MnCl2 promoted an increase in the enzymatic activity (above 100%). The enzyme was found to be stable and showed high activity when exposed to polar solvents such as dimethyl sulfoxide, dimethylformamide, and methanol. The use of an integrated strategy of purification combining simple purification methods such as ATPS and precipitation was herein reported for the first time for esterase. This strategy proved to be an interesting approach to partially purify the esterase produced under submerged fermentation by A. pullulans. Furthermore, the enzyme showed potential to be applied in industrial biocatalytic processes using high temperature and different salts or solvents. Also, the production of esterase using olive mill wastewater as substrate demonstrated to be a suitable alternative to reduce and valorize agro-industrial residues., ACL acknowledges his post-doctoral grant and research funds from CAPES and CNPq (Brazilian funding agencies). SCS and LRR acknowledge their grants SFRH/BPD/88584/2012 and SFRH/BSAB/142873/ 2018, respectively, from the Portuguese Foundation for Science and Technology (FCT). The study also received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the Project MultiBiorefinery - Multi‐purpose strategies for broadband agro‐forest and fisheries by‐products valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403) and the Project Lignozymes - Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-010145-FEDER-029773). The authors also acknowledge financial support from the BioTecNorte operation (NORTE-01-0145-FEDER-000004) fundedbytheEuropeanRegionalDevelopment Fundunderthescopeof Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with Kd values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis. © 2021, The Author(s)., Tis study was supported by the 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. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Joaquim Barbosa (JB) and Diana A. Sousa (DAS) acknowledge FCT for the Grants SFRH/BD/51109/2010 and PD/BD/139083/2018, respectively., info:eu-repo/semantics/publishedVersion

Innovation of pastry products towards higher nutritional and commercial value remains a challenge to the growing field of healthy food. In this study, the prebiotic supplementation and sugar reduction were explored in a widely consumed pastry product with a low level of innovation. The prebiotic potential of commercial agave inulin and galacto-oligosaccharides (GOS) was evaluated and compared by an in vitro model using human fecal inocula. Rice muffins containing 100% of sugar or 75% of sugar supplemented with 0.8% GOS were produced and compared with commercial rice muffins regarding their physical and textural properties. GOS fermentation led to the highest production of lactate and short-chain fatty acids, besides the most significant reduction of the final pH value and of the ammonia and methane production. Inulin presented a higher selectivity towards Lactobacillaceae (51 ± 1% of all), while GOS are more efficient to stimulate Bifidobacteriaceae growth (65 ± 7% of all)., This study was supported by the 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-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte; COMPETE 2020 (POCI-01-0145-FEDER-006684), FoSynBio (POCI-01-0145-FEDER-029549) and NewFood (NORTE-01-0246-FEDER000043). CA and BBC acknowledge their grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from FCT. RM acknowledges the financial support to the Xunta de Galicia and FEDER by the project (EDD431B 2019/01)., info:eu-repo/semantics/publishedVersion

"Available online 07 January 2021", Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs sterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma110 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi., This work was supported by national funds through the Portuguese Foundation for Science and Technology (FCT I.P.) under the scope of the strategic funding of “Contrato-Programa” UIDB/04050/2020 and UIDB/ 04469/2020 unit; by 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; and by the Servicio para el Control de la Esterilización, Laboratorio de Microbiología Oral (CN-16-036). Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral programme in Applied and Environmental Microbiology (DP_AEM)., info:eu-repo/semantics/publishedVersion

The control of the intracellular pH is vital for the survival of all organisms. Membrane transporters, both at the plasma and intracellular membranes, are key players in maintaining a finely tuned pH balance between intra- and extracellular spaces, and therefore in cellular homeostasis. V-ATPase is a housekeeping ATP-driven proton pump highly conserved among prokaryotes and eukaryotes. This proton pump, which exhibits a complex multisubunit structure based on cell type-specific isoforms, is essential for pH regulation and for a multitude of ubiquitous and specialized functions. Thus, it is not surprising that V-ATPase aberrant overexpression, mislocalization, and mutations in V-ATPase subunit-encoding genes have been associated with several human diseases. However, the ubiquitous expression of this transporter and the high toxicity driven by its off-target inhibition, renders V-ATPase-directed therapies very challenging and increases the need for selective strategies. Here we review emerging evidence linking V-ATPase and both inherited and acquired human diseases, explore the therapeutic challenges and opportunities envisaged from recent data, and advance future research avenues. We highlight the importance of V-ATPases with unique subunit isoform molecular signatures and disease-associated isoforms to design selective V-ATPase-directed therapies. We also discuss the rational design of drug development pipelines and cutting-edge methodological approaches toward V-ATPase-centered drug discovery. Diseases like cancer, osteoporosis, and even fungal infections can benefit from V-ATPase-directed therapies., European Regional Development Fund, Grant/Award Number: BioTecNorte operation (NORTE‐01‐0145‐FEDER‐000004); Fundação para a Ciência e a Tecnologia, Grant/Award Numbers: PhD fellowship (PD/BD/128032/2016), Strategic funding of UIDB/04469/2020 unit and of “Contrato‐Programa” UIDB/04050/2020, info:eu-repo/semantics/publishedVersion

The potential use of alternative culture media towards the development of a sustainable bioprocess to produce lipases by Diutina rugosa is clearly demonstrated. First, a synthetic medium containing glucose, peptone, yeast extract, oleic acid, and ammonium sulfate was proposed, with lipase activity of 143 U/L. Then, alternative culture media formulated with agro-industrial residues, such as molasses, corn steep liquor (CSL), and olive mill waste (OMW), were investigated. An experimental design was conducted, and only CSL concentration was found to have a positive effect in lipase production. The highest lipase activity (561 U/L) was produced on a mixture of molasses (5 g/L), CSL (6 g/L), OMW (0.5\\% v/v), 0.5 g/L of ammonium sulfate, and 3 g/L of peptone at 24 h of cultivation. Lipase production was also carried out in a 1-L bioreactor leading to a slightly higher lipase activity at 24 h of cultivation. The semi-purified enzyme exhibits an optimum temperature and pH of 40 \textdegreeC and 7.0, respectively. Finally, the media cost per unit of lipase produced (UPC) was influenced by the medium components, specially by the inducer used. The lowest UPC was obtained when the agro-industrial residues were combined and used at the improved concentrations., The study is funded by CAPES, CNPq, and FUNCAP (from Brazil) for the financial support that made this work possible. In addition, the study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, 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, and the Project LIGNOZYMES (POCI-01-0145- FEDER-029773)., info:eu-repo/semantics/publishedVersion

A levan-type fructooligosaccharide was produced by a Paenibacillus strain isolated from Brazilian crude oil, the purity of which was 98.5% after precipitation with ethanol and dialysis. Characterization by FTIR, NMR spectroscopy, GC-FID and ESI-MS revealed that it is a mixture of linear (26) fructosyl polymers with average degree of polymerization (DP) of 18 and branching ratio of 20. Morphological structure and physicochemical properties were investigated to assess levan microstructure, degradation temperature and thermomechanical features. Thermal Gravimetric Analysis highlighted degradation temperature of 218°C, Differential Scanning Calorimetry (DSC) glass transition at 81.47°C, and Dynamic Mechanical Analysis three frequency-dependent transition peaks. These peaks, corresponding to a first thermomechanical transition event at 86.60°C related to the DSC endothermic event, a second at 170.9°C and a third at 185.2°C, were attributed to different glass transition temperatures of oligo and polyfructans with different DP. Levan showed high morphological versatility and technological potential for the food, nutraceutical, and pharmaceutical industries., São Paulo Research Foundation (FAPESP) within Grants 2020/13271-6 and 2018/25511-1. Additionally, the authors acknowledge the financial support by National Council for Scientific and Technological Development – CNPq and by Coordination for the Improvement of Higher Education Personnel (CAPES), Finance Code 001. In addition, this study was supported by the Portuguese Science Foundation (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and the Project FOSYNBIO (POCI-01-0145-FEDER-029549)., info:eu-repo/semantics/publishedVersion

Furanocoumarins are plant secondary metabolites used to treat several skin disorders, such as psoriasis and vitiligo, and also with other potential therapeutic activities. Furanocoumarins are extracted from plants where they accumulate in low amounts over long growth periods. In addition, their extraction and purification are difficult in an environmentally unfriendly and expensive process. Hence, new sustainable and greener production schemes able to overcome such limitations ought to be developed. While the heterologous production of simple coumarins has been demonstrated, the biosynthesis of more complex furanocoumarins remains greatly unexplored. Although several important steps of the pathway have been elucidated in the last decade, the complete pathway has not been completely unravelled. In this paper, we review the natural conversion of amino acids into furanocoumarins, as well as the heterologous expression of each enzyme of the pathway. We also explore the challenges that need to be addressed so that their heterologous production can become a viable alternative., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 – North Portugal Regional Program. In addition, this research has been carried out at the Biomass and Bioenergy Research Infrastructure (BBRI) – LISBOA-010145-FEDER 022059, supported by Operational Program for Competitiveness and Internationalization (PORTUGAL2020), the Lisbon Portugal Regional Operational Program (Lisboa2020), and Norte2020 under the Portugal 2020 Partnership Agreement, through the ERDF., info:eu-repo/semantics/publishedVersion

Aflatoxins are hepatotoxic and carcinogenic fungal secondary metabolites that usually contaminate crops and represent a serious health hazard for humans and animals worldwide. In this work, the effect of rhamnolipids (RLs) produced by Pseudomonas aeruginosa #112 on the growth and aflatoxins production by Aspergillus flavus MUM 17.14 was studied in vitro. At concentrations between 45 and 1500 mg/L, RLs reduced the mycelial growth of A. flavus by 2340% and the production of aflatoxins by 93.999.5%. Purified mono-RLs and di-RLs exhibited a similar inhibitory activity on fungal growth. However, the RL mixture had a stronger inhibitory effect on aflatoxins production at concentrations up to 190 mg/L, probably due to a synergistic effect resulting from the combination of both congeners. Using transmission electron microscopy, it was demonstrated that RLs damaged the cell wall and the cytoplasmic membrane of the fungus, leading to the loss of intracellular content. This disruptive phenomenon explains the growth inhibition observed. Furthermore, RLs down-regulated the expression of genes aflC, aflE, aflP and aflQ involved in the aflatoxins biosynthetic pathway (6.4, 44.3, 38.1 and 2.0-fold, respectively), which is in agreement with the almost complete inhibition of aflatoxins production. Overall, the results herein gathered demonstrate for the first time that RLs could be used against aflatoxigenic fungi to attenuate the production of aflatoxins, and unraveled some of their mechanisms of action., This study was supported by the 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. The authors also acknowledge the support of the i3S Scientific Platform Histology and Electron Microscopy (HEMS), member of the PPBI (PPBI POCI-01-0145-FEDER-022122). A.I. Rodrigues and L. Abrunhosa acknowledge FCT for the grant SFRH/BD/111600/2015 and the Assistant Research contract CEECIND/00728/2017, respectively., info:eu-repo/semantics/publishedVersion

This work reports for the first time a colorimetric based biosensor using nanocellulose (NC) based supports drop-deposited onto a cellulose paper substrate for glucose detection in point-of-care. For this purpose, microcrystalline cellulose (MCC) samples were oxidized with 2,2,6,6-tetramethylpiperidine-N-oxyl radical (TEMPO), sodium hypochlorite, and potassium bromide, to produce NC that corresponded to carboxyl- NC. For the characterization, we used several methods: TEM, FTIR and conductometric titration. In all samples, the primary alcohol groups were selectively oxidized into carboxyl groups, provided the sodium hypochlorite is added dropwise and the reaction is performed at constant pH?10. Carboxyl- NC was further casted on a cellulose substrate and used as support for glucose oxidase (GOx), horseradish peroxidase (HRP) and 2,2?-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) reactions, aiming to yield a coloured detection system for glucose. The sensing system was generated by integrating GOx on the carboxyl- NC /cellulose substrate. Upon reaction with glucose, the enzyme produced hydrogen peroxide, which was converted into a blue-coloured product by reaction with HRP and the chromogenic reagent ABTS. The test-strip was calibrated by incubating it in different concentrations of glucose. The colours obtained were further analysed by a suitable image analysis software. Linear response for glucose ranged 1.5 to 13.0?mM. Overall, this new test-strip used renewable material for glucose determination, which is an advantage when compared to other systems that require more complex technological approaches. Moreover, it was found that Carboxyl- NC improved the colour homogeneity of the test-strip and the intrinsic linear response of concentration range., The Portuguese Science and Technology Foundation, I.P. (FCT), isacknowledged for the financial support through the project reference P2020 -PTDC/AAG-TEC/5400/2014 (also POCI-01-0145-FEDER-016637, FEDER, COMPETE, POCI) and POCTEP/INTERREG is also acknowledged through the project 2QBioneuro, Impulso de una red deI + i en química biológica para diagnóstico y tratamiento de enfermedades neurológicas. MCCGC acknowledges FCT for the PhD grant SFRH/BD/131959/2017. The Portuguese Foundation for Science andTechnology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorteoperation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund underthe scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.lwt.2020.109322., Until now the prebiotic potential of pure trisaccharide raffinose on human health assessed through high-throughput sequencing remains poorly investigated. In this work, an in vitro model using human fecal inocula of two healthy volunteers (D1 and D2) was used to study the prebiotic potential of raffinose and compare it with the well-stablished and commercial prebiotic lactulose. The intestinal microbiota showed preference for raffinose as substrate showing the highest consumption value at 48h (96.0±0.9% D1 and 95.3±0.7% D2). The fermentation of raffinose decreased the medium pH, the ammonia concentration and the relative amount of Proteobacteria, while increasing the total production of lactate and short chain fatty acids (129.9±2.6mmol/L D1 and 179.6±0.6mmol/L D2), CO2 (10.8±0.8 mmol/Lmedium D1 and 5.2±0.3 mmol/Lmedium D2) and the relative amount of Bifidobacterium and Lactobacillus. This study suggests that raffinose holds potential functional properties for human health., CA an BBC acknowledge her grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from Portuguese Foundation for Science andTechnology (FCT). The study receivedfinancial support from FCT underthe scope of the strategic funding of UID/BIO/04469/2020 unit;COMPETE 2020 (POCI-01-0145-FEDER-006684), through nationalfunds and where applicable co-financed by the FEDER, within thePT2020 Partnership Agreement; the Projects FoSynBio (POCI-01-0145-FEDER-029549) and NewFood–Food Technologies Valorization(NORTE-324 01-0246-FEDER-000043). The authors also acknowledgeBioTecNorte operation (NORTE-01-0145-FEDER-000004) funded bythe European Regional Development Fund under the scope ofNorte2020 -Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

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

Sugar fatty acid esters (SFAEs) are composed of one or more fatty acid chains linked to a carbohydrate molecule. They are commonly used as non-ionic and biodegradable surfactants and emulsifiers. However, SFAEs also present other interesting features such as tasteless, odorless, non-toxicity, and bioactivity which make them suitable for industrial applications in the food, pharmaceutical, and cosmetics industries. Traditionally, SFAEs are obtained from chemical synthesis despite the recognized disadvantages associated to this process. More recently, the synthesis of SFAEs using enzymes as biocatalysts proved to be a suitable and greener alternative. However, the use of organic solvents as reaction medium has been pointed out as one of the greatest limitations of the enzymatic process. To overcome this issue, ionic liquids (ILs) have been proposed as alternative solvents. ILs are frequently described as “tunable” solvents due to the possibility of combining different cations and anions to obtain the desired physicochemical properties. Based on their unique characteristics, ILs have been recognized as suitable solvents for biocatalytic processes involving different enzymes and reactions. Nevertheless, the selection of the suitable IL for the enzymatic synthesis of SFAEs is considered a key aspect in the bioprocess since compounds chemically different (sugars, fatty acids, and enzymes) are simultaneously present in the reaction medium. Ideally, the IL used as solvent should facilitate the solubilization of the substrates and not negatively affect the enzymatic activity and stability. The enzymatic synthesis of SFAEs can occur though two different approaches, namely esterification and transesterification. Several ILs and biocatalysts have been successfully reported for the synthesis of different types of SFAEs. Considering the enormous versatility of combining different sugars with fatty acids, it is expected that novel and promising SFAEs could be synthesized in the future using ILs as reaction medium.

Brewers spent grain (BSG) is an inexpensive and abundant brewery by-product that can be used to produce prebiotic arabino-xylooligosaccharides (AXOS). In this study, Bacillus subtilis 3610 was used, for the first time, to produce AXOS through direct fermentation of BSG. Additionally, the microorganism was genetically modified to improve the AXOS production. The xylanase gene xyn2 from Trichoderma reesei coupled with a secretion tag endogenous to B. subtilis was cloned in pDR111 and integrated into its chromosome. After optimization by experimental design, AXOS with a degree of polymerization ranging from 2 to 6 were obtained. The maximum production yield expressed in xylose equivalents per amount of BSG (54.2 ± 1.1 mg/g) represents an increase of 33% comparing to the wild type. When compared with the enzymatic hydrolysis process, single-step fermentation with B. subtilis proved to be a very promising low-cost strategy for the simultaneous production of AXOS and valorization of BSG., CA, SCS, EC and LRR acknowledge their grants (PD/BD/105870/ 2014, SFRH/BPD/88584/2012, SFRH/BPD/70589/2010 and SFRH/ BSAB/142873/2018) from Portuguese Foundation for Science and Technology(FCT).Thestudyreceived financialsupportfromFCTunder the scope of the strategic funding of UID/BIO/04469/2013 unit; COMPETE2020 (POCI-01-0145-FEDER-006684); QOPNAresearch Unit (FCT UID/QUI/00062/2013), through national founds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the projects MultiBiorefinery (POCI-01-0145-FEDER016403); FoSynBio (POCI-01-0145-FEDER-029549) and Lignozymes (POCI-01-0145-FEDER-029773). The authors also acknowledge financial support from the BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

"Available online 21 November 2017", The antimicrobial and anti-adhesive activities of the cell-bound biosurfactants, produced by Lactobacillus pentosus (PEB), characterized as glycolipopeptide macromolecules, were evaluated against several microorganisms present in the skin microflora, envisaging its potential use as a natural ingredient in cosmetic and personal care formulations. Their performance was compared with another cell-bound biosurfactants also characterized as glycolipopeptides produced by Lactobacillus paracasei (PAB). At concentrations of 50 mg/mL, the PEB showed an important antimicrobial activity against Pseudomonas aeruginosa (85% when extracted with phosphate buffer (PB) and 100% when extracted with phosphate buffer saline (PBS)), Streptococcus agalactiae (100% for both extracts), Staphylococcus aureus (67% when extracted with PBS and 100% when extracted with PB), Escherichia coli (72% when extracted with PB and 89% when extracted with PBS), Streptococcus pyogenes (about 85% for both extracts) and Candida albicans (around 70% for both extracts), comparable with that obtained for the PAB. However, at lower concentrations the PAB exhibited in general higher antimicrobial activities. Biosurfactants produced by both microorganisms also showed significant anti-adhesive properties against all the microorganisms under study, except for E. coli and C. albicans (less than 30%). Overall, these cell-bound biosurfactants could be used as potential antimicrobial and anti-adhesive agents in cosmetic and pharmaceutical formulations., 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, COMPETE 2020 (POCI01-0145-FEDER-006684), the Project MultiBiorefinery − Multipurposestrategies for broadband agro-forest and fisheries byproducts valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403) and the project RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462), as well as X. Vecino post-doctoral grant (SFRH/BPD/101476/2014). Also, L. Rodríguez-López acknowledges to the Spanish Ministry of Education, Culture and Sport for her pre-doctoral fellowship (FPU15/00205)., info:eu-repo/semantics/publishedVersion

Cosmetic and personal care products including toothpaste, shampoo, creams, makeup, among others, are usually formulated with petroleum-based surfactants, although in the last years the consume trend for green products is inducing the replacement of surface-active agents in these formulations by natural surfactants, so-called biosurfactants. In addition to their surfactant capacity, many biosurfactants can act as good emulsifiers, which is an extra advantage in the preparation of green cosmetic products. In this work, a biosurfactant obtained from Lactobacillus paracasei was used as a stabilizing agent in oil-in-water emulsions containing essential oils and natural antioxidant extract. In the presence of biosurfactant, maximum percentages of emulsion volumes (EV = 100%) were observed, with droplets sizes about 199 nm. These results were comparable with the ones obtained using sodium dodecyl sulfate (SDS), a synthetic well known surfactant with high emulsify capacity. Moreover, the biosurfactant and emulsions cytotoxicity was evaluated using a mouse fibroblast cell line. Solutions containing 5 g/L of biosurfactant presented cell proliferation values of 97%, whereas 0.5 g/L of SDS showed a strong inhibitory effect. Overall, the results herein gathered are very promising towards the development of new green cosmetic formulations., 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, COMPETE 2020 (POCI-01-0145-FEDER-006684) and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), as well as X. Vecino post-doctoral grant (SFRH/BPD/101476/2014). Additionally, the authors acknowledge the financial support from Spanish Ministry of Economy and Competitiveness (FEDER funds) under the project CTM2015-68904. Also, A. Ferreira acknowledges to the Region Aquitaine Limousin Poitou-Charentes for her Erasmus + internship., info:eu-repo/semantics/publishedVersion

Studies on the specific and nonspecific interactions of biosurfactants with proteins are broadly relevant given the potential applications of biosurfactant/protein systems in pharmaceutics and cosmetics. The aim of this study was to evaluate the interactions of divalent counterions with the biomolecular anionic biosurfactant surfactin-C15 through molecular modeling, surface tension and dynamic light scattering (DLS), with a specific focus on its effects on biotherapeutic formulations. The conformational analysis based on a semi-empirical approach revealed that Cu2+ ions can be coordinated by three amide nitrogens belonging to the surfactin-C15 cycle and one oxygen atom of the aspartic acid from the side chain of the lipopeptide. Backbone oxygen atoms mainly involve Zn2+, Ca2+ and Mg2+. Subsequently, the interactions between metal-coordinated lipopeptide complexes and bovine serum albumin (BSA) were extensively investigated by fluorescence spectroscopy and molecular docking analysis. Fluorescence results showed that metal-lipopeptide complexes interact with BSA through a static quenching mechanism. Molecular docking results indicate that the metal-lipopeptide complexes are stabilized by hydrogen bonding and van der Waals forces. The biosurfactant-protein interaction properties herein described are of significance for metal-based drug discovery hypothesizing that the association of divalent metal ions with surfactin allows its interaction with bacteria, fungi and cancer cell membranes with effects that are similar to those of the cationic peptide antibiotics., This work was supported by the National Science Centre, Poland, projects 2018/02/X/NZ6/02201 and 2017/26/E/NZ9/00975. Publication supported by Wrocław Centre of Biotechnology, program the Leading National Research Centre (KNOW) for years 2014–2018., info:eu-repo/semantics/publishedVersion

Colorectal cancer is frequently diagnosed at an advanced stage due to the absence of early clinical indicators. Hence, the identification of new targeting molecules is crucial for an early detection and development of targeted therapies. This study aimed to identify and characterize novel peptides specific for the colorectal cancer cell line RKO using a phage-displayed peptide library. After four rounds of selection plus a negative step with normal colorectal cells, CCD-841-CoN, there was an obvious phage enrichment that specifically bound to RKO cells. Cell-based enzyme-linked immunosorbent assay (ELISA) was performed to assess the most specific peptides leading to the selection of the peptide sequence CPKSNNGVC. Through fluorescence microscopy and cytometry, the synthetic peptide RKOpep was shown to specifically bind to RKO cells, as well as to other human colorectal cancer cells including Caco-2, HCT 116 and HCT-15, but not to the normal non-cancer cells. Moreover, it was shown that RKOpep specifically targeted human colorectal cancer cell tissues. A bioinformatics analysis suggested that the RKOpep targets the monocarboxylate transporter 1, which has been implicated in colorectal cancer progression and prognosis, proven through gene knockdown approaches and shown by immunocytochemistry co-localization studies. The peptide herein identified can be a potential candidate for targeted therapies for colorectal cancer., 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 and the Project FCOMP-01–0124-FEDER-021053 (PTDC/SAU-BMA/121028/2010). Débora Ferreira is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Franklin L. Nobrega, Sara Granja and Ligia R Rodrigues acknowledge FCT for the grants SFRH/BD/86462/2012, SFRH/BPD/117858/2016 and SFRH/BSAB/142991/2018, respectively. Catarina Barbosa-Matos also acknowledge her research grant UMINHO/BI/395/2018, info:eu-repo/semantics/publishedVersion

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2018.09.088., The global demand of prebiotics such as xylooligosaccharides (XOS) has been growing over the years, motivating the search for different production processes with increased efficiency. In this study, a cloned Bacillus subtilis 3610, containing the xylanase gene xyn2 of Trichoderma reesei coupled with an endogenous secretion tag, was selected for XOS production through direct fermentation of beechwood xylan. A mixture of XOS with a degree of polymerization ranging from 4-6 was obtained, presenting high stability after a static in vitro digestion (98.5±0.2%). The maximum production yield expressed as total XOS per amount of xylan (306±4mg/g) was achieved after 8h of fermentation operating under one-time impulse fed-batch. The optimal conditions found were pH 6.0 and 42.5°C, using 2.5g/L of initial concentration of xylan increased up to 5.0g/L at 3h. Xylopentaose was the major oligosaccharide produced, representing 47% of the total production yield., CA, SCS, ACP, EC and LRR acknowledge their grants (PD/BD/ 105870/2014, SFRH/BPD/88584/2012, SFRH/BPD/ 101181/2014, SFRH/BPD/70589/2010 and SFRH/BSAB/142873/2018) from Portuguese Foundation for Science and Technology (FCT). The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit; COMPETE 2020 (POCI-010145-FEDER-006684); QOPNA research Unit (FCT UID/QUI/00062/ 2013), through national founds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Project MultiBiorefinery (POCI-01-0145-FEDER-016403); the Project FoSynBio (POCI-01-0145-FEDER-029549) and Lignozymes (POCI-01-0145FEDER-029773). The authors also acknowledge 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

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.foodchem.2018.07.080., Xylooligosaccharides (XOS) are prebiotic nutraceuticals that can be sourced from lignocellulosic biomass, such as agro-residues. This study reports for the first time an optimization study of XOS production from agro-residues by direct fermentation using two Trichoderma species. A total of 13 residues were evaluated as potential substrates for single-step production. The best results were found for Trichoderma reesei using brewers spent grain (BSG) as substrate. Under optimal conditions (3 days, pH 7.0, 30°C and 20 g/L of BSG), a production yield of 38.3 ± 1.8 mg/g (xylose equivalents/g of BSG) was achieved. The obtained oligosaccharides were identified as arabino-xylooligosacharides (AXOS) with degree of polymerization from 2 to 5. One-step fermentation proved to be a promising strategy for AXOS production from BSG, presenting a performance comparable with the use of commercial enzymes. This study provides new insights towards the bioprocess integration, enabling further developments of low-cost bioprocesses for the production of these valuable compounds., 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-FEDER006684) and under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project MultiBiorefinery – Multi purpose strategies for broadband agro forest and fisheries by products valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403) the Project Lignozymes (POCI-010145-FEDER-029773) and the Project FoSynBio (POCI-01-0145FEDER-029549). The authors also acknowledge financial support from BioTecNorte (NORTE-01-0145-FEDER-000004) Project BioInd – Biotechnology and Bioengineering for improved Industrial and AgroFood processes(NORTE-07–0124-FEDER-000028) funded by the European Regional Development Fund under the scope of Norte2020 Programa Operacional Regional do Norte. CA and SCS acknowledge their grants (PD/BD/105870/2014 SFRH/BPD/88584/2012) from FCT., info:eu-repo/semantics/publishedVersion

An integrated process enabling the simultaneous production and purification of fructo-oligosaccharides (FOS) was explored. A co-culture fermentation with Aspergillus ibericus (used as FOS producer strain) and Saccharomyces cerevisiae YIL162W (for small saccharides removal) was optimized. Inoculation conditions of S. cerevisiae, fermentative broth composition, temperature and pH were optimized by experimental design. Yeast extract concentration and temperature were the most significant variables affecting FOS purity. Co-culture fermentations with simultaneously inoculation of the strains, run under 30°C, initial pH 6.0 and 17gL1 yeast extract led to FOS mixtures with 97.4±0.2% (w/w) purity. The fermentations conducted in bioreactor, at a 0.8 vvm aeration rate, yielded 0.70±0.00gFOS.ginitial GF1 at 45h fermentation, with a FOS content of 133.7±0.1gL1. A purity of FOS up to 93.8±0.7% (w/w) was achieved. The one-step fermentation proved to be efficient, economical and fast., Clarisse Nobre and Lígia R. Rodrigues acknowledgethe Portuguese Foundation for Science and Technology (FCT) for the Grants reference SFRH/BPD/87498/2012 and SFRH/BSAB/142873/2018. This study was supported by the 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 also, Project ColOsH 02/SAICT/ 2017 (POCI-01-0145-FEDER-030071), MultiBiorefinery SAICTPAC/0040/2015 (POCI-01-0145-FEDER-016403), Biomass and Bioenergy Research Infrastructure 01/SAICT/2016 (PINFRA/22059/2016) and Project FoSynBio 02/SAICT/2017 (POCI-01-0145-FEDER-029549)., info:eu-repo/semantics/publishedVersion

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.bej.2021.107954., Aureobasidium pullulans URM 7059 produced esterase using residual glycerol from biodiesel as the sole carbon source. The culture medium containing residual glycerol (0.1% v/v), (NH4)2SO4 (4g/L), and yeast extract (8g/L) resulted in the highest esterase production using shake-flasks. The enzyme exhibited a molar mass of 50kDa and was stable at neutral pH and temperatures below 30°C. The cations Cu2+ and Al3+ did not affect the esterase activity, while Ca2+ promoted the highest activity loss. The enzyme kinetic parameters were determined using different substrates (p-nitrophenylcaprylate and p-nitrophenylbutyrate). Km and Vmax were 1.4mM and 218µmolmin-1 for p-NPC, and 1.55mM and 76.7µmolmin-1 for p-NPB. The esterase production was further evaluated using stirred tank and 2-L airlift bioreactors. The airlift reactor operating at the highest air flow rate (8L/min) increased the enzyme productivity 3-fold compared to the shake-flasks experiments. However, the crude enzymatic extract showed 3 active protein bands by zymography with molecular masses of 172kDa, 66kDa, and 40kDa approximately, suggesting that the pattern of enzyme production changed due to aeration. The crude enzyme degraded the MACO-Sty biopolymer in 14 days, being stable in a wide range of pH (7.0 9.0) and temperatures (40°C 80°C). The results suggest that this enzyme is a promising catalyst in remediation processes., This study 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), projects MultiBiorefinery (POCI-01-0145-FEDER016403) and Lignozymes (POCI-01-0145-FEDER-029773), all funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. In Brazil, this study was financed in part by the Coordenaçao ˜ de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The authors also thank the CNPq and FUNCAP for financial support., info:eu-repo/semantics/publishedVersion

Breast cancer is the most common type of cancer affecting women. Despite the good prognosis when detected early, significant challenges remain in the treatment of metastatic breast cancer. The recruitment of the vacuolar H+-ATPase (V-H+-ATPase) to the plasma membrane, where it mediates the acidification of the tumor microenvironment (TME), is a recognized feature involved in the acquisition of a metastatic phenotype in breast cancer. Therefore, inhibitors of this pump have emerged as promising anticancer drugs. Lactoferrin (Lf) is a natural pro-apoptotic iron-binding glycoprotein with strong anticancer activity whose mechanism of action is not fully understood. Here, we show that bovine Lf (bLf) preferentially induces apoptosis in the highly metastatic breast cancer cell lines Hs 578T and MDA-MB-231, which display a prominent localisation of V-H+-ATPase at the plasma membrane, but not in the lowly metastatic T-47D or in the non-tumorigenic MCF-10-2A cell lines. We also demonstrate that bLf decreases the extracellular acidification rate and causes intracellular acidification in metastatic breast cancer cells and, much like the well-known proton pump inhibitors concanamycin A and bafilomycin A1, inhibits V-H+-ATPase in sub-cellular fractions. These data further support that bLf targets V-H+-ATPase and explain the selectivity of bLf for cancer cells, especially for highly metastatic breast cancer cells. Altogether, our results pave the way for more rational in vivo studies aiming to explore this natural non-toxic compound for metastatic breast cancer therapy., This study was supported by national funds through FCT I.P and the European Community fund ERDF, through Program COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI), under the scope of the strategic programmes UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and UID/BIO/04469/2013; by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, ERDF through project RECI/BBB EBI/0179/2012 (FCOMP-01-0124-FEDER-027462); as well as by FCT through the program PIDDAC (project FCT-ANR/ BEX-BCM/0175/2012).

Bacterial cellulose (BC) films from two distinct sources (obtained by static culture with Gluconacetobacter xylinus ATCC 53582 (BC1) and from a commercial source (BC2)) were modified by bovine lactoferrin (bLF) adsorption. The functionalized films (BC+bLF) were assessed as edible antimicrobial packaging, for use in direct contact with highly perishable foods, specifically fresh sausage as a model of meat products. BC+bLF films and sausage casings were characterized regarding their water vapour permeability (WVP), mechanical properties, and bactericidal efficiency against two food pathogens, Escherichia coli and Staphylococcus aureus. Considering their edibility, an in vitro gastrointestinal tract model was used to study the changes occurring in the BC films during passage through the gastrointestinal tract. Moreover, the cytotoxicity of the BC films against 3T3 mouse embryo fibroblasts was evaluated. BC1 and BC2 showed equivalent density, WVP and maximum tensile strength. The percentage of bactericidal efficiency of BC1 and BC2 with adsorbed bLF (BC1+bLF and BC2+bLF, respectively) in the standalone films and in inoculated fresh sausages, was similar against E. coli (mean reduction 69 % in the films per se versus 94 % in the sausages) and S. aureus (mean reduction 97 % in the films per se versus 36 % in the case sausages). Moreover, the BC1+bLF and BC2+bLF films significantly hindered the specific growth rate of both bacteria. Finally, no relevant cytotoxicity against 3T3 fibroblasts was found for the films before and after the simulated digestion. BC films with adsorbed bLF may constitute an approach in the development of bio-based edible antimicrobial packaging systems., The authors would like to acknowledge Portuguese Foundation for Science and Technology (Fundação para a Ciência e Tecnologia) for the research grants: Jorge Padrão SFRH/BD/64901/2009, Sara Gonçalves SFRH/BD/63578/2009, João Pedro Silva SFRH/BPD/ 64958/2009, Ana Cristina Pinheiro SFRH/BPD/101181/2014. V. Sencadas thanks support from the COST Action MP1206: “Electrospun nano-fibres for bio inspired composite materials and innovative industrial applications” and MP1301: “New Generation Biomimetic and Customized Implants for Bone Engineering”. The authors would also like to thank the co-funded by the Programa Operacional Regional do Norte (ON.2 e O Novo Norte), QREN, FEDER Projects “BioHealth e Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124- FEDER-000027; “BioInd e Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07- 0124-FEDER-000028; Strategic Project PEST-C/FIS/UI607/2014; Matepro eOptimizing Materials and Processes”, ref. NORTE-07- 0124-FEDER-000037; Strategic Project PEst-OE/EQB/LA0023/2013 and project ref. RECI/BBB-EBI/0179/2012 (project number FCOMP- 01-0124-FEDER-027462). Finally, the authors thank the Fundação para a Ciência e Tecnologia for the strategic funding from the UID/ BIO/04469/2013 unit.

Fructo-oligosaccharides (FOS) obtained by fermentation of sucrose may be purified at large-scale by continuous chromatography (Simulated Moving Bed: SMB). In order to improve the efficiency of the subsequent SMB purification, the optimization of the fermentative broth composition in salts and sugars was investigated. Fermentations conducted at reduced amount of salts, using Aureobasidium pullulans whole cells, yielded 0.63 ± 0.03 g of FOS per gram of initial sucrose. Additionally, a microbial treatment was proposed to reduce the amount of small saccharides in the mixture. Two approaches were evaluated, namely a co-culture of A. pullulans with Saccharomyces cerevisiae; and a two-step fermentation in which FOS were first synthesized by A. pullulans and then the small saccharides were metabolised by S. cerevisiae. Assays were performed in 100 mL shaken flasks and further scaled-up to a 3 L working volume bioreactor. Fermentations in two-step were found to be more efficient than the co-culture ones. FOS were obtained with a purity of 81.6 ± 0.8% (w/w), on a dry weight basis, after the second-step fermentation with S. cerevisiae. The sucrose amount was reduced from 13.5 to 5.4% in total sugars, which suggests that FOS from this culture broth will be more efficiently separated by SMB., The financial support from the F.R.S.-FNRS, the Belgium National Fund for the Scientific Research, (PDR: T.0196.13) is gratefully acknowledged. Clarisse Nobre acknowledges to Fundacao para a Ciencia e a Tecnologia (Portugal) and POPH/FSE for the Post-Doc Grant received (reference SFRH/BPD/87498/2012). The authors acknowledge the financial support from the Strategic funding of UID/BIO/04469/2013 unit and project ref. RECI/BBB-EBI/0179/2012 (project number FCOMP-01-0124-FEDER-027462) funded by Fundacao para a Ciencia e a Tecnologia.

Lignocellulosic materials require pretreatment to remove lignin enabling the enzyme access to the cellulose. This work used multivariate analysis to investigate the acid and alkali pretreatments of vine pruning followed by enzymatic hydrolysis. The best acid pretreatment conditions were H2SO4 1.5%, 120 °C for 30 min, removing 68.7% of hemicellulose, enabling 95.8% of cellulose recovery. However, this treatment was not enough to allow the enzyme hydrolysis. A second step of treatment with NaOH 3.0% at 120 °C without agitation for 60 min led to a material with 75.0% of cellulose and 25.0% of lignin. However, the lowest glucose yield (80.86% and 32.26 g L?1 of glucose) was obtained after the enzyme hydrolysis of this material. The highest glucose yield (98.72% with 35.06 g L?1) was obtained using a pretreated material containing 68.1% of cellulose and 31.9% of lignin obtained after a milder condition (NaOH 2% at 100 °C), thus showing that not all the lignin need to be removed to obtain a high saccharification yield. A less severe pretreatment with no adverse effect on the glucose yield with the advantage of preserving the non-cellulose biomass fractions was effective for vine prune valorization., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, BioTecNorte operation (NORTE-01-0145-FEDER000004), the projects Multibiorefinery (POCI-01-0145-FEDER-016403), FoSynBio (POCI-01-0145-FEDER-029549) and Lignozymes (POCI-01- 0145-FEDER-029773) funded by the European Regional Development Fund under the scope of Norte 2020. In Brazil, this study was funded in part by the Coordenaçao ~ de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq) and Fundaçao Cearense de Apoio ao Desenvolvimento Científico e Tecnologico (FUNCAP). The authors would like to acknowledge the Central Analytical (Physical Department) of Federal University of Ceara for conducting the SEM analysis and the Centro de Tecnologia Canavieira – CTC/Brazil for the support. E. Gudina and L. Rodrigues acknowledge FCT for the Post-doctoral (CEB-BPD/01/2015/07) and sabbatical (SFRH/BSAB/142991/2019) grants, respectively., info:eu-repo/semantics/publishedVersion

Polyphenols are plant secondary metabolites with diverse biological and potential therapeutic activities such as antioxidant, anti-inflammatory and anticancer, among others. However, their extraction from the native plants is not enough to satisfy the increasing demand for this type of compounds. The development of microbial cell factories to effectively produce polyphenols may represent the most attractive solution to overcome this limitation and produce high amounts of these bioactive molecules. With the advances in the synthetic biology field, the development of efficient microbial cell factories has become easier, largely due to the development of the molecular biology techniques and by the identification of novel isoenzymes in plants or simpler organisms to construct the heterologous pathways. Furthermore, efforts have been made to make the process more profitable through improvements in the host chassis. In this review, advances in the production of polyphenols by genetically engineered Saccharomyces cerevisiae as well as by synthetic biology and metabolic engineering approaches to improve the production of these compounds at industrial settings are discussed., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 – North Portugal Regional Program. In addition, this research has been carried out at the Biomass and Bioenergy Research Infrastructure (BBRI)–LISBOA-010145-FEDER-022059, supported by Operational Program for Competitiveness and Internationalization (PORTUGAL2020), the Lisbon Portugal Regional Operational Program (Lisboa2020), and Norte2020 under the Portugal 2020 Partnership Agreement, through the ERDF. JR is recipient of a fellowship supported by a doctoral advanced training (SFRH/BD/138325/2018) funded by FCT., info:eu-repo/semantics/publishedVersion

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.carbpol.2019.115460., Xylooligosaccharides (XOS) are emergent prebiotics exhibiting high potential as food ingredients. In this work, in vitro studies were performed using human fecal inocula from two healthy donors (D 1 and D2) to evaluate the prebiotic effect of commercial lactulose and XOS produced in a single-step by recombinant Bacillus subtilis 3610. The fermentation of lactulose led to the highest production of lactate (D1: 33.7±0.5mM; D2:19.7±0.3mM) and acetate (D1: 77.5±0.6mM; D2: 81.0±0.7mM), while XOS led to the highest production of butyrate (D1: 9.0±0.6mM; D2: 10.5±0.8mM) and CO2 (D1: 8.92±0.02mM; D2: 11.4±0.3mM). Microbiota analysis showed a significant decrease in the relative abundance of Proteobacteria for both substrates and an increase in Bifidobacterium and Lactobacillus for lactulose, and Bacteroides for XOS., CA and BBC acknowledge their grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from the Portuguese Foundation for Science and Technology (FCT). The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2019 unit; COMPETE 2020 (POCI-01-0145-FEDER-006684), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Project FoSynBio (POCI-01-0145FEDER-029549), and NewFood (NORTE-01-0246-FEDER-000043). The authors also acknowledge BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110598., The interactions between two types of quaternary ammonium surfactants (N,N,N-trimethyl-2-(dodecanoyloxy)ethaneammonium bromide (DMM-11) and N,N,N-trimethyl-2-(dodecanoyloxy)propaneammonium bromide (DMPM-11)) and hen egg white lysozyme were studied through several techniques, including isothermal titration calorimetry (ITC), circular dichroism (CD) and fluorescence spectroscopy, and surface tension measurement. The average number of surfactants interacting with each molecule of lysozyme was calculated from the biophysical results. Moreover, the CD results showed that the conformation of lysozyme changed in the presence of DMM-11 and DMPM-11. The studies drew a detailed picture on the physicochemical nature of interactions between both surfactants and lysozyme. Both DMM-11 and DMPM-11, with and without lysozyme were studied against three target microorganisms, including Gram-negative (Escherichia coli) and Gram-positive (Enterococcus hirae and Enterococcus faecalis) bacteria. The results revealed a broad spectrum of antibacterial nature of surfactant/lysozyme complexes, as well as their effect on the membrane damage, hence providing the basis to further explore DMM-11 and DMPM-11 combined with lysozyme as possible antibacterial tools., The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2019 unit; COMPETE 2020 (POCI-01-0145-FEDER-006684), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement. The authors also acknowledge BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fundunder the scope of Norte2020 - Programa Operacional Regional do Norte. ITC measurements were performed at the Laboratory of Elemental Analysis and Structural Research located at the Faculty of Pharmacy and the Division of Laboratory Diagnostics, Wroclaw Medical University, supported by the ERDF Project within the Innovation Economy Operational Programme POIG.02.01.00-14-122/ 09., info:eu-repo/semantics/publishedVersion

The molecular interactions between two single-chain lysosomotropic surfactants DMM-11 (2-Dodecanoyloxyethyl)trimethylammonium bromide) and DMPM-11 (2-Dodecanoyloxypropyl)trimethylammonium bromide) with a small heme-protein (cytochrome c (cyt-c)) in Hepes buffer (pH = 7.4) were extensively investigated by surface tension, dynamic light scattering (DLS), circular dichroism (CD) and fluorescence spectroscopy in combination with molecular dynamic simulation techniques. The results demonstrated that surfactants can destroy the hydrophobic cavity of cyt-c, make the α-helical become loose and convert it into the β-sheet structure. The interactions between surfactants and cyt-c are mainly hydrophobic. Molecular modelling approaches were also used to gather a deeper insight on the binding of lysosomotropic surfactants with cyt-c and the in silico results were found to be in good agreement with the experimental ones. This study provides a molecular basis for the applications of protein-surfactant complexes in biological, food, pharmaceutical, industrial and cosmetic systems, Polish-Portugal Executive Program for years 2017–2018 sponsored by the Polish Ministry of Science and Higher Education and by the Portuguese Foundation for Science and Technology (FCT). Additionally, FCT supported the study under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). T. Janek was supported by the National Science Centre, Poland, projects 2017/26/E/NZ9/00975 and 2018/02/X/NZ6/02201. L. R. Rodrigues acknowledges FCT for the grant SFRH/BSAB/142873/2018. E. J. Gudiña was supported by the grant UMINHO/BPD/39/2015 funded by FCT under the scope of the project UID/BIO/04469/2013, info:eu-repo/semantics/publishedVersion

The present study aimed to explore the interactions of divalent counterions with biomolecular amphisin using circular dichroism (CD), ultravioletvisible (UVVis) and density functional theory (DFT). The binding mode of interactions between metal-amphisin complexes and bovine serum albumin (BSA) were studied using fluorescence spectroscopy. The results showed that Cu2+ is coordinated by one oxygen atom of the aspartic acid side chain and three amide nitrogen atoms, whereas Zn2+, Ca2+ and Mg2+ favour the association with backbone oxygen atoms of the amphisin. On the other hand, the aggregation of amphisin induced by divalent counterions was studied by dynamic light scattering (DLS). Our results revealed that the self-assembly process of amphisin can be controlled by the addition of metal ions. The results of CD spectra demonstrated that the binding of divalent counterions to the lipopeptide induces conformational changes in amphisin. Further studies using fluorescence spectroscopy showed that the metal-lipopeptide systems could interact with some functional groups of BSA, increasing the microenvironment around Trp residues of BSA. Thus, the interaction data acquired herein for the interesting class of complexes will be of significance in metal-based drug discovery and developmental research., This work was supported by Polish-Portugal Executive Program for years 2017–2018. Lígia Rodrigues acknowledges the Portuguese Foundation for Science and Technology (FCT) for the financial support under the scope of the strategic funding of SFRH/BSAB/142873/2018, UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). Żaneta Czyżnikowska gratefully acknowledges the allotment of the CPU time in Wroclaw Center of Networking and Supercomputing (WCSS)., info:eu-repo/semantics/publishedVersion

Prostate cancer and osteosarcoma are the second most common type of cancer affecting men and the fifth most common malignancy among adolescents, respectively. The use of non-toxic natural or natural-derived products has been one of the current strategies for cancer therapy, owing to the reduced risks of induced-chemoresistance development and absence of secondary effects. In this perspective, lactoferrin (Lf), a natural protein derived from milk, emerges as a promising anticancer agent due to its well-recognized cytotoxicity and anti-metastatic activity. Here, we aimed to ascertain the potential activity of bovine Lf (bLf) against highly metastatic cancer cells. The bLf effect on prostate PC-3 and osteosarcoma MG-63 cell lines, both displaying plasmalemmal V-ATPase, was studied and compared with the breast cancer MDA-MB-231 and the non-tumorigenic BJ-5ta cell lines. Cell proliferation, cell death, intracellular pH, lysosomal acidification and extracellular acidification rate were evaluated. Results show that bLf inhibits proliferation, induces apoptosis, intracellular acidification and perturbs lysosomal acidification only in highly metastatic cancer cell lines. In contrast, BJ-5ta cells are insensitive to bLf. Overall, our results establish a common mechanism of action of bLf against highly metastatic cancer cells exhibiting plasmalemmal V-ATPase. This study opens promising perspectives for further research on the anticancer role of Lf, which ultimately will contribute to its safer and more rational application in the human therapy of these life-threatening cancers., This study was supported by national funds through Fundação para a Ciência e Tecnologia (FCT) under the scope of the projects: UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569), UID/ BIO/04469/2013 (POCI-01-0145-FEDER-006684), FCT-ANR/ BEX-BCM/0175/2012, PEstOE/BIA/UI4050/2014, RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and PTDC/ SAU-BMA/121028/2010., info:eu-repo/semantics/publishedVersion

A great demand for prebiotics is driving the search for new sources of fructo-oligosaccharides (FOS) producers and for FOS with differentiated functionalities. In the present work, FOS production by a new isolated strain of Aspergillus ibericus was evaluated. The temperature of fermentation and initial pH were optimized in shaken flask to yield a maximal FOS production, through a central composite experimental design. FOS were produced in a one-step bioprocess using the whole cells of the microorganism. The model (R2 = 0.918) predicted a yield of 0.56, experimentally 0.53 ± 0.03 gFOS.ginitial sucrose1 was obtained (37.0 °C and a pH of 6.2). A yield of 0.64 ± 0.02 gFOS.ginitial sucrose1 was obtained in the bioreactor, at 38 h, with a content of 118 ± 4 g.L1 in FOS and a purity of 56 ± 3%. The chemical structure of the FOS produced by A. ibericus was determined by HPLC and NMR. FOS were identified as 1-kestose, nystose, and 1F-fructofuranosylnystose. In conclusion, A. ibericus was found to be a good alternative FOS producer., Clarisse Nobre acknowledges the Portuguese Foundation forScience and Technology (FCT) for her Post-Doc Grant [ref. SFRH/BPD/87498/ 2012] and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124FEDER-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., info:eu-repo/semantics/publishedVersion

The bifidogenic potential of fructo-oligosaccharides (FOS) produced by a newly isolated strain Aspergillus ibericus was studied. Their activity was compared to FOS produced by Aureobasidium pullulans and to a non-microbial commercial FOS sample (Raftilose® P95). FOS fermentability by a number of probiotic bacteria and their hydrolytic resistance to the simulated harsh conditions of the digestive system was evaluated. Aspergillus ibericus FOS sample effectively promoted probiotic bacteria growth. Overall, microbial-derived FOS promoted greater cellular growth compared to the commercial sample. FOS fermentation was both substrate and strain specific. The FOS structural differences identified may explain their distinct assimilation by the probiotics. [Fru(26)Glc] (possibly blastose) and a reducing trisaccharide (possibly [Fru(26)Glc(12)Fru], neokestose) were only found in microbial-derived FOS samples, while Raftilose® P95 was richer in inulobiose/inulotriose. 1-Kestose and nystose were only slightly hydrolyzed in the presence of gastric and intestinal fluid. FOS synthesized by Aspergillus exhibited great potential as food ingredients with likely prebiotic features., Clarisse Nobre, Sérgio Sousa, Elisabete Coelho and Ana Pinheiro acknowledge the Portuguese Foundation for Science and Technology (FCT) for their Post-Doc and Doc Grants (SFRH/BPD/87498/2012, SFRH/BD/105304/2014, SFRH/BPD/70589/2010, and SFRH/BPD/101181/2014, respectively). This study was supported by the 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, also QOPNA research Unit (FCT UID/QUI/00062/2013) through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement, project Essence (POCI-01-0247-FEDER-010887) for financial support of Post-Doc grant of Elisabete Coelho and, multi‐purpose strategies for broadband agro‐forest and fisheries by‐products valorisation: a step forward for a truly integrated biorefinery SAICTPAC/0040/2015 (POCI-01-0145-FEDER-016403), Biomass and Bioenergy Research Infrastructure (01/SAICT/2016), also CBQF research centre by National Funds from FCT through project UID/Multi/50016/2013., info:eu-repo/semantics/publishedVersion

The increase of antibiotic resistant microorganisms urged the development and synthesis of novel antimicrobial biomaterials to be employed in a broad range of applications, ranging from food packaging to medical devices. This work describes the production and characterization of a protein-based electrospun fibrous membranes bearing antimicrobial properties. Its composition is exclusively comprised of proteins, with fish gelatine as structural matrix and bovine lactoferrin (bLF) as the active antimicrobial agent. The bLF bactericidal effect was determined against clinical isolates of Escherichia coli and Staphylococcus aureus through microdilution assays. Two distinctive methods were used to incorporate bLF into the fish gelatine nanofibres: (i) as a filler in the electrospinning formulation with concentrations of 2, 5 and 10 (wt%), and cross-linked with glutaraldehyde vapour, in order to achieve stability in aqueous solution; and (ii) through adsorption in a solution with 40 mg mL1 bLF. Fourier transform infrared spectroscopy analysis showed that the structure of both proteins remained intact through the electrospinning blending and cross-linking procedure. Remarkable antibacterial properties were obtained with membranes containing 5% and 10% bLF with a bacterial reduction of approximately 90% and 100%, respectively., Portuguese Foundation for Science and Technology (FCT) Strategic Project PEst-OE/EQB/LA0023/ 2013 UID/BIA/04050/2013, UID/BIO/04469/2013, and for the research grants funding through POPH/FSE. FEDER funds through the “Programa Operacional Fatores de Competitividade – COMPETE” and by national funds arranged by FCT FCT, project ref- erences PEST-C/FIS/UI607/2014, Matepro – Optimizing Materials and Processes”, ref. NORTE-07-0124-FEDER-000037”, RECI/BBB- EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the project “BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124-FEDER-000028, co-funded by the “Programa Operacional Regional do Norte” (ON.2 – O Novo Norte), under the “Quadro de Referência Estratégico Nacional” (QREN), through the “Fundo Europeu de Desenvolvimento Regional” (FEDER). COST Action MP1206 “Electrospun Nano-fibres for bio inspired composite materials and innovative industrial applications”.

Electrochemical aptasensors may be used to detect protein biomarkers related to tumor activity. Osteopontin (OPN), a protein present in several body fluids, has been suggested as a potential biomarker since its overexpression seems to be associated with breast cancer progression and metastasis. In this work, a simple and label-free voltammetric aptasensor for the detection of OPN, using an RNA aptamer previously reported to have affinity for human OPN as the molecular recognition element, and the ferro/ferricyanide solution as a redox probe, was developed. The RNA aptamer was synthetized and immobilized in a working microelectrode gold surface (diameter of 0.8 mm) of a screen-printed strip with a silver pseudo-reference electrode and a gold counter electrode. The electrochemical behavior of the electrode surface after each preparation step of the aptasensor was studied using cyclic voltammetry and square wave voltammetry. The resulting voltammetric aptasensor was used to detect OPN in standard solutions. Cyclic voltammetry results showed that the aptasensor has reasonable detection and quantification limits (3.7±0.6 nM and 11±2 nM, respectively). Indeed, the detection limit falls within the osteopontin levels reported in the literature for patients with metastatic breast cancer. Moreover, the aptasensor is able to selectively detect the target protein in the presence of other interfering proteins, except for thrombin. Considering the overall results, a possible application of the aptasensor for cancer prognosis may be foreseen in a near future., FCT and FEDER under Program PT2020 (Project UID/EQU/50020/2013); by the Strategic Project PEst-OE/EQB/LA0023/2013 and by the project ref. RECI/BBB-EBI/ 0179/2012 (project number FCOMP-01-0124-FEDER-027462) funded by FCT, Fundação para a Ciência e a Tecnologia(FCT) through the PhD grant SRFH/BD/65021/2009.

Curcuminoids, components of the rhizome of turmeric, show several beneficial biological activities, including anticarcinogenic, antioxidant, anti-inflammatory, and antitumor activities. Despite their numerous pharmaceutically important properties, the low natural abundance of curcuminoids represents a major drawback for their use as therapeutic agents. Therefore, they represent attractive targets for heterologous production and metabolic engineering. The understanding of biosynthesis of curcuminoids in turmeric made remarkable advances in the last decade, and as a result, several efforts to produce them in heterologous organisms have been reported. The artificial biosynthetic pathway (e.g., in Escherichia coli) can start with the supplementation of the amino acid tyrosine or phenylalanine or of carboxylic acids and lead to the production of several natural curcuminoids. Unnatural carboxylic acids can also be supplemented as precursors and lead to the production of unnatural compounds with possibly novel therapeutic properties. In this paper, we review the natural conversion of curcuminoids in turmeric and their production by E. coli using an artificial biosynthetic pathway. We also explore the potential of other enzymes discovered recently or already used in other similar biosynthetic pathways, such as flavonoids and stilbenoids, to increase curcuminoid yield and activity., We acknowledge financial support from the Strategic Project PEst-OE/EQB/LA0023/2013, project reference RECI/BBB-EBI/0179/2012 (project number FCOMP-01-0124-FEDER-027462), project SYNBIOBACTHER (PTDC/EBB-BIO/102863/2008), and a doctoral grant (SFRH/BD/51187/ 2010) to J. L. Rodrigues, funded by Fundacao para a Ciencia e a Tecnologia. We thank the MIT-Portugal Program for support given to J. L. Rodrigues.

Cellulosic sugars extracted from vineyard pruning waste (VPW) were used as a low-cost carbon source for biosurfactant production by Lactobacillus paracasei. The results obtained showed that when glucose from VPW was used, the biosurfactant was a glycolipopeptide, whereas when it was replaced by lactose the biosurfactant produced was a glycoprotein. Additionally, it was found that the extraction process, either with phosphate-buffer or phosphate-buffer saline, influenced the biosurfactant chemical structure and emulsion capacity. Overall, these results highlight the possibility of producing biosurfactants à la carte with the same strain but changing the carbon source, increasing its potential in different industrial applications., 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, COMPETE 2020 (POCI-01-0145-FEDER-006684) and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), as well as X. Vecino post-doctoral grant (SFRH/BPD/101476/2014). The authors also acknowledge financial support from BioTecNorte operation (NORTE-01-0145-FEDER-000004) and the project MultiBiorefinery (POCI-01-0145-FEDER-016403) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and the Post-doctoral grant CEB-BPD/01/2015/07 from the project UID/BIO/04469/2013, funded by FCT, to E. Gudiña. Additionally, the authors acknowledge the financial support from Spanish Ministry of Economy and Competitiveness (FEDER funds under the project CTM2015-68904) and L. Rodríguez-López acknowledges to the Spanish Ministry of Education, Culture and Sport for her pre-doctoral fellowship (FPU15/00205)., info:eu-repo/semantics/publishedVersion

The interactions between two cationic lysosomotropic surfactants (2-dodecanoyloxyethyl)trimethylammonium bromide (DMM-11) and (2-dodecanoyloxypropyl)trimethylammonium bromide (DMPM-11) with bovine serum albumin (BSA) in Hepes buffer (pH = 7.4) were systematically studied by surface tension, fluorescence and circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). Furthermore, the size of the micellar aggregates and the polydispersity indexes of both cationic surfactants were studied by dynamic light scattering technique (DLS). The hydrodynamic radii, micellar volumes and aggregation numbers were calculated using a method based on density functional theory (DFT). The results showed that, in both cases, the surface tension was modified upon addition of BSA, and the critical micelle concentration (CMC) values of DMM-11 and DMPM-11 were higher in the presence of BSA. The fluorescence intensity of BSA decreased significantly as the concentration of both cationic surfactants increased and this effect was attributed to the formation of surfactant-BSA complexes. Synchronous fluorescence spectrometry showed the binding-induced conformational changes in BSA. Finally, CD and DLS results revealed the occurrence of changes in the secondary structure of the protein in the presence of both surfactants. In conclusion, understanding the interactions between lysosomotropic surfactants and BSA is required to explore their potential applications in medicine., Author Żaneta Czyżnikowska gratefully acknowledge the allotment of the CPU time in Wroclaw Center of Networking and Supercomputing (WCSS). All the ITC experiments were performed by Joanna Gałęzowska in the Laboratory of Elemental Analysis and Structural Research, Faculty of Pharmacy and the Division of Laboratory Diagnostics, Wroclaw Medical University, supported by the ERDF Project within the Innovation Economy Operational Programme POIG.02.01.00-14-122/09”. Ligia R. Rodrigues and Eduardo J. Gudiña acknowledge the Portuguese Foundation for Science and Technology (FCT) for the financial support 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. The authors also thank the FCT for the financial support under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). This work was supported by Polish-Portugal Executive Program for years 2017-2018., info:eu-repo/semantics/publishedVersion

In this study, the biosurfactant production by an Aureobasidium thailandense LB01 was reported for the first time. Different agro-industrial by-products (corn steep liquor, sugarcane molasses, and olive oil mill wastewater) were evaluated as alternative low-cost substrates. The composition of the culture medium was optimized through response surface methodology. The highest biosurfactant production (139 ± 16 mg/L) was achieved using a culture medium containing yeast extract (2 g/L); olive oil mill wastewater (1.5%, w/w); glucose (6 g/L) and KH2PO4 (1 g/L) after 48 h of fermentation. The partially purified biosurfactant exhibited a critical micelle concentration of 550 mg/L, reducing the surface tension of water up to 31.2 mN/m. Its molecular structure was found to be similar to a lauric acid ester. The biosurfactant exhibited a better performance than the chemical surfactant sodium dodecyl sulfate (SDS) in oil dispersion assays, thus suggesting its potential application in bioremediation., The authors acknowledge the Biotechnology laboratory (UFC) and Doctor Tatiana Nunes, as well as CAPES, CNPq and FUNCAP for the financial support. 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 under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project MultiBiorefinery − Multi-purpose strategies for broadband agro-forest and fisheries by-products valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403). The authors also acknowledge financial support from BioTecNorte operation (NORTE-01-0145-FEDER-000004) and Project BioInd − Biotechnology and Bioengineering for improved Industrial and Agro-Food processes (NORTE-07–0124-FEDER-000028) funded by the European Regional Development Fund under the scope of Norte2020 − Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

In this work, the antifungal activity of rhamnolipids produced by Pseudomonas aeruginosa #112 was evaluated against Aspergillus niger MUM 92.13 and Aspergillus carbonarius MUM 05.18. It was demonstrated that the di-rhamnolipid congeners were responsible for the antifungal activity exhibited by the crude rhamnolipid mixture, whereas mono-rhamnolipids showed a weak inhibitory activity. Furthermore, in the presence of NaCl (from 375 mM to 875 mM), the antifungal activity of the crude rhamnolipid mixture and the purified di-rhamnolipids was considerably increased. Dynamic Light Scattering studies showed that the size of the structures formed by the rhamnolipids increased as the NaCl concentration increased, being this effect more pronounced in the case of di-rhamnolipids. These results were confirmed by Confocal Scanning Laser Microscopy, which revealed the formation of giant vesicle-like structures (in the µm range) by self-assembling of the crude rhamnolipid mixture in the presence of 875 mM NaCl. In the case of the purified mono- and di-rhamnolipids, spherical structures (also in the µm range) were observed at the same conditions. The results herein obtained demonstrated a direct relationship between the rhamnolipids antifungal activity and their aggregation behaviour, opening the possibility to improve their biological activities for application in different fields., 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, COMPETE 2020 (POCI-01–0145-FEDER-006684) and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). The authors also acknowledge financial support from BioTecNorte operation (NORTE-01-0145-FEDER-000004) and the project MultiBiorefinery (POCI-01-0145-FEDER-016403) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. A. I. Rodrigues was supported by the doctoral Grant SFRH/111600/2015 provided by FCT. E. J. Gudiña was supported by the Post-Doctoral grant CEBBPD/01/2015/07 from the project UID/BIO/04469/2013, funded by FCT., info:eu-repo/semantics/publishedVersion

A DNA aptamer with affinity and specificity for human osteopontin (OPN), a potential breast cancer biomarker, was selected using the SELEX process, considering its homology rate and the stability of its secondary structures. This aptamer exhibited a satisfactory affinity towards OPN, showing dissociation constants lower than 2.5 nM. It was further used to develop a simple, label-free electrochemical aptasensor against OPN. The aptasensor showed good sensitivity towards OPN in standard solutions, being the square wave voltammetry (SWV), compared to the cyclic voltammetry, the most sensitive technique with detection and quantification limits of 1.4 ± 0.4 nM and 4.2 ± 1.1 nM, respectively. It showed good reproducibility and acceptable selectivity, exhibiting low signal interferences from other proteins, as thrombin, with 2.610 times lower current signals-off than for OPN. The aptasensor also successfully detected OPN in spiked synthetic human plasma. Using SWV, detection and quantification limits (1.3 ± 0.1 and 3.9 ± 0.4 nM) within the OPN plasma levels reported for patients with breast cancer (0.44.5 nM) or with metastatic or recurrent breast cancer (0.98.4 nM) were found. Moreover, preliminary assays, using a sample of human plasma, showed that the aptasensor and the standard ELISA method quantified similar OPN levels (2.2 ± 0.7 and 1.7 ± 0.1 nM, respectively). Thus, our aptasensor coupled with SWV represents a promising alternative for the detection of relevant breast cancer biomarkers., The authors acknowledge the financial support from the Strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and from project BioTecNorte (project number NORTE-01-0145-FEDER-000004). This work was also financially supported by Project POCI-01–0145-FEDER-006984 – Associate Laboratory LSRE-LCM and by Project UID/QUI/00616/2013 – CQ-VR both funded by FEDER - Fundo Europeu de Desenvolvimento Regional through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia, Portugal. S. Meirinho also acknowledges the research grant provided by Project UID/EQU/50020/2013., info:eu-repo/semantics/publishedVersion

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bej.2017.05.015., Hydroxycinnamic acids and curcumin are compounds with great therapeutic potential, including anticancer properties. In this study, p-coumaric acid, caffeic acid and curcumin were produced in Escherichia coli. Their production was induced by heat using the dnaK and ibpA heat shock promoters. The ribosome binding site (RBS) used was tested and further optimized for each gene to assure an efficient translation. p-Coumaric acid was successfully produced from tyrosine and caffeic acid was produced either from tyrosine or p-coumaric acid using tyrosine ammonia lyase (TAL) from Rhodotorula glutinis, 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris. The highest p-coumaric acid production obtained was 2.5 mM; caffeic acid production reached 370 M. Regarding curcumin, 17 M was produced using 4-coumarate-CoA ligase (4CL1) from Arabidopsis thaliana, diketide-CoA synthase (DCS) and curcumin synthase 1 (CURS1) from Curcuma longa. Stronger RBSs and/or different induction conditions should be further evaluated to optimize those production levels. Herein it was demonstrated that the biosynthetic pathway of p-coumaric acid, caffeic acid and curcumin in E. coli can be triggered by using heat shock promoters, suggesting its potential for the development of new industrial bioprocesses or even new bacterial therapies., FundingThis study was supported by the Portuguese Foundationfor Science and Technology (FCT) under the scope of thestrategic funding of UID/BIO/04469/2013 unit and COMPETE2020 (POCI-01-0145-FEDER-006684) and under the scope ofthe Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). The authors also acknowledge financial support fromBioTecNorte operation (NORTE-01-0145-FEDER-000004) and theproject MultiBiorefinery (POCI-01-0145-FEDER-016403) funded by the European Regional Development Fund under the scope ofNorte2020 − Programa Operacional Regional do Norte and the Post-doctoral grant (UMINHO/BPD/37/2015) to J. L. Rodrigues funded byFCT., info:eu-repo/semantics/publishedVersion