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" Available online 22 May 2022", This study explores the structural characterization, antioxidant and prebiotic activities of hydrolysates containing xylooligosaccharides (XOS) produced by different strategies: direct fermentation of beechwood xylan (FermBX) and enzymatic treatment of beechwood (EnzBX) and rice husk (EnzRH) xylans. EnzBX and EnzRH showed XOS with a backbone of (1 4)-linked-xylopyranosyl residues and branches of arabinose, galactose, and uronic acids. FermBX presented the highest content of total phenolic compounds (14 mg GAE/g) and flavonoids (0.6 mg QE/g), which may contribute to its antioxidant capacity 39.1 mol TE/g (DPPH), 45.7 mol TE/g (ABTS), and 79.9 mol Fe II/g (FRAP). The fermentation of hydrolysates decreased the abundance of microorganisms associated with intestinal diseases from Eubacteriales, Desulfovibrionales and Methanobacteriales orders, while stimulating the growth of organisms belonging to Bacteroides, Megamonas and Limosilactobacillus genera. The production of short-chain fatty acids, ammonia, and CO2 suggested the prebiotic potential. In conclusion, hydrolysates without previous purification and obtained from non-chemical approaches demonstrated promising biological activities for further food applications., This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001; by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) – Grant numbers 423285/2018-1 and 304857/2018-1; by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of 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; by the COMPETE 2020 (POCI-01-0145- FEDER-006684); by the FoSynBio (POCI-01-0145-FEDER-029549); and LAQV/REQUIMTE (UIDB/50006/2020, UIDP/50006/2020) through national founds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement. GG acknowledges the Universidade Federal do Rio Grande for the financial support from Postgraduate Student Mobility Program (PROPESP/FURG). BC, CA, and SPS acknowledge their grants (SFRH/BD/132324/2017, 2020.00293 CEECIND, and SFRH/BD/136471/2018) from FCT. LB also acknowledge FCT for the junior research contract (CEECIND/03280/2020). EC thanks the research contract (CDL-CTTRI-88-ARH/2018 – REF. 049-88-ARH/2018) funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19., info:eu-repo/semantics/publishedVersion

Lactoferrin (Lf) is a versatile natural milk-derived protein that exhibits multiple interesting biological activities. Since it is safe for human administration and currently manufactured using low cost and well-established large-scale processes, the Lf scientific community has been devoted at dissecting its mechanisms of action towards its more rational and efficient use for various applications. Emerging literature has identified proton pumping ATPases as molecular targets of Lf in different cellular models linked to distinct activities of this natural protein. Information on this subject has not been systematically analysed before, hence herein we review the current state of art on the effect of Lf on proton pumping ATPases. Though structurally different, we propose that Lf holds a proton pump inhibitor (PPI)-like activity based on the functional resemblance with the classical inhibitors of the stomach H+/K+-ATPase. The downstream events and outcomes of the PPI-like activity of Lf, as well as its impact for the development of improved Lf applications are also discussed., FCT -Fundação para a Ciência e a Tecnologia (UIDB/04050/2020), 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

Consumers are conscientiously changing their eating preferences toward healthier options, such as functional foods enriched with pre- and probiotics. Prebiotics are attractive bioactive compounds with multidimensional beneficial action on both human and animal health, namely on the gastrointestinal tract, cardiometabolism, bones or mental health. Conventionally, prebiotics are non-digestible carbohydrates which generally present favorable organoleptic properties, temperature and acidic stability, and are considered interesting food ingredients. However, according to the current definition of prebiotics, application categories other than food are accepted, as well as non-carbohydrate substrates and bioactivity at extra-intestinal sites. Regulatory issues are considered a major concern for prebiotics since a clear understanding and application of these compounds among the consumers, regulators, scientists, suppliers or manufacturers, health-care providers and standards or recommendation-setting organizations are of utmost importance. Prebiotics can be divided in several categories according to their development and regulatory status. Inulin, galactooligosaccharides, fructooligosaccharides and lactulose are generally classified as well established prebiotics. Xylooligosaccharides, isomaltooligosaccharides, chitooligosaccharides and lactosucrose are classified as "emerging" prebiotics, while raffinose, neoagaro-oligosaccharides and epilactose are "under development." Other substances, such as human milk oligosaccharides, polyphenols, polyunsaturated fatty acids, proteins, protein hydrolysates and peptides are considered "new candidates." This chapter will encompass actual information about the non-established prebiotics, mainly their physicochemical properties, market, legislation, biological activity and possible applications. Generally, there is a lack of clear demonstrations about the effective health benefits associated with all the non-established prebiotics. Overcoming this limitation will undoubtedly increase the demand for these compounds and their market size will follow the consumer's trend.

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

The updated definition of prebiotic expands the range of potential applications in which emerging xylooligosaccharides (XOS) can be used. It has been demonstrated that XOS exhibit prebiotic effects at lower amounts compared to others, making them competitively priced prebiotics. As a result, the industry is focused on developing alternative approaches to improve processes efficiency that can meet the increasing demand while reducing costs. Recent advances have been made towards greener and more efficient processes, by applying process integration strategies to produce XOS from costless lignocellulosic residues and using genetic engineering to create microorganisms that convert these residues to XOS. In addition, collecting more in vivo data on their performance will be key to achieve regulatory claims, greatly increasing XOS commercial value., CA, SCS and LRR acknowledge their grants (UMINHO/BPD/4/2019, SFRH/BPD/88584/2012 and SFRH/BSAB/142873/2018) from University of Minho and 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 Lignozymes (POCI-01-0145-FEDER-029773). 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.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

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). The authors also acknowledge financial support from BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. D. Ferreira is recipient of a fellowship (UMINHO/BD/21/2016) supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020. J. L. Rodrigues acknowledges the post-doctoral grant (UMINHO/BPD/37/2015) funded by FCT.

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

Early breast cancer detection remains a challenge and comes at high costs for governments, health institutions, and the patients. Risk factors (age, genetics and geographical variations) and disease heterogeneity are complex and not always easy to understand using the conventional diagnosis methodologies (e.g. mammography, X-rays, ultrasounds and magnetic resonance imaging (MRI)). This chapter provides insight into the current developments on breast cancer and the role of biosensors for its rapid detection as a potential solution to the limitations of current approaches for the detection and differentiation of the different types of breast cancer.

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

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

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

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

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

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

Synthetic biology in combination with systems biology uses engineering principles to leverage the power of biology. The impact and potential of systems and synthetic biology in the prevention, diagnosis and treatment of diseases are growing. In this chapter, we will revise some of the tools, methods and applications within these fields that can be useful in biomedical engineering and human health, such as genome editing and the development of quorum sensing mechanisms, among others. We will discuss remarkable advances made, for example, in vaccine development, diagnosis and treatment of cancer and infectious diseases, microbiome engineering and cell therapy. Lastly, the challenges and perspectives of these fields will be debated.

The development of bioinspired sensors has emerged and grown in recent decades, given their broad and exciting applications to pharmaceutical formulations, medical diagnosis and health care. These sensor-based technologies, owing to their function and to the elements involved in their design, potentially generate fast, selective, sensitive, safe and green analysis solutions. Their applications have evolved into mainly two fields. The first comprises sensor array systems like the electronic tongues (E-tongues), which are innovative (bio)sensing devices that, combined with chemometric tools, provide highly specialized responses, enabling the discrimination of thousands of possible targets. The second line of action involves the application of biosensors, namely aptasensors, which are highly specific against a target biomarker. In this chapter, the concept, design and application of electrochemical sensor approaches, such as E-tongues and aptasensors, for (bio)sensing applications, will be discussed and the main trends in medical and pharmaceutical applications will be highlighted. info:eu-repo/semantics/publishedVersion

The field of synthetic biology combines relevant expertise from various science, engineering, and computing disciplines to take the best of biology for the benefit of humans and the environment. Herein we review some of the tools and methods of synthetic biology, such as the use of engineering principles and genome editing tools. In addition, we discuss the advantages of synthetic biology and how it can be used to improve industrial biotechnology, for example, in the production of biofuels, drugs, and other value-added chemicals. Finally, we approach the perception that the population in general has about synthetic biology and how the scientists working in this field are trying to change it by creating containment measures, some of them using synthetic biology itself, to increase biosecurity and biosafety.

In this work, oil mill wastewater (OMW), a residue generated during olive oil extraction, was evaluated as an inducer of rhamnolipid production. Using a medium containing as sole ingredients corn steep liquor (10%, v/v), sugarcane molasses (10%, w/v) and OMW (25%, v/v), Pseudomonas aeruginosa #112 produced 4.5 and 5.1 g of rhamnolipid per liter in flasks and reactor, respectively, with critical micelle concentrations as low as 13 mg/l. Furthermore, in the medium supplemented with OMW, a higher proportion of more hydrophobic rhamnolipid congeners was observed comparing with the same medium without OMW. OMW is a hazardous waste which disposal represents a serious environmental problem; therefore, its valorization as a substrate for the production of added-value compounds such as rhamnolipids is of great interest. This is the first report of rhamnolipid production using a mixture of these three agro-industrial by-products, which can be useful for the sustainable production of rhamnolipids., 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). The authors also thank the FCT for the financial support under the scope of the Project RECI/BBBEBI/ 0179/2012 (FCOMP-01-0124-FEDER-027462). E.J. Gudiña was supported by the Post-Doctoral fellowship CEB-BPD/01/2015/07 from the project UID/BIO/04469/2013, financed by FCT. A.I. Rodrigues was supported by the doctoral Grant SFRH/BD/111600/2015 provided by FCT.

The commercialization of safe foods is a main concern and should be ensured throughout the entire food supply chain. So, fast, sensitive, and reliable analytical methods are needed to identify food-related specific hazards, ensuring consumers' protection and avoiding health problems. This chapter is focused on the emerging (bio)sensors to detect the heat-stable allergen gliadin in gluten-free foods. A brief overview concerning gliadin and its relation to celiac disease is provided. Several works related to the development of biosensors for gliadin detection are presented. Moreover, the use of electronic tongues to detect gliadin or gluten is reviewed and further investigated in an attempt to explain the possible detection mechanism based on gliadin-lipid membranes interactions. The challenges, concerns, and potential application of these (bio)sensors are discussed. Finally, some future perspectives are included regarding the applicability of electronic tongue within this research field. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) and the European Community fund FEDER, through Program COMPETE, under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462); under the Strategic funding of UID/BIO/04469/2013 unit; and under the Program PT2020 (Project UID/EQU/50020/2013). info:eu-repo/semantics/publishedVersion

"Available online 28 June 2016", The interaction of natural lipopeptide pseudofactin II with a series of doubly charged metal cations was examined by matrixassisted laserdesorption ionizationtime of flight (MALDI-TOF) mass spectrometry and molecular modelling. The molecular modelling for metal-pseudofactin II provides information on the metalpeptide binding sites. Overall, Mg2+, Ca2+ and Zn2+ favor the association with oxygen atoms spanning the peptide backbone, whereas Cu2+ is coordinated by three nitrogens. Circular dichroism (CD) results confirmed that Zn2+ and Cu2+ can disrupt the secondary structure of pseudofactin II at high concentrations, while Ca2+ and Mg2+ did not essentially affect the structure of the lipopeptide. Interestingly, our results showed that the addition of Zn2+ and Cu2+ helped smaller micelles to form larger micellar aggregates. Since pseudofactin II binds metals, we tested whether this phenomena was somehow related to its antimicrobial activity against Staphylococcus epidermidis and Proteus mirabilis. We found that the antimicrobial effect of pseudofactin II was increased by supplementation of culture media with all tested divalent metal ions. Finally, by using Gram-positive and Gram-negative bacteria we showed that the higher antimicrobial activity of metal complexes of pseudofactin II is attributed to the disruption of the cytoplasmic membrane., Fundação para a Ciência e Tecnologia (FCT) for the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684)

Polysaccharides and oligosaccharides can improve quality and enhance nutritional value of final food products due to their technological and nutritional features ranging from their capacity to improve texture to their effect as dietary fibers. For this reason, they are among the most studied ingredients in the food industry. The use of natural polysaccharides and oligosaccharides as food additives has been a reality since the food industry understood their potential technological and nutritional applications. Currently, the replacement of traditional ingredients and/or the synergy between traditional ingredients and polysaccharides and oligosaccharides are perceived as promising approaches by the food industry. Traditionally, polysaccharides have been used as thickening, emulsifying, and stabilizing agents, however, at this moment polysaccharides and oligosaccharides claim health and nutritional advantages, thus opening a new market of nutritional and functional foods. Indeed, their use as nutritional food ingredients enabled the food industry to develop a countless number of applications, e.g., fat replacers, prebiotics, dietary fiber, and antiulcer agents. Based on this, among the scientific community and food industry, in the last years many research studies and commercial products showed the possibility of using either new or already used sources (though with changed properties) of polysaccharides for the production of food additives with new and enhanced properties. The increasing interest in such products is clearly illustrated by the market figures and consumption trends. As an example, the sole market of hydrocolloids is estimated to reach $7 billion in 2018. Moreover, oligosaccharides can be found in more than 500 food products resulting in a significant daily consumption. A recent study from the Transparency Market Research on Prebiotic Ingredients Market reported that prebiotics' demand was worth $2.3 billion in 2012 and it is estimated to reach $4.5 billion in 2018, growing at a compound annual growth rate of 11.4% between 2012 and 2018. The entrance of this new generation of food additives in the market, often claiming health and nutritional benefits, imposes an impartial analysis by the legal authorities regarding the accomplishment of requirements that have been established for introducing novel ingredients/food, including new poly- and oligosaccharides. This chapter deals with the potential use of polysaccharides and oligosaccharides as food additives, as well as alternative sources of these compounds and their possible applications in food products. Moreover, the regulation process to introduce novel polysaccharides and oligosaccharides in the market as food additives and to assign them health claims is discussed.

"Available online 2 September 2014", Bovine lactoferrin (bLF) was shown to efficiently inhibit the growth of MCF-7, T-47D, MDA-MB-231 and Hs578T breast cancer cells in a concentration-dependent manner. However, apoptosis was only induced in MCF-7 cells, which was associated with the mitochondria membrane depolarisation and a decrease of Bcl-2 levels. bLF led to the cycle arrest of MCF-7 cells at G1/G0 phase, as well as a significant decrease in the expression of CDC25c. The possibility that the observed anticancer effects could be due to the high exogenous bLF concentrations in the culture media was excluded. Moreover, bLF was shown to restrain the colony formation of MCF-7 cells, although it promoted cell migration. This later effect was unspecific and related to the presence of a high protein concentration in the culture or medium. The results gathered in this work provide valuable insights for the evaluation and further study of the potential of bLF in cancer therapy., Zhang Y. acknowledges the Erasmus Mundus External Cooperation window (Bridging the Gap) for supporting his PhD grant. The authors acknowledge the financial support from the Strategic Projects PEst-OE/EQB/LA0023/2013 and PEst-OE/AGR/UI4033/2014, as well as the project ref. RECI/BBB-EBI/0179/2012 (project number FCOMP-01-0124-FEDER-027462) funded by Fundacao para a Ciencia e a Tecnologia, Portugal.

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids., Fonds Europeen de Developpement Economique et Regional. COMPETE Program, Fonds Europeen de Developpement Economique et Regional. ON2 Program, Fundacao para a Ciencia e a Tecnologia (Project FCOMP-01-0124-FEDER-027462), Fundacao para a Ciencia e a Tecnologia (PEst-OE/EQB/LA0023/2013), Fundacao para a Ciencia e a Tecnologia (NORTE-07-0124-FEDER-000028), Fundacao para a Ciencia e a Tecnologia (NORTE-07-0124-FEDER-000027), Fundacao para a Ciencia e a Tecnologia (Grant SFRH/BD/51187/2010), Fundacao para a Ciencia e a Tecnologia (SYNBIOBACTHER Project PTDC/EBB-BIO/102863/2008)

Curcumin is a natural compound obtained from turmeric, and is well known for its pharmacological effects. In this work, we design a heterologous pathway for industrial production of curcumin in Escherichia coli. A kinetic model of the pathway is then developed and connected to a kinetic model of the central carbon metabolism of E. coli. This model is used for optimization of the mutant strain through a rational design approach, and two manipulation targets are identified for overexpression. Dynamic simulations are then performed to compare the curcumin production profiles of the different mutant strains. Our results show that it is possible to obtain a significant improvement in the curcumin production rates with the proposed mutants. The kinetic model here developed can be an important framework to optimize curcumin production at an industrial scale and add value to its biomedical potential., Research supported by the Portuguese Foundation for Science and Technology (FCT), through the project "SYNBIOBACTHER - Synthetic biology approaches to engineer therapeutic bacteria" (PTDC/EBB-BIO/102863/2008).

Biosurfactants have recently emerged as promising molecules for their structural novelty, versatility, and diverse properties that are potentially useful for many therapeutic applications. Mainly due to their surface activity, these molecules interact with cell membranes of several organisms and/or with the surrounding environments, and thus can be viewed as potential cancer therapeutics or as constituents of drug delivery systems. Some types of microbial surfactants, such as lipopeptides and glycolipids, have been shown to selectively inhibit the proliferation of cancer cells and to disrupt cell membranes causing their lysis through apoptosis pathways. Moreover, biosurfactants as drug delivery vehicles offer commercially attractive and scientifically novel applications. This review covers the current state-ofthe- art in biosurfactant research for therapeutic purposes, providing new directions towards the discovery and development of molecules with novel structures and diverse functions for advanced applications., The authors acknowledge Fundacao para a Ciencia e a Tecnologia (FCT) (Portugal) and the Department of Science and Technology (DST) (India) for financial support of the project 'MEDSURF - The potential use of biosurfactants for medical applications' developed under the scope of a bilateral agreement between Portugal and India.

A method for the separation and fractionation of the major whey proteins from a whey protein concentrate (WPC80) by anion-exchange chromatography coupled to a Fast Protein Liquid Chromatography (FPLC) system is proposed. The method is based on the use of an ionic column (Mono Q) and a salt gradient elution by increasing the ionic strength of the elution buffer (Tris–HCl 20 mM plus 0 to 1 M NaCl). The proposed method was found to be suitable to fractionate the major whey proteins from the WPC80 in different fractions, namely one fraction containing all the α-Lactalbumin and immunoglobulins; another fraction containing all the bovine serum albumin; and two distinct fractions each containing a different variant of β-Lactoglobulin. A 60.5% (w/w) recovery of the two main β-Lactoglobulin variants was obtained.

A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.

Food-grade galacto-oligosaccharides (GOS) are commercially available as transparent syrups or dried powders. Food powders can be found in an amorphous metastable state which is very sensitive to changes in temperature and moisture content. In this work the impact of water content on thermal behavior and relative humidity on water sorption behavior of amorphous GOS powders were studied. Results from differential scanning calorimetry (DSC) and sorption isotherms suggest that GOS mixture studied, with high content of oligosaccharides, has low ability to crystallize. A dramatic decrease in the stability of GOS powders occurred above critical water content (12–14 g/100 g) and corresponding critical water activity (0.55–0.62). Above these conditions GOS powder lost its amorphous character, collapsed and shrank, as the powder became a transparent “solution-like” material. The knowledge about the physicochemical changes, acquired during the present study, should be used to a proper control of processing and storage conditions to achieve and maintain optimum powder quality with desired properties., Agência da Inovação (ADI) - Project BIOLIFE – PRIME 03/347, Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BDE/15510/2004

Poly(dimethyl siloxane) elastomer, (PDMS) is widely used as a biomaterial. However, PDMS is very hydrophobic and easily colonized by several bacteria and yeasts. Consequently, surface modification has been used to improve its wettability and reduce bacterial adhesion. The aim of this work was to modify the PDMS surface in order to improve its hydrophilicity and bacterial cell repulsion to be used as a biomaterial. Plasma was used to activate the PDMS surface and sequentially promote the attachment of a synthetic surfactant, Pluronic® F-68, or a polymer, Poly(ethylene glycol) methyl methacrylate, PEGMA. Bare PDMS, PDMS argon plasma activated, PDMS coated with Pluronic® F-68 and PEGMA-grafted PDMS were characterized by contact angle measurements, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The influence of the surface modifications on blood compatibility of the materials was evaluated by thrombosis and haemolysis assays. The cytotoxicity of these materials was tested for mouse macrophages. After modification, AFM results suggest the presence of a distinct layer at the surface and by the contact angle measures it was observed an increase of hydrophilicity. XPS analysis indicates an increase of the oxygen content at the surface as a result of the modification. All the studied materials revealed no toxicity and were found to be non-haemolytic or in some cases slightly haemolytic. Therefore, plasma was found to be an effective technique for the PDMS surface modification., The present study was developed under the scope of the BIOSURFA - PTDC/SAU-BEB/73498/2006 project funded by the Portuguese Science and Technology Foundation (Fundacao para a Ciencia e Tecnologia - FCT).

In this study, the crude biosurfactant produced by a Lactobacillus paracasei strain isolated in a Portuguese dairy industry was characterized. The minimum surface tension (41.8mN/m) and the critical micelle concentration (2.5 mg/ml) obtained were found to be similar to the values previously reported for biosurfactants isolated from other lactobacilli. The biosurfactant was found to be stable to pH changes over a range from 6 to 10, being more effective at pH 7, and showed no loss of surface activity after incubation at 60 ◦C for 120 h. Although the biosurfactant chemical composition has not been determined yet, a fraction was isolated through acidic precipitation, which exhibited higher surface activity as compared with the crude biosurfactant. Furthermore, this isolated biosurfactant showed antimicrobial and anti-adhesive activities against several pathogenic microorganisms. In addition, L. paracasei exhibited a strong autoaggregating phenotype, which was maintained after washing and resuspending the cells in PBS, meaning that this attribute must be related to cell surface components and not to excreted factors. The autoaggregation ability exhibited by this strain, together with the antimicrobial and anti-adhesive properties observed for this biosurfactant opens the possibility for its use as an effective probiotic strain., Fundação para a Ciência e a Tecnologia (FCT)under the scope of the Project BIOSURFA: Biosurfactants application for microbial adhesion inhibition in medical devices.

The ability of Aspergillus japonicus ATCC 20236 to colonize different synthetic materials (polyurethane foam, stainless steel sponge, vegetal fiber, pumice stones, zeolites, and foam glass) and to produce fructooligosaccharides (FOS) from sucrose (165 g/L) is described. Cells were immobilized in situ by absorption, through direct contact with the carrier particles at the beginning of fermentation. Vegetal fiber was the best immobilization carrier as A. japonicus grew well on it (1.25 g/g carrier), producing 116.3 g/L FOS (56.3 g/L 1-kestose, 46.9 g/L 1-nystose, and 13.1 g/L 1-β-fructofuranosyl nystose) with 69% yield (78% based only in the consumed sucrose amount), giving also elevated activity of the β-fructofuranosidase enzyme (42.9 U/mL). In addition, no loss of material integrity, over a 2 day-period, was found. The fungus also immobilized well on stainless steel sponge (1.13 g/g carrier), but in lesser extents on polyurethane foam, zeolites, and pumice stones (0.48, 0.19, and 0.13 g/g carrier, respectively), while on foam glass no cell adhesion was observed. When compared with the FOS and β-fructofuranosidase production by free A. japonicus, the results achieved using cells immobilized on vegetal fiber were closely similar. It was thus concluded that A. japonicus immobilized on vegetal fiber is a potential alternative for high production of FOS at industrial scale., Fundação para a Ciência e a Tecnologia (FCT)

Monitoring the industrial production of galacto-oligosaccharides (GOS) requires a fast and accurate methodology able to quantify, in real time, the substrate level and the product yield. In this work, a simple, fast and inexpensive UV spectrophotometric method, together with partial least squares regression (PLS) and artificial neural networks (ANN), was applied to simultaneously estimate the products (GOS) and the substrate (lactose) concentrations in fermentation samples. The selected multiple models were trained and their prediction abilities evaluated by cross-validation and external validation being the results obtained compared with HPLC measurements. ANN models, generated from absorbance spectra data of the fermentation samples, gave, in general, the best performance being able to accurately and precisely predict lactose and total GOS levels, with standard error of prediction lower than 13 g kg 1 and coefficient of determination for the external validation set of 0.93–0.94, showing residual predictive deviations higher than five, whereas lower precision was obtained with the multiple model generated with PLS. The results obtained show that UV spectrophotometry allowed an accurate and non-destructive determination of sugars in fermentation samples and could be used as a fast alternative method for monitoring GOS production., Fundação para a Ciência e a Tecnologia (FCT) - Bolsa de doutouramento SFRH/BDE/15510/2004, Agência da Inovação – Programa IDEIA (Potugal)

Isolation and characterization of the surface active components from the crude biosurfactant produced by Streptococcus thermophilus A was studied. A fraction rich in glycolipids was obtained by the fractionation of crude biosurfactant using hydrophobic interaction chromatography. Molecular (by Fourier transform infrared spectroscopy) and elemental compositions (by X-ray photoelectron spectroscopy) were determined. Critical micelle concentration achieved was 20 g/l, allowing for a surface tension value of 36 mJ/m2. Moreover, this glycolipid rich fraction was found to be an anti-adhesive and antimicrobial agent against several bacterial and yeast strains isolated from explanted voice prostheses. Further purification steps should be carefully analyzed as each purification step will increase the costs and decreases the amounts of biosurfactants recovered., Fundação para a Ciência e a Tecnologia(FCT) CACTI (Spain) ; University of Groningen, The Netherlands.

Screening of biosurfactant-producing ability of four Lactobacillus strains was performed, being shown that for all the tested strains biosurfactant production occurred mainly in the first 4 h. The Lactobacillus strains showed zones of clearing in the blood agar with a diameter