Detection of confinement and jumps in single molecule membrane trajectories

We propose a novel variant of the algorithm by Simson et al. [R. Simson, E.D. Sheets, K. Jacobson, Biophys. J. 69, 989 (1995)]. Their algorithm was developed to detect transient confinement zones in experimental single particle tracking trajectories of diffusing membrane proteins or lipids. We show that our algorithm is able to detect confinement in a wider class of confining potential shapes than Simson et al.'s one. Furthermore it enables to detect not only temporary confinement but also jumps between confinement zones. Jumps are predicted by membrane skeleton fence and picket models. In the case of experimental trajectories of $\mu$-opioid receptors, which belong to the family of G-protein-coupled receptors involved in a signal transduction pathway, this algorithm confirms that confinement cannot be explained solely by rigid fences.Comment: 4 pages, 3 figure

Application of experimental design methodology to optimize antibiotics removal by walnut shell based activated carbon

Three-level Box-Behnken experimental design with three factors (pH, temperature and antibiotic initial concentration) combined with response surface methodology (RSM) was applied to study the removal of Metronidazole and Sulfamethoxazole by walnut shell based activated carbon. This methodology enabled to identify the effects of the different factors studied and their interactions in the response of each antibiotic. The relationship between the independent variable (sorption capacity) and the dependent variables (pH, temperature and antibiotic concentration) was adequately modelled by second-order polynomial equation. The pH factor exerted a significant but distinct influence on the removal efficiency of both antibiotics. The removal of Metronidazole is favoured by increasing pH values, with the maximum value obtained for pH 8 - upper limit of the study domain; while Sulfamethoxazole displays a maximum value around 5.5, with a decrease in the extent of adsorption as the pH increases. The best conditions, predicted by the model, for the removal of the antibiotic Sulfamethoxazole (106.9 mg/g) are obtained at a temperature of 30 °C, initial concentration of 40 mg/L and a pH value of 5.5. For the antibiotic Metronidazole, the highest removal value (127 mg/g) is expected to occur at the maximum levels attributed to each of the factors (pH = 8, Cin = 40 mg/L, T = 30 °C). The results of isotherm experiments (at 20 °C and pH 6) displayed a good agreement with the models predictions. The maximum sorption capacity, estimated by the Langmuir model, was 107.4 mg/g for Metronidazole and 93.5 mg/g for Sulfamethoxazole.This work was financially supported by the projects POCI-01-0145-FEDER-006939 (LEPABE), POCI-010145-FEDER-007265 (REQUIMTE/LAQV) funded by the European Regional Development Fund (ERDF), through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds, through FCT - Fundação para a Ciência e a Tecnologia (LEPABE - UID/EQU/00511/2013; REQUIMTE/LAQV - UID/QUI/50006/2013) and NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, supported by North Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

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

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

Fructo-oligosaccharides: production, characterization and purification

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

Optimization of Whey Protein-Based Films Incorporating Foeniculum vulgare Mill: Essential Oil

Petroleum-based plastics used in food packaging are not biodegradable. They accumulate in the environment in large amounts, causing a decrease in soil fertility, jeopardizing marine habitats, and causing serious problems to human health. Whey protein has been studied for applications in food packaging, either because of its abundant availability or because it confers transparency, flexibility, and good barrier properties to packaging materials. Taking advantage of whey protein to produce new food packaging materials is a clear example of the so-called circular economy. The present work focuses on optimizing the formulation of whey protein concentrate-based films to enhance their general mechanical properties applying the Box–Behnken experimental design. Foeniculum vulgare Mill. (fennel) essential oil (EO) was incorporated into the optimized films, which were then further characterized. The incorporation of fennel EO in the films leads to a significant increase (p 90%). The results of the bioactive activities of the optimized films showed their ability to be applied as active materials for food packaging to improve the shelf-life of food products and also to prevent foodborne diseases associated with the growth of pathogenic microorganisms.info:eu-repo/semantics/publishedVersio

Nuclear microprobe: the tool to characterize new materials for energy conversion [Poster]

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Vaccinations in prisons: A shot in the arm for community health

From the first day of imprisonment, prisoners are exposed to and expose other prisoners to various communicable diseases, many of which are vaccine-preventable. The risk of acquiring these diseases during the prison sentence exceeds that of the general population. This excess risk may be explained by various causes; some due to the structural and logistical problems of prisons and others to habitual or acquired behaviors during imprisonment. Prison is, for many inmates, an opportunity to access health care, and is therefore an ideal opportunity to update adult vaccination schedules. The traditional idea that prisons are intended to ensure public safety should be complemented by the contribution they can make in improving community health, providing a more comprehensive vision of safety that includes public health

Fructo-oligosaccharides separation and purification by simulated moving bed chromatography

The interest on oligosaccharides such as fructo-oligosaccharides (FOS) has strongly increased in recent years for food and pharmaceutical applications, mainly due to their improved technological and functional properties. FOS can be produced by fermentative processes from sucrose, and can be found in mixture with other mono- and di-saccharides and salts, at the end of the process [1]. Unlike FOS, the small saccharides (SGF), namely fructose, glucose and sucrose in the mixture, are known to be cariogenic, caloric and do not present prebiotic activity. The purification of FOS from the other sugars can represent and important increment on the economic value of the final product, which can be further used in diabetic and dietetic food [2]. Different strategies have been developed for this purpose, including microbial treatment [3], ultra and nano-filtration, activated charcoal systems [4], or ion-exchange chromatography [5]. Ion exchange resins may be then used in batch or continuous chromatographic processes, as Simulated Moving Bed (SMB) chromatography, to purify sugars. A screening of different commercial resins was previously done in order to select the most suitable to separate the oligosaccharides [5]. The resin Diaion 535Ca showed an increased recovery yield and purity of FOS (92 and 90%, respectively). In the present work, the separation process was implemented in the SMB, using the selected resin, namely. Equilibrium adsorption isotherms were determined by the Retention Time Method (RTM), for each single sugar. The resin was afterwards packed in eight SMB columns, and tested in the pilot plant. Different operation parameters, including switching time, extra time, internal flow-rates and operating pump flow-rates for feed, raffinate, desorbent, eluent and recycling streams, were tested in the plant. The separation of fructose from glucose and FOS from the SGF was evaluated. Firstly, the separation of a binary sugar mixture of fructose/sucrose (~ 50/50%) was performed followed by the separation of FOS from a fermentative broth. Fructose was purified from 53 to 76% and sucrose from 47 to 77%. FOS and SGF were purified from 50 to 67%. The implementation of UV detectors between the SMB columns allowed following the sugar concentration profile online during the separation process. The accurate selection of the operating parameters was made using the concentration signal obtained and showed to be a crucial step for an improved separation

Microbial treatment approaches for high-purity fructo-oligosaccharides production

The production of high-purity fructooligosaccharides (FOS), known as prebiotics, by sucrose fermentation using whole microbial cells has been recently explored. At the end of the fermentation process, FOS are present in mixture with small saccharides that are known to have an inhibitory effect of transfructosylating enzymes and to decrease the prebiotic activity of the mixture. This issue can be overcome by reducing the small saccharides present in FOS broth, which can be done using a combined microbial treatment, among others, improving as well the further purification of FOS by Simulated Moving Bed (SMB) chromatography. The main goal of this work was the use of combined microbial treatment approaches to improve FOS production and enhance a high purity FOS content. Aureobasidium pullulans and Saccharomyces cerevisiae were used combined to produce FOS and reduce the small sugars in the culture, respectively. FOS-producing microorganism was used free, immobilized to a non-conventional carrier or encapsulated in Ca-alginate beads, in mixture with the non-oligosaccharides consuming microorganism, free or encapsulated in Ca-alginate beads. A factorial design, considering three different variables, was performed, to select the microbial treatment approach through which increased FOS levels and yields can be obtained. The 38 assays were performed in shaken-flasks and the most suitable one was scaled-up to a 3L bioreactor. The inoculation time of S. cerevisiae showed to be the most relevant variable for FOS production, and the use of immobilized A. pullulans, mixed with encapsulated S. cerevisiae inoculated after 20h of fermentation, was the best combination, with statistical relevance (p<0.01), to obtain enhanced FOS concentration, percentage in the medium, yield and productivity. Results in bioreactor showed a higher fermentation time (20 to 25h) needed to obtain an increased maximal production of FOS (around 132 g.L-1) and yielded 0.70 ± 0.05 g of FOS per gram of initial sucrose. Also, the approach selected improved the percentage of FOS in the medium throughout the fermentation time, providing a pre-purified broth, with lower levels of mono-saccharides for further purification by SMB

Síntese de frutooligossacarídeos a partir da β-frutofuranosidase obtida de Penicillium citreonigrum URM 4459

O objetivo deste trabalho foi sintetizar frutooligossacarídeos (FOS) a partir da β-frutofuranosidase (FTase) produzida em biorreator utilizando células livres de Penicillium citreonigrum. Para isso, o fungo foi cultivado em meio rico em sacarose (20% p/v) a 150 rpm, pH 6,5, 28ºC durante 61 h. Ao fim da fermentação, o sobrenadante obtido por filtração foi utilizado para determinação da atividade extracelular e síntese enzimática de FOS utilizando sacarose a 60% (p/v). A máxima atividade da β- frutofuranosidase extracelular produzida em biorreator (232,16 U/mL) foi obtida após 38 h de fermentação (produtividade de 6,11 U/mL.h). A concentração máxima de FOS foi observada após 24h de reação, correspondendo a um consumo de 30% (m/m) da sacarose inicial. O rendimento máximo de FOS obtido foi de 20% (m/m) em relação à concentração inicial de sacarose, resultando em níveis elevados de GF2 (18%, m/m) e baixos rendimentos de GF3 (1,5%, m/m) e GF4 (0,6%, m/m). Os resultados aqui apresentados sugerem utilização do fungo P. citreonigrum em processos que visem à produção de kestose por via enzimática. No entanto, estudos mais aprofundados são necessários para otimizar os parâmetros reacionais, e assim aumentar o rendimento do FOS