Utilização de diferentes meios de cultura na identificação e recuperação de bactérias lácticas

Foram isoladas várias colónias bacterianas provenientes de amostras de queijo em duas fases de maturação (3 e 45 dias) inoculadas nos meios de cultura de de Man, Rogosa e Sharpe Agar (MRSA), M17 Agar, Kanamicina Esculina Azida Agar (KA3), APT Agar e Dextrose Triptona Agar (DTA). Com base nas suas características culturais, morfológicas e bioquímicas foram identificados os diferentes géneros de bactérias lácticas (BAL). Uma pequena percentagem (13,96%) dos isolados revelou não ser BAL. O meio KA3 permitiu o crescimento quase exclusivo de Enterococcus. No meio MRSA, além de Lactobacillus, cresceram também Enterococcus e algumas não BAL, provenientes principalmente de amostras com 3 dias de maturação. O meio M17, onde cresceu a maior parte de Lactococcus provenientes, principalmente, de queijos com 3 dias de maturação, foi também aquele que permitiu o crescimento de maior número de não BAL. O meio DTA foi o que revelou menor selectividade, permitindo o crescimento de vários géneros de BAL provenientes das diferentes amostras, podendo a sua selectividade ser alterada por diferentes condições de incubação, como a temperatura. O meio APT, moderadamente selectivo, permitiu a proliferação de Enterococcus e de Lactobacillus em números apreciáveis. Dos diferentes meios avaliados, o meio KA3 foi o mais eficaz no isolamento de Enterococcus. O meio MRSA, mesmo permitindo o crescimento de algumas não BAL, foi útil para o isolamento de Lactobacillus. Apesar de o meio M17 ter consentido o crescimento de muitas não BAL, revelou-se o mais eficaz para o isolamento de Lactococcus

Estimação da função de distribuição bivariada na presença de censura

Dissertação de mestrado em EstatísticaEm muitos estudos longitudinais, os indivíduos experimentam eventos recorrentes. Este tipo de dados é frequentemente observado em investigação médica, em engenharia e áreas afins. Dados dois tempos provenientes de eventos sequenciais, vários métodos foram desenvolvidos para a estimação de quantidades de interesse, como a função de distribuição bivariada, as funções de distribuição marginais ou a função de distribuição condicional. Na maioria destes métodos admite-se que os indivíduos que entram no estudo continuam em observação até ao fim do estudo. No entanto em muitas aplicações isso não ocorre, e os indivíduos não são observados até ao final do estudo por várias razões. Neste trabalho é estudada a função de distribuição bivariada na presença de censura pela direita. Relativamente à organização da presente dissertação, a mesma foi dividida em cinco secções. Na primeira secção são resumidos os fundamentos teóricos mais relevantes de forma a facilitar ao leitor com alguns conhecimentos nos domínios da Matemática ou da Estatística a compreensão das secções seguintes. Assim são introduzidos conceitos fundamentais nas áreas da análise de sobrevivência, da estimação pontual e da estatística multivariada. Na segunda secção é introduzido o problema da estimação da função de distribuição bivariada para tempos sequenciais na presença de censura pela direita. Quatro estimadores distintos são apresentados. A terceira secção apresenta uma extensão para o software estatístico R, desenvolvida no âmbito da investigação que esteve na origem da escrita desta dissertação. A extensão mencionada foi desenvolvida com o objectivo de proporcionar resultados numéricos e gráficos em relação a cada um dos quatro estimadores apresentados nesta dissertação. De modo a demonstrar as potencialidades da extensão desenvolvida, são apresentados exemplos de utilização numérica e gráfica, acompanhados de possíveis interpretações. A quarta secção descreve detalhadamente um estudo de simulação envolvendo os quatro estimadores abordados nesta dissertação. Nesta secção cada um dos estimadores é analisado em relação às propriedades desejáveis das funções de distribuição bivariadas. O comportamento de cada um dos estimadores em relação ao enviesamento, ao desvio padrão e ao erro quadrático médio é estudado recorrendo a gráficos e tabelas. De modo a identificar o estimador mais eficiente, os quatro estimadores são comparados dois a dois em termos da eficiência relativa. Na quinta e última secção são apresentadas as principais conclusões obtidas do estudo de simulação descrito na secção anterior.In many longitudinal studies, the individuals experience recurrent events. This type of data is frequently observed in medical research, engineering and related fields. Given two times coming from sequential events, several methods have been developed for the estimation of quantities of interest, such as the bivariate distribution function, the marginal distribution functions, and the conditional distribution function. Most of these methods admit that the individuals entering the study remain in observation until the end of the study. However, in many applications that doesn’t happen, and the individuals aren’t observed until the end of the study for many reasons. In this work the bivariate distribution function under right censoring is studied. Pertaining to the organization of the present dissertation, the same was divided in five sections. In the first section the most relevant theoric fundaments are resumed. So that a reader with some background in Mathematics or Statistics can easily understand the contents of the following sections. In this way fundamental concepts in the areas of survival analysis, point estimation and multivariate statistics are introduced. The second section introduces the problem of estimation of the bivariate distribution function for sequentially ordered events under right censoring. Four distinct estimators are presented. In the third section, a package for the statistical software R, developed in the framework of the research that led to this dissertation, is presented. This software package was developed with the purpose of producing numerical and graphical output for each of the four estimators discussed in this dissertation. To demonstrate some of the possibilities of the software package, numerical and graphical examples are given accompanied by possible interpretations. The fourth section describes the details of a simulation study involving each one of the four estimators discussed in this dissertation. Each one of the estimators is analyzed in respect of the desirable properties of the bivariate distribution functions. With the aid of graphics and tables, the behavior of each one of the estimators is studied in respect to bias, standard deviation and mean square error. To identify the most efficient estimator, the four estimators are compared by means of the relative efficiency. The fifth and last section presents the main conclusions of the simulation study described in the previous section.Fundação para a Ciência e a Tecnologia (FCT) - Projeto PTDC/MAT/104879/2008FEDER através do Programa Operacional Factores de Competitividade - COMPET

Decolourization of paprika dye effluent with hydrogen peroxide produced by glucose oxidase

Hydrogen peroxide was produced from bran by a two-step process using cellulase/xylanase and glucose oxidase, sequentially. The decolourization efficiency of the produced reagent was tested using paprika oil dye (effluent from industrial source) and high levels of colour removal (96%) were achieved after saponification pre-treatment and hydrogen peroxide application. The method is economically and environmentally advantageous since lower energy and chemical input are needed and wastewater pollution is considerably reduced. At the same time, the utilization of waste materials was successfully achieved.The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. Carla Silva would like to acknowledge the Portuguese Fundacao para a Ciencia e a Tecnologia (FCT) for funding under the scholarship SFRH/BPD/46515/2008

Interfacial stabilization of enzymes in microemulsions

One of the major constrains to the use of enzymes in industrial processes is their insufficient stability under processing conditions, namely high temperatures, presence of ultrasounds, among others. Herein, we investigated the use of oil-in-water proteinaceous (BSA) microemulsions as a novel methodology for the stabilization of laccase from ascomycete Micelliophthora thermophila. The immobilization of laccase onto the produced microemulsions benefitiated its stability under ultrasonic conditions. The half life time of immobilized laccase was 2.4-fold higher (from 23 to 56 minutes) than laccase in the free form. This technique show promising potentialities for the stabilization of enzymes used onto a variety of processes, namely textile bleaching, surface hydrolysis, among others

Protein-based nanoformulations for α-Tocopherol encapsulation

Nanoparticles of bovine serum albumin (BSA) and silk fibroin (SF) with entrapped α-tocopherol were produced via ultrasonic emulsification. Populations with particle size of 200300 nm and highly negatively charged were obtained for all the tested formulations. Entrapment efficiencies of around 99% revealed the effective encapsulation of α-tocopherol into the produced nanoformulations. Generally, these nanodevices did not induce significant cytotoxicity to human skin keratinocytes for all the concentrations tested. The developed formulations showed free radical scavenging of ABTS.+ ability resulting from the synergistic effect between proteins in formulation and the entrapped tocopherol. Overall, the results contribute for the establishment of BSA:VO and BSA:SF:VO as biodegradable and non-toxic nanoformulations for the functionalization of textile devices and controlled delivery of tocopherol into the skin.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 COMPETE2020 (POCI-01-0145FEDER-006684) and BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by European Regional Development Fund under the scope of Norte 2020-Programa Operacional Regional do Norte. Artur Ribeiro thanks FCT for the SFRH\BPD\98388\2013 grant. Fatemeh Shahmoradi would like to acknowledge the Iran Ministry of Science, Research and Technology (MSRT) for the monetary support.info:eu-repo/semantics/publishedVersio

Enzymatic synthesis of poly(catechin)-antibiotic conjugates : an antimicrobial approach for indwelling catheters

Biofilm formation in urinary indwelling catheters is one of the most critical issues that patients face. Catheters were coated with poly(catechin)-antibiotic conjugates with enhanced antimicrobial properties. Catechin was conjugated with two antibiotics, namely trimethoprim (TMP) and sulfamethoxazole (SMZ) via activation with N,N′-disuccinimidyl carbonate (DSC) and subsequent coupling to molecules containing α-amine moieties. Silicone and polyurethane catheters were functionalized in situ through laccase oxidation of catechin-antibiotic conjugates. Four antimicrobial coatings were produced, namely with poly(catechin), poly(catechin)- TMP, poly(catechin)-SMZ and poly(catechin)-TMP-SMZ. The bacterial adhesion reduction was tested on the functionalized devices using gram-negative and gram-positive strains. The most significant reduction in adhesion was observed with poly(catechin)-TMP (gram-negative −85 % and grampositive −87 %) and with poly(catechin)-TMP-SMZ (gramnegative −85 % and gram-positive −91 %). The cytotoxicity to mammalian cells was tested by indirect contact for 5 days and revealed that all the tested coatings supported more than 90 % of viable cells. A promising approach for the increase of the indwelling catheter lifespan was developed aiming to reduce catheter-associated chronic infections.The authors would like to acknowledge Pronefro (Portugal) and Degania (Israel) for supplying polyurethane and silicone catheters, respectively. The author Idalina Goncalves would like to acknowledge the NOVO project (FP7-HEALTH-2011.2.3.1-5) for funding. This work was partly supported by FEDER through POFC-COMPETE and by Portuguese funds from Fundacao para a Ciencia e a Tecnologia (FCT) through the project PEst-OE/BIA/UI4050/2014. The authors Carla Silva and Teresa Matama would like to acknowledge FCT for their scholarships SFRH/BPD/46515/2008 and SFRH/BPD/47555/2008

Influence of mechanical agitation on cutinases and protease activity towards polyamide substrates

Two polyamide 6,6 substrates with different constructions, namely a model substrate and a fabric, were hydrolyzed using native cutinase and L182A cutinase mutant (from Fusarium solani pisi) and a protease (subtilisin from Bacillus sp.). The catalytic efficiency of these enzymes, measured in terms of hydrolysis products release, was measured for both substrates and the protease released five times more amines to the bath treatment. The L182A cutinase mutant showed higher activity when compared with the native enzyme. All enzymes have shown activity additive effects with higher levels of mechanical agitation for polyamide fabrics. The results achieved are of paramount importance on the design of a process of enzymatic functionalization of polyamide.European Community - Biosyntex Project, G5RD-CT-2000-30110Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/22490/200

Coloured and low conductive fabrics by in situ laccase-catalysed polymerization

Coloured and conductive fabrics were obtained through in situ laccase polymerization of catechol and p-phenylenediamine under high-pressure homogenization. Both monomers, catechol and p-phenylenediamine, were polymerized by different laccase forms, namely native, PEGylated and Epoxy-PEGylated. All the catalysts were placed inside a textile fabric bag which served simultaneously as enzyme support and as substrate for coating with the newly produced polymers. The PEGylated laccase forms gave rise to higher amount of oligomers/polymers and higher colouration level of polyethylene terephthalate (PET), cotton and wool fabrics compared to native laccase. Both functional polymers were able to confer conductivity to the substrates however in a different extent. Fabrics coated with poly(p-phenylenediamine) present higher conductivity, rather due to its polymerized structure than to the amount of polymer produced by enzyme catalysis. Herein a green approach was presented to produce polyphenols with increased fixation onto different textile substrates. These substrates reach high levels of colouration and good fastness behaviour after washing.Chinese Government Scholarship under China Scholarship Council [grant No. 201606790036]; and by the Chinese Foundation Key projects of governmental cooperation in international scientific and technological innovation [grant No. 2016 YFE0115700]; and 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 BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This work was also supported by the European Union through the European Regional Development Fund (ERDF) under the Competitiveness Operational Program [grant COP-A1.1.4-E-2015nr.30/01.09.2016]; and by the Project “Search-ON2: Revitalization of HPC infrastructure of UMinho” [grant NORTE-07-0162-FEDER-000086), co-funded by the North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). The work was also supported by the FCT- Fundação para a Ciência e a Tecnologia [grant SFRH/BD/121673/2016 and IF/00186/2015]info:eu-repo/semantics/publishedVersio

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 24 full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL−1 of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor

Sonochemical and hydrodynamic cavitation reactors for laccase/hydrogen peroxide cotton bleaching

Article in pressThe main goal of this work is to develop a novel and environmental-friendly technology for cotton bleaching with reduced processing costs. This work exploits a combined laccase–hydrogen peroxide process assisted by ultrasound. For this purpose, specific reactors were studied, namely ultrasonic power generator type K8 (850 kHz) and ultrasonic bath equipment Ultrasonic cleaner USC600TH (45 kHz). The optimal operating conditions for bleaching were chosen considering the highest levels of hydroxyl radical production and the lowest energy input. The capacity to produce hydroxyl radicals by hydrodynamic cavitation was also assessed in two homogenizers, EmulsiFlex®-C3 and APV-2000. Laccase nanoemulsions were produced by high pressure homogenization using BSA (bovine serum albumin) as emulsifier. The bleaching efficiency of these formulations was tested and the results showed higher whiteness values when compared to free laccase. The combination of laccase–hydrogen peroxide process with ultrasound energy produced higher whiteness levels than those obtained by conventional methods. The amount of hydrogen peroxide was reduced 50% as well as the energy consumption in terms of temperature (reduction of 40 °C) and operating time (reduction of 90 min).The author Idalina Goncalves would like to acknowledge the Cottonbleach project (FP7-SME-2008-2; 243529-2-cottonbleach) for the funding. The author Carla Silva would like to acknowledge FCT - Fundacao para a Ciencia e a Tecnologia for the grant SFRH/BPD/46515/2008