Isolamento e caracterização de bactérias do ácido láctico, produtoras de bacteriocinas e sua aplicação no fabrico de queijo fresco

Dissertação de Mestrado em Tecnologia e Segurança Alimentar

Q192R polymorphism of the paraoxonase-1 gene as a risk factor for obesity in Portuguese women

Introduction - Obesity became a major public health problem as a result of its increasing prevalence worldwide. Paraoxonase-1 (PON1) is an esterase able to protect membranes and lipoproteins from oxidative modifications. At the PON1 gene, several polymorphisms in the promoter and coding regions have been identified. The aims of this study were i) to assess PON1 L55M and Q192R polymorphisms as a risk factor for obesity in women; ii) to compare PON1 activity according to the expression of each allele in L55M and Q192R polymorphisms; iii) to compare PON1 activity between obese and normal-weight women. Materials and methods - We studied 75 healthy (35.9±8.2 years) and 81 obese women (34.3±8.2 years). Inclusion criteria for obese subjects were body mass index ≥30 kg/m2 and absence of inflammatory/neoplasic conditions or kidney/hepatic dysfunction. The two PON1 polymorphisms were assessed by real-time PCR with TaqMan probes. PON1 enzymatic activity was assessed by spectrophotometric methods, using paraoxon as a substrate. Results - No significant differences were found for PON1 activity between normal and obese women. Nevertheless, PON1 activity was greater (P<0.01) for the RR genotype (in Q192R polymorphism) and for the LL genotype (in L55M polymorphism). The frequency of allele R of Q192R polymorphism was significantly higher in obese women (P<0.05) and was associated with an increased risk of obesity (odds ratio=2.0 – 95% confidence interval (1.04; 3.87)). Conclusion - 55M and Q192R polymorphisms influence PON1 activity. The allele R of the Q192R polymorphism is associated with an increased risk for development of obesity among Portuguese Caucasian premenopausal women.

Estudo da obtenção de gelatina a partir de raspa Wet-Blue da indústria de curtumes

Mestrado em Engenharia Química. Ramo Tecnologias de Protecção Ambiental.

Produção de bioetanol a partir de um resíduo orgânico proveniente da central de compostagem da LIPOR

Mestrado em Engenharia Química

Desenvolvimento de processo de depilação de pele caprina sem destruição do pêlo e avaliação do seu impacto ambiental

O meio ambiente é uma grande preocupação mundial existindo cada vez mais imposições legais no sentido de o proteger. Torna-se assim necessário que as indústrias adoptem e desenvolvam processos alternativos mais limpos. A indústria de curtumes transforma a pele animal em couro, material resistente à putrefacção e com estabilidade térmica suficiente para ser manufacturado nas indústrias do calçado, estofos, vestuário e marroquinaria. A transformação referida é efectuada através duma série de processos entre os quais o caleiro, processo que visa depilar a pele e promover o relaxamento da estrutura fibrilar, tem um papel importante. O processo de caleiro produz um efluente bastante poluente. Tendo em conta esse facto, este trabalho teve como objectivo desenvolver processos de depilação de pele caprina sem destruição do pêlo, com vista à redução da quantidade de sulfureto e sulfidrato de sódio utilizada e da carga poluente do efluente. Além disso, o processo permite a recuperação do pêlo e este pode ser reaproveitado como fertilizante orgânico, hidrolisado de queratina, compostagem, etc. Todo o trabalho foi realizado tomando por base um processo de caleiro padrão utilizado industrialmente, alterando-o de forma a obter um processo de caleiro óptimo de depilação sem destruição do pêlo com e sem utilização de enzimas. Numa primeira fase, desenvolveu-se um processo de depilação sem destruição do pêlo normalmente utilizado para a pele de bovino que foi adaptado com resultados positivos, designado de processo sem enzimas. Numa segunda fase, desenvolveu-se um processo enzimático em que se utilizou uma protease (Erhavit DMC), uma lipase (Defat 50) e uma amilase (Mayzme SD-L); este processo deu bons resultados tal como o anterior. A redução da quantidade de sulfureto de sódio foi de 33% e 53% para o processo sem enzimas e processo com enzimas, respectivamente, em relação ao processo padrão. O efeito dos processos na carga poluente, à escala piloto, comprova uma redução de 37% nos SST, 37% nos SDT, 60% na CQO, 9% no S2- e 73% no Azoto para o processo sem enzimas e uma redução de 93% nos SST, 63% nos SDT, 69% na CQO, 69% no S2- e 83% no Azoto para o processo com enzimas, quando comparados com o processo padrão. . Uma análise de custos, com incidência apenas nos agentes químicos utilizados, permitiu concluir a existência de uma redução de custos anuais de 30% para o processo com enzimas e de 13% para o processo sem enzimas, quando comparados com o processo padrão. Como conclusão, os processos desenvolvidos apresentam um grande potencial para serem explorados industrialmente.

Utilização de co-solventes na produção de biodiesel a partir de óleo residual e etanol por catálise enzimática

A constante e sistemática subida de preço dos combustíveis fósseis e as contínuas preocupações com o meio ambiente determinaram a procura de soluções ambientalmente sustentáveis. O biodiesel surge, então, como uma alternativa para essa problemática, bem como uma solução para resíduos líquidos e gordurosos produzidos pelo ser humano. A produção de biodiesel tem sido alvo de extensa atenção nos últimos anos, pois trata-se de um combustível biodegradável e não poluente. A produção de biodiesel pelo processo de transesterificação usando álcoois de cadeia curta e catalisadores químicos, nomeadamente alcalinos, tem sido aceite industrialmente devido à sua elevada conversão. Recentemente, a transesterificação enzimática tem ganho adeptos. No entanto, o custo da enzima permanece uma barreira para a sua aplicação em grande escala. O presente trabalho visa a produção de biodiesel por transesterificação enzimática a partir de óleo residual de origem vegetal. O álcool usado foi o etanol, em substituição do metanol usado convencionalmente na catálise homogénea, pois a atividade da enzima é inibida pela presença deste último. As maiores dificuldades apresentadas na etanólise residem na separação das fases (Glicerol e Biodiesel) após a reação bem como na menor velocidade de reação. Para ajudar a colmatar esta desvantagem foi estudada a influência de dois cosolventes: o hexano e o hexanol, na proporção de 20% (v/v). Após a escolha do co-solvente que permite obter melhor rendimento (o hexano), foi elaborado um planeamento fatorial no qual se estudou a influência de três variáveis na produção de biodiesel por catálise enzimática com etanol e co-solventes: a razão molar óleo/álcool (1:8, 1:6 e 1:4), a quantidade de co-solvente adicionado (30, 20 e 10%, v/v) e o tempo de reação (48, 36 e 24h). A avaliação do processo foi inicialmente seguida pelo rendimento da reação, a fim de identificar as melhores condições, sendo substituída posteriormente pela quantificação do teor de ésteres por cromatografia em fase gasosa. O biodiesel com teor de ésteres mais elevado foi produzido nas condições correspondentes a uma razão molar óleo:álcool de 1:4, com 5g de Lipozyme TL IM como catalisador, 10% co-solvente (hexano, v/v), à temperatura de 35 ºC durante 24h. O rendimento do biodiesel produzido sob estas condições foi de 73,3%, traduzido em 64,7% de teor de ésteres etílicos. Contudo o rendimento mais elevado que se obteve foi de 99,7%, para uma razão óleo/álcool de 1:8, 30% de co-solvente (hexano, v/v), reação durante 48h a 35 ºC, obtendo-se apenas 46,1% de ésteres. Por fim, a qualidade do biodiesel foi ainda avaliada, de acordo com as especificações da norma EN 14214, através das determinações de densidade, viscosidade, ponto de inflamação, teor de água, corrosão ao cobre, índice de acidez, índice de iodo, teor de sódio (Na+) e potássio (K+), CFPP e poder calorífico. Na Europa, os ésteres etílicos não têm, neste momento, norma que os regule quanto à classificação da qualidade de biodiesel. Contudo, o biodiesel produzido foi analisado de acordo com a norma europeia EN14214, norma esta que regula a qualidade dos ésteres metílicos, sendo possível concluir que nenhum dos parâmetros avaliados se encontra em conformidade com a mesma.

Glucose-6-Phosphate dehydrogenase deficiency in children from 0 to 14 years hospitalized at the Pediatric Hospital David Bernardino, Luanda, Angola

The Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic defect in the world. The most common clinical manifestations are acute hemolytic anemia associated with drugs, infections, neonatal jaundice and hemolytic non-spherocytic chronic anemia. The main aim of this study was to determine the frequency of major genetic variants of G6PD leading to enzyme deficiency in children from 0 to 14 years at a Pediatric Hospital in Luanda, Angola. A cross-sectional and descriptive analytical study covered a total of 194 children aged from 0 to 14 years, of both genders and hospitalized at the Pediatric Hospital David Bernardino, Luanda between November and December, 2011. The G202A, A376G and C563T mutations of the G6PD gene were determined by real-time PCR with Taqman probes. The disabled A-/A- genotype was detected in 10 girls (10.9%). Among the boys, 21 (20.6%) presented the genotype A-. Considering all the samples, the A- variant was observed in 22.4% of cases. The Mediterranean mutation was not detected in the Angolan sample. Furthermore, no association was found between genotype and anemia, nutritional state and mucosa color. A significant association, however, was observed with jaundice. Based on the results obtained, there is a clear need to identify those with the disabled genotype in the Angolan population in order to avoid cases of drug-induced anemia, particularly in the treatment of malaria, so prevalent in Angola.

Towards a reliable technology for antioxidant capacity and oxidative damage evaluation: Electrochemical (bio)sensors

To counteract and prevent the deleterious effect of free radicals the living organisms have developed complex endogenous and exogenous antioxidant systems. Several analytical methodologies have been proposed in order to quantify antioxidants in food, beverages and biological fluids. This paper revises the electroanalytical approaches developed for the assessment of the total or individual antioxidant capacity. Four electrochemical sensing approaches have been identified, based on the direct electrochemical detection of antioxidant at bare or chemically modified electrodes, and using enzymatic and DNA-based biosensors.

Biosensor based on multi-walled carbon nanotubes paste electrode modified with laccase for pirimicarb pesticide quantification

This study focused on the development of a sensitive enzymatic biosensor for the determination of pirimicarb pesticide based on the immobilization of laccase on composite carbon paste electrodes. Multi- walled carbon nanotubes(MWCNTs)paste electrode modified by dispersion of laccase(3%,w/w) within the optimum composite matrix(60:40%,w/w,MWCNTs and paraffin binder)showed the best performance, with excellent electron transfer kinetic and catalytic effects related to the redox process of the substrate4- aminophenol. No metal or anti-interference membrane was added. Based on the inhibition of laccase activity, pirimicarb can be determined in the range 9.90 ×10- 7 to 1.15 ×10- 5 molL 1 using 4- aminophenol as substrate at the optimum pH of 5.0, with acceptable repeatability and reproducibility (relative standard deviations lower than 5%).The limit of detection obtained was 1.8 × 10-7 molL 1 (0.04 mgkg 1 on a fresh weight vegetable basis).The high activity and catalytic properties of the laccase- based biosensor are retained during ca. one month. The optimized electroanalytical protocol coupled to the QuEChERS methodology were applied to tomato and lettuce samples spiked at three levels; recoveries ranging from 91.0±0.1% to 101.0 ± 0.3% were attained. No significant effects in the pirimicarb electro- analysis were observed by the presence of pro-vitamin A, vitamins B1 and C,and glucose in the vegetable extracts. The proposed biosensor- based pesticide residue methodology fulfills all requisites to be used in implementation of food safety programs.

Molinate quantification in environmental water by a glutathione-S-transferase based biosensor

A glutathione-S-transferase (GST)based biosensor was developed to quantify the thiocarbamate herbicide molinate in environmental water.The biosensor construction was based on GST immobilization onto a glassy carbon electrode via aminosilane–glutaraldehyde covalent attachment. The principle supporting the use of this biosensor consists of the GST inhibition process promoted by molinate. Differential pulse voltammetry was used to obtain a calibration curve for molinate concentration, ranging from 0.19 to 7.9 mgL -1 and presenting a detection limit of 0.064 mgL- 1. The developed biosensor is stable,and reusable during 15 days.The GST-based biosensor was successfully applied to quantify molinate in rice paddy field floodwater samples. The results achieved with the developed biosensor were in accordance with those obtained by high performance liquid chromatography. The proposed device is suitable for screening environmental water analysis and, since no sample preparation is required, it can be used in situ and in real-time measurements.

Laccase–Prussian blue film–graphene doped carbon paste modified electrode for carbamate pesticides quantification

A novel enzymatic biosensor for carbamate pesticides detection was developed through the direct immobilization of Trametes versicolor laccase on graphene doped carbon paste electrode functionalized with Prussianblue films (LACC/PB/GPE). Graphene was prepared by graphite sonication-assisted exfoliation and characterized by transmission electron microscopy and X-ray photoelectron spectro- scopy. The Prussian blue film electrodeposited onto graphene doped carbon paste electrode allowed considerable reduction of the charge transfer resistance and of the capacitance of the device.The combined effects of pH, enzyme concentration and incubation time on biosensor response were optimized using a 23 full-factorial statistical design and response surface methodology. Based on the inhibition of laccase activity and using 4-aminophenol as redox mediator at pH 5.0,LACC/PB/GPE exhibited suitable characteristics in terms of sensitivity, intra-and inter-day repeatability (1.8–3.8% RSD), reproducibility (4.1 and 6.3%RSD),selectivity(13.2% bias at the higher interference: substrate ratios tested),accuracy and stability(ca. twenty days)for quantification of five carbamates widely applied on tomato and potato crops.The attained detection limits ranged between 5.2×10−9 mol L−1(0.002 mg kg−1 w/w for ziram)and 1.0×10−7 mol L−1 (0.022 mg kg−1 w/w for carbofuran).Recovery values for the two tested spiking levels ranged from 90.2±0.1%(carbofuran)to 101.1±0.3% (ziram) for tomato and from 91.0±0.1%(formetanate)to 100.8±0.1%(ziram)for potato samples.The proposed methodology is appropriate to enable testing pesticide levels in food samples to fit with regulations and food inspections.

Regulation of histone H2A.Z expression is mediated by sirtuin 1 in prostate cancer

Histone variants seem to play a major role in gene expression regulation. In prostate cancer, H2A.Z and its acetylated form are implicated in oncogenes’ upregulation. SIRT1, which may act either as tumor suppressor or oncogene, reduces H2A.Z levels in cardiomyocytes, via proteasome-mediated degradation, and this mechanism might be impaired in prostate cancer cells due to sirtuin 1 downregulation. Thus, we aimed to characterize the mechanisms underlying H2A.Z and SIRT1 deregulation in prostate carcinogenesis and how they interact. We found that H2AFZ and SIRT1 were up- and downregulated, respectively, at transcript level in primary prostate cancer and high-grade prostatic intraepithelial neoplasia compared to normal prostatic tissues. Induced SIRT1 overexpression in prostate cancer cell lines resulted in almost complete absence of H2A.Z. Inhibition of mTOR had a modest effect on H2A.Z levels, but proteasome inhibition prevented the marked reduction of H2A.Z due to sirtuin 1 overexpression. Prostate cancer cells exposed to epigenetic modifying drugs trichostatin A, alone or combined with 5-aza-2’-deoxycytidine, increased H2AFZ transcript, although with a concomitant decrease in protein levels. Conversely, SIRT1 transcript and protein levels increased after exposure. ChIP revealed an increase of activation marks within the TSS region for both genes. Remarkably, inhibition of sirtuin 1 with nicotinamide, increased H2A.Z levels, whereas activation of sirtuin 1 by resveratrol led to an abrupt decrease in H2A.Z. Finally, protein-ligation assay showed that exposure to epigenetic modifying drugs fostered the interaction between sirtuin 1 and H2A.Z. We concluded that sirtuin 1 and H2A.Z deregulation in prostate cancer are reciprocally related. Epigenetic mechanisms, mostly histone post-translational modifications, are likely involved and impair sirtuin 1-mediated downregulation of H2A.Z via proteasome-mediated degradation. Epigenetic modifying drugs in conjunction with enzymatic modulators are able to restore the normal functions of sirtuin 1 and might constitute relevant tools for targeted therapy of prostate cancer patients

Biodegradação enzimática de aminas aromáticas tóxicas

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica

Experimental design methodologies for the identification of Michaelis-Menten type kinetics

The main objective of this work was to investigate the application of experimental design techniques for the identification of Michaelis-Menten kinetic parameters. More specifically, this study attempts to elucidate the relative advantages/disadvantages of employing complex experimental design techniques in relation to equidistant sampling when applied to different reactor operation modes. All studies were supported by simulation data of a generic enzymatic process that obeys to the Michaelis-Menten kinetic equation. Different aspects were investigated, such as the influence of the reactor operation mode (batch, fed-batch with pulse wise feeding and fed-batch with continuous feeding) and the experimental design optimality criteria on the effectiveness of kinetic parameters identification. The following experimental design optimality criteria were investigated: 1) minimization of the sum of the diagonal of the Fisher information matrix (FIM) inverse (A-criterion), 2) maximization of the determinant of the FIM (D-criterion), 3) maximization of the smallest eigenvalue of the FIM (E-criterion) and 4) minimization of the quotient between the largest and the smallest eigenvalue (modified E-criterion). The comparison and assessment of the different methodologies was made on the basis of the Cramér-Rao lower bounds (CRLB) error in respect to the parameters vmax and Km of the Michaelis-Menten kinetic equation. In what concerns the reactor operation mode, it was concluded that fed-batch (pulses) is better than batch operation for parameter identification. When the former operation mode is adopted, the vmax CRLB error is lowered by 18.6 % while the Km CRLB error is lowered by 26.4 % when compared to the batch operation mode. Regarding the optimality criteria, the best method was the A-criterion, with an average vmax CRLB of 6.34 % and 5.27 %, for batch and fed-batch (pulses), respectively, while presenting a Km’s CRLB of 25.1 % and 18.1 %, for batch and fed-batch (pulses), respectively. As a general conclusion of the present study, it can be stated that experimental design is justified if the starting parameters CRLB errors are inferior to 19.5 % (vmax) and 45% (Km), for batch processes, and inferior to 42 % and to 50% for fed-batch (pulses) process. Otherwise equidistant sampling is a more rational decision. This conclusion clearly supports that, for fed-batch operation, the use of experimental design is likely to largely improve the identification of Michaelis-Menten kinetic parameters.

Kinetic behavior of Desulfovibrio gigas aldehyde oxidoreductase encapsulated in reverse micelles

Biochemical and Biophysical Research Communications 308 (2003) 73–78