Contributo para a determina����o simult��nea, por cromatografia l��quida de alta resolu����o, de carotenoides, vitamina A e vitamina E em amostras compostas por diferentes matrizes alimentares

Trabalho Final de Mestrado para obten����o do grau de Mestre em Engenharia Qu��mica e Biol��gica

Aplica����o de filmes de suberina na produ����o de materiais bio-funcionais

Trabalho Final de Mestrado para obten����o do grau de Mestre em Engenharia Qu��mica e Biol��gica

Valida����o de um m��todo de cromatografia de alta efici��ncia para determina����o de conservantes em g��neros aliment��cios

Trabalho Final de Mestrado para obten����o do grau de Mestre em Engenharia Qu��mica e Biol��gica

Determina����o do teor de compostos arom��ticos polic��clicos de ��leos minerais isolantes para transformadores

Mestrado em Engenharia Qu��mica - Ramo Optimiza����o Energ��tica na Ind��stria Qu��mica

Desenvolvimento de M��todos de Extra����o de Res��duos de Antimicrobianos e Quantifica����o em Amostras Alimentares

O leite �� um alimento complexo, pela sua composi����o rico em ��gua, prote��nas, l��pidos, vitaminas e minerais. Devido ao seu alto valor nutricional �� fundamental para a amamenta����o de crian��as e animais em crescimento, pois fornece componentes fundamentais para o desenvolvimento e manuten����o da sa��de. Os antimicrobianos s��o amplamente utilizados como uma medida terap��utica no tratamento de infe����es bacterianas, profilaxia e como promotores de crescimento (aditivos). A presen��a de res��duos de antimicrobianos no leite pode representar riscos para a sa��de humana, como rea����es al��rgicas em indiv��duos hipersens��veis e resist��ncias. Os objetivos deste estudo s��o o desenvolvimento de novos m��todos de limpeza e de pr��-concentra����o para amostras de leite, por meio de extra����o em fase s��lida (SPE), com a finalidade de realizar uma melhor identifica����o e quantifica����o de antimicrobiana por Cromatografia L��quida de Alta Performance (HPLC). Todos os m��todos desenvolvidos s��o de f��cil execu����o, com taxas de recupera����o dos agentes antimicrobianos vi��veis, com uma percentagem de recupera����o a partir de 85%. O m��todo cromatogr��fico utilizado para a dete����o e quantifica����o (HPLC-DAD) t��m os limites de dete����o (LD) entre 2.43ng / mL e 1.62ng / mL e os limites de quantifica����o (LQ) entre 7,36 ng / mL e 4.92 ng / mL, o que significa este m��todo vai de encontro ��s diretrizes estipuladas pela Uni��o Europeia para os agentes antimicrobianos estudados. A combina����o dos m��todos propostos de limpeza e pr��-concentra����o por SPE e multirres��duo por HPLC-DAD permite, por conseguinte, a dete����o e quantifica����o de res��duos de antibi��ticos no leite, tornando esta uma alternativa importante e ��til no processo de controlo de qualidade para a ind��stria de alimentos e outras ��rea.

Desenvolvimento de m��todos de extra����o de res��duos de antimicrobianos e quantifica����o em amostras alimentares

O leite �� um alimento complexo, pela sua composi����o rico em ��gua, prote��nas, l��pidos, vitaminas e minerais. Devido ao seu alto valor nutricional �� fundamental para a amamenta����o de crian��as e animais em crescimento, pois fornece componentes fundamentais para o desenvolvimento e manuten����o da sa��de. Os antimicrobianos s��o amplamente utilizados como uma medida terap��utica no tratamento de infe����es bacterianas, profilaxia e como promotores de crescimento (aditivos). A presen��a de res��duos de antimicrobianos no leite pode representar riscos para a sa��de humana, como rea����es al��rgicas em indiv��duos hipersens��veis e resist��ncias. Os objetivos deste estudo s��o o desenvolvimento de novos m��todos de limpeza e de pr��- concentra����o para amostras de leite, por meio de extra����o em fase s��lida (SPE), com a finalidade de realizar uma melhor identifica����o e quantifica����o de antimicrobiana por Cromatografia L��quida de Alta Performance (HPLC). Todos os m��todos desenvolvidos s��o de f��cil execu����o, com taxas de recupera����o dos agentes antimicrobianos vi��veis, com uma percentagem de recupera����o a partir de 85%. O m��todo cromatogr��fico utilizado para a dete����o e quantifica����o (HPLC-DAD) t��m os limites de dete����o (LD) entre 2.43ng / mL e 1.62ng / mL e os limites de quantifica����o (LQ) entre 7,36 ng / mL e 4.92 ng / mL, o que significa este m��todo vai de encontro ��s diretrizes estipuladas pela Uni��o Europeia para os agentes antimicrobianos estudados. A combina����o dos m��todos propostos de limpeza e pr��-concentra����o por SPE e multirres��duo por HPLC-DAD permite, por conseguinte, a dete����o e quantifica����o de res��duos de antibi��ticos no leite, tornando esta uma alternativa importante e ��til no processo de controlo de qualidade para a ind��stria de alimentos e outras ��rea.

Valoriza����o da Dr��che Cervejeira: Fermenta����o de Pentoses

O presente trabalho tem como objetivo a otimiza����o da etapa de fermenta����o dos a����cares obtidos a partir da dr��che cervejeira para produ����o do bioetanol atrav��s da utiliza����o das leveduras Pichia stipitis NCYC 1541 e Kluyveromyces marxianus NCYC 2791 como agentes fermentativos. O meio de cultura usado para manter as culturas destas leveduras foi Yeast Extract Peptone Dextrose (YEPD). O principal prop��sito deste trabalho foi o de encontrar alternativas aos combust��veis f��sseis, pautando-se por solu����es inofensivas para o meio ambiente e sustent��veis. Assim, o trabalho est�� dividido em quatro etapas: 1) carateriza����o qu��mica e biol��gica da dr��che; 2) pr��-tratamento ��cido e hidr��lise enzim��tica para primeiramente quebrar as mol��culas de lenhina que envolvem os pol��meros de celulose e hemicelulose e em seguida romper as liga����es polim��ricas destas macromol��culas por a����o enzim��tica e transforma-las em a����cares simples, respetivamente, obtendo-se ent��o a glucose, a maltose, a xilose e a arabinose; e, por ��ltimo, 3) otimiza����o da etapa de fermenta����o da glucose, maltose e das pentoses que constitui a condi����o essencial para se chegar �� s��ntese do bioetanol de um modo eficiente e sustent��vel e 4) a recupera����o do bioetanol produzido por destila����o fracionada. A quantifica����o dos a����cares libertados no processo foi feita recorrendo a an��lises por cromatografia l��quida de alta efici��ncia (HPLC). Neste estudo foram identificados e quantificados cinco a����cares: Arabinose, Glucose, Maltose, Ribose e Xilose. Na etapa de pr��-tratamento e hidr��lise enzim��tica foram usados os ��cidos clor��drico (HCl) e n��trico (HNO3) com a concentra����o de 1% (m/m), e as enzimas Glucanex 100g e Ultraflo L. Foram testadas seis condi����es de pr��-tratamento e hidr��lise enzim��tica, alterando os par��metros tempo de contacto e raz��o enzimas/massa de dr��che, respetivamente, e mantendo a temperatura (50 ��C), velocidade de agita����o (75 rpm) e concentra����o dos ��cidos (1% (m/m)). No processamento de 25 g de dr��che seca com 0,5 g de Glucanex, 0,5 mL de Ultraflo e um tempo de rea����o de 60 minutos para as enzimas foi obtida uma efici��ncia de 15%, em hidrolisado com 6% da celulose. Realizou-se a fermenta����o do hidrolisado resultante do pr��-tratamento ��cido e hidr��lise enzim��tica de dr��che cervejeira e de meios sint��ticos preparados com os a����cares puros, usando as duas estirpes selecionadas para este estudo: Pichia stipitis NCYC 1541 e Kluyveromyces marxianus NYCY 2791. As efici��ncias de fermenta����o dos a����cares nos meios sint��ticos foram superiores a 80% para ambas as leveduras. No entanto, as efici��ncias de fermenta����o do hidrolisado da dr��che foram de 45,10% pela Pichia stipitis e de 36,58 para Kluyveromyces marxianus, para um tempo de fermenta����o de 72 horas e �� temperatura de 30 ��C. O rendimento te��rico em ��lcool no hidrolisado da dr��che �� de 0,27 g/g, tr��s vezes maior do que o real (0,0856 g/g), para Pichia stipitis e de 0,19 g/g seis vezes maior do que o real (0,0308 g/g), para a Kluyveromyces marxianus.

Pre-treatment of different types of lignocellulosic biomass using ionic liquids

Disserta����o apresentada na Faculdade de Ci��ncias e Tecnologia da Universidade Nova de Lisboa para obten����o do grau Mestre em Biotecnologia

Design and characterization of novel biopolymeric structure using biocompatible ionic liquids

The main objective of this thesis was the development of polymeric structures from the dissolution of FucoPol, a bacterial exopolysaccharide (EPS), in a biocompatible ionic liquid, choline acetate. The FucoPol was produced by the bacteria Enterobacter A47 using glycerol as carbon source at controlled temperature and pH (30��C and 7, respectively). At the end of 3 days it was produced 7 g/L of FucoPol. The net yield of Fucopol in glycerol (YP/S) was 0.22 g/g and the maximum productivity 2.37 g/L.d This polymer was characterized about its composition in sugars and acyl groups (by High-Performance Liquid Chromatography - HPLC), containing fucose (35 % mol), galactose (21 % mol), glucose (29 % mol), rhamnose (3% mol) and glucuronic acid (12% mol) as well as acetate (14.28 % mol), pyruvate (2.15 % mol) and succinate (1.80 % mol). Its content of water and ash was 15% p/p and 2% p/p, respectively, and the chemical bonds (determined by Infrared Spectroscopy - FT-IR) are consistent to the literature reports. However, due to limitations in Differential Scanning Calorimetry (DSC) equipment it was not possible to determine the glass transition temperature. In turn, the ionic liquid showed the typical behavior of a Newtonian fluid, glass transition temperature (determined by DSC) -98.03��C and density 1.1031 g/cm3. The study of chemical bonds by FT-IR showed that amount of water (8.80%) influenced the visualization of the bands predicted to in view of their chemical structure. After the dissolution of the FucoPol in the ionic liquid at different temperatures (50, 60, 80 and 100 �� C) it was promoted the removal of this by the phase inversion method using deionized water as a solvent, followed by drying in an oven at 70 �� C. The mixtures before and after the phase inversion method were characterized through the studies mentioned above. In order to explore possible application field���s biocompatibility assays and collage on balsa wood tests were performed. It was found that the process of washing with water by the phase inversion method was not totally effective in removing the biocompatible ionic liquid, since all FucoPol ��� IL mixtures still contained ionic liquid in their composition as can be seen by the DSC results and FT-IR. In addition, washing the mixtures with water significantly altered the composition of FucoPol. However, these mixtures, that developed a viscous behavior typical of a non-Newtonian fluid (shear-thinning), have the potential to be applied in the biomedical field as well as biological glues.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor ��-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to ���in-cell��� Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.

Polyphenols and sugars recovery from autohydrolysis of pineapple waste

[Excerpt] The aim of this research was to evaluate the influence of temperature, time and mass/ volume ratio on the release of sugars and polyphenols using an autohydrolysis procedure from pineapple waste. A Box-Bhenken design was used with three factors (time, temperature and mass/volume ratio) and three levels was used. All treatments were performed in triplicate. Nine central points were used. For autohydrlosysis treatments, an oil bath was used [1]. After autohydrolysis, liquid phases or hydrolysates were analyzed for glucose and fructose concentration by high performance liquid chromatography (HPLC) [2]. The FolinCiocalteu assay was used to measure total polyphenols of hydrolysates [3] and HPLC to identify these molecules [4]. (...)

Determinaci�� de diferents compostos bioactius en ingredients naturals per a l'elaboraci�� de bombons funcionals amb beneficis cardiovasculars

Durant les darreres d��cades, i degut, principalment, a un canvi en els h��bits alimentaris, hi ha hagut un augment a nivell mundial de malalties cr��niques (l���obesitat, malalties cardiovasculars, etc.). En els pa��sos mediterranis hi ha menys incid��ncia d���aquestes malalties i sembla ser que aix�� es deu a l���anomenada dieta mediterr��nia. La dieta mediterr��nia es caracteritza per una combinaci�� d���oli d���oliva com a grassa principal, verdures, hortalisses i fruites en abund��ncia, lleguminoses, fruits secs, formatges i iogurt, peix, pa, pasta, cereals i els seus derivats i un consum moderat de vi i carns. Aquest model alimentari, ric en tocoferols, fitosterols i fitoestanols que ajuden a reduir el contingut de colesterol en sang, fa que en les poblacions mediterr��nies hi hagi menys incid��ncia de malalties cardiovasculars. Aquests compostos inhibeixen el deteriorament oxidatiu dels olis, actuen com agent antipolimeritzaci�� per olis de fregir. Tenen capacitat de reduir els nivells de colesterol, evitant la incid��ncia de malalties cardiovasculars. Els fitoesterols y fitoestanols es poden trobar en forma lliure o esterificada amb ��cids grassos, ��cids fen��lics i glucosa. Els objectius d��� aquest treball han estat, primer en el desenvolupament de m��todes d'an��lisi r��pids, fiables i robusts dels tocoferols, fitoesterols i fitoestanols i la seva aplicaci�� en fruits sec, oli de seg��, oli de pinyol de ra��m i productes que els continguin. El primer m��tode va estar basat en la cromatograf��a l��quida (HPLC-DAD) amb extracci�� en fase s��lida (SPE) com t��cnica alternativa a la saponificaci�� para la determinaci�� de fitoesterols lliures. Aquest m��tode va estar aplicada a mostres de bombons que contenia fitoesterols. El segon m��tode va estar basat en la cromatografia de gasos (GCFID) amb aponificaci�� i SPE per quantificar fitoesterols i fitoestanols lliures, esterificats i totals. En els documents annexos es descriuen a profunditat els m��todes desenvolupats.

Profile of organic acid concentrations in the digestive gland and hemolymph of Biomphalaria glabrata under estivation

Using high performance liquid chromatography (HPLC) analysis it was possible to determine simultaneously the concentration of organic acids (pyruvate, lactate, succinate, fumarate, malate, acetate, propionate, acetoacetate, and ��-hydroxybutyrate) in the digestive gland and the extracellular concentration of these same acids in the hemolymph of estivating Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. After a 7 day period of estivation, there was a significant increase in the tissue levels of lactate, succinate, malate and acetate compared to non-estivating snails. After 14 days of estivation, the levels of lactate and acetate were also significantly elevated. The hemolymph concentrations of pyruvate and acetate increased significantly after 7 days and acetate concentrations continued to be significantly increased up to 14 days of estivation. The other organic acids studied, such as ketone body acetoacetate and ��-hydroxybutyrate or the volatile acid propionate, did not accumulate. Their tissue concentrations, however, increased on the 7th day of estivation and reached normal levels within two weeks of estivation for some of them. One should take into consideration how the reduction in metabolism can be handled under aerobic conditions, and what role anaerobic pathways may play in both energy formation and redox balance processes.

Plasma angiotensin-(1-8) octapeptide measurement to assess acute angiotensin-converting enzyme inhibition with captopril administered parenterally to normal subjects.

The blood pressure (BP), heart rate (HR), and humoral effects of single intravenous (i.v.) doses of the angiotensin-converting enzyme (ACE) inhibitor captopril was investigated in five normotensive healthy volunteers. Each subject received at 1-week intervals a bolus dose of either captopril (1, 5, and 25 mg) or its vehicle. The study was conducted in a single-blind fashion, and the order of treatment phases was randomized. The different doses of captopril had no acute effect on BP and HR. They induced a dose-dependent decrease in plasma ACE activity and plasma angiotensin II levels. The angiotensin-(1-8) octapeptide was isolated by solid-phase extraction and high-performance liquid chromatography (HPLC) prior to radioimmunoassay (RIA). All three doses of captopril reduced circulating angiotensin II levels within 15 min of drug administration. Only with the 25-mg dose was the angiotensin II concentration below the detection limit at 15 min and still significantly reduced 90 min after drug administration. Simultaneous and progressive decreases in plasma aldosterone levels were observed both with ACE inhibition and during vehicle injection, but the relative fall was more pronounced after captopril administration. No adverse reaction was noticed. These results demonstrate that captopril given parenterally blocks the renin-angiotensin system in a dose-dependent manner. Only with the dose of 25 mg was the inhibition of plasma-converting enzyme activity and the reduction of plasma angiotensin II sustained for at least 1 1/2 h.

Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease.

Introduction. Fibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia. Methods. We studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q10 levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX��� and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker��� Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells. Results. We found reduced levels of coenzyme Q10, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy. Conclusions. These findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.