Valorização da Drêche Cervejeira: Fermentação de Pentoses

O presente trabalho tem como objetivo a otimização da etapa de fermentação dos açúcares obtidos a partir da drêche cervejeira para produção do bioetanol através da utilização 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 propósito deste trabalho foi o de encontrar alternativas aos combustíveis fósseis, pautando-se por soluções inofensivas para o meio ambiente e sustentáveis. Assim, o trabalho está dividido em quatro etapas: 1) caraterização química e biológica da drêche; 2) pré-tratamento ácido e hidrólise enzimática para primeiramente quebrar as moléculas de lenhina que envolvem os polímeros de celulose e hemicelulose e em seguida romper as ligações poliméricas destas macromoléculas por ação enzimática e transforma-las em açúcares simples, respetivamente, obtendo-se então a glucose, a maltose, a xilose e a arabinose; e, por último, 3) otimização da etapa de fermentação da glucose, maltose e das pentoses que constitui a condição essencial para se chegar à síntese do bioetanol de um modo eficiente e sustentável e 4) a recuperação do bioetanol produzido por destilação fracionada. A quantificação dos açúcares libertados no processo foi feita recorrendo a análises por cromatografia líquida de alta eficiência (HPLC). Neste estudo foram identificados e quantificados cinco açúcares: Arabinose, Glucose, Maltose, Ribose e Xilose. Na etapa de pré-tratamento e hidrólise enzimática foram usados os ácidos clorídrico (HCl) e nítrico (HNO3) com a concentração de 1% (m/m), e as enzimas Glucanex 100g e Ultraflo L. Foram testadas seis condições de pré-tratamento e hidrólise enzimática, alterando os parâmetros tempo de contacto e razão enzimas/massa de drêche, respetivamente, e mantendo a temperatura (50 ºC), velocidade de agitação (75 rpm) e concentração dos ácidos (1% (m/m)). No processamento de 25 g de drêche seca com 0,5 g de Glucanex, 0,5 mL de Ultraflo e um tempo de reação de 60 minutos para as enzimas foi obtida uma eficiência de 15%, em hidrolisado com 6% da celulose. Realizou-se a fermentação do hidrolisado resultante do pré-tratamento ácido e hidrólise enzimática de drêche cervejeira e de meios sintéticos preparados com os açúcares puros, usando as duas estirpes selecionadas para este estudo: Pichia stipitis NCYC 1541 e Kluyveromyces marxianus NYCY 2791. As eficiências de fermentação dos açúcares nos meios sintéticos foram superiores a 80% para ambas as leveduras. No entanto, as eficiências de fermentação do hidrolisado da drêche foram de 45,10% pela Pichia stipitis e de 36,58 para Kluyveromyces marxianus, para um tempo de fermentação de 72 horas e à temperatura de 30 °C. O rendimento teórico em álcool no hidrolisado da drêche é de 0,27 g/g, três 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.

CHARACTERIZATION OF MOLLUSCICIDAL COMPONENT OF Moringa oleifera LEAF AND Momordica charantia FRUITS AND THEIR MODES OF ACTION IN SNAIL Lymnaea acuminata

SUMMARY The molluscicidal activity of the leaf powder of Moringa oleifera and lyophilized fruit powder of Momordica charantia against the snail Lymnaea acuminata was time and concentration dependent. M. oleifera leaf powder (96 h LC50: 197.59 ppm) was more toxic than M. charantia lyophilized fruit powder (96 h LC50: 318.29 ppm). The ethanolic extracts of M. oleifera leaf powder and Momordica charantia lyophilized fruit powder were more toxic than other organic solvent extracts. The 96 h LC50 of the column purified fraction of M. oleifera leaf powder was 22.52 ppm, while that of M. charantia lyophilized fruit powder was 6.21 ppm. Column, thin layer and high performance liquid chromatography analysis show that the active molluscicidal components in M. oleifera leaf powder and lyophilized fruit of M. charantia are benzylamine (96 h LC50: 2.3 ppm) and momordicine (96 h LC50: 1.2 ppm), respectively. Benzylamine and momordicine significantly inhibited, in vivo and in vitro, the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activities in the nervous tissues of L. acuminata. Inhibition of AChE, ACP and ALP activity in the nervous tissues of L. acuminata by benzylamine and momordicine may be responsible for the molluscicidal activity of M. oleifera and M. charantia fruits, respectively.

Valorização das águas corticeiras: extracção de antioxidantes

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

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

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau Mestre em Biotecnologia

Ionic liquids as catalysts of lignocellulosic biomass processing

The present work is devoted to study the pre-treatment of lignocellulosic biomass, especially wheat straw, by the application of the acidic ionic liquid (IL) such as 1-butyl-3-methylimidazolium hydrogen sulphate. The ability of this IL to hydrolysis and conversion of biomass was scrutinised. The pre-treatment with hydrogen sulphate-based IL allowed to obtain a liquor rich in hemicellulosic sugars, furans and organic acids, and a solid fraction mainly constituted by cellulose and lignin. Quantitative and qualitative analyses of the produced liquors were made by capillary electrophoresis and high-performance liquid chromatography. Pre-treatment conditions were set to produce xylose or furfural. Specific range of temperatures from 70 to 175 °C and residence times from 20.0 to 163.3 min were studied by fixing parameters such as biomass/IL ratio (10 % (w/w)) and water content (1.25 % (w/w)) in the pre-treatment process. Statistical modelling was applied to maximise the xylose and furfural concentrations. For the purpose of reaction condition comparison the severity factor for studied ionic liquid was proposed and applied in this work. Optimum conditions for xylose production were identified to be at 125 °C and 82.1 min, at which 16.7 % (w/w) xylose yield was attained. Furfural was preferably formed at higher pre-treatment temperatures and longer reaction time (161 °C and 104.5 min) reaching 30.7 % (w/w) maximum yield. The influence of water content on the optimum xylose formation was also studied. Pre-treatments with 5 and 10 % (w/w) water content were performed and an increase of 100 % and 140 % of xylose yield was observed, respectively, while the conversion into furfural maintained unchanged.

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 (30ºC 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) -98.03ºC 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.

Validação de limpeza de equipamentos para produção de fórmulas líquidas e pastosas

A utilização de equipamentos multiproduto é uma prática bastante comum em indústria farmacêutica. No entanto, a esta prática podem associar-se riscos de transferência de contaminantes sendo de extrema importância garantir a eficácia dos procedimentos de limpeza estabelecidos. Esta dissertação tem como objetivo a validação de limpeza dos equipamentos do setor das Formas Líquidas e Pastosas (FLP), dos Laboratórios Atral, SA por forma a evidenciar a eficácia e eficiência destes procedimentos na remoção de substâncias ativas (SA), excipientes e agentes de limpeza (tipos de contaminantes). Tendo em conta a diversidade de produtos e equipamentos do setor, recorreu-se a uma análise de risco para identificação de equipamentos e produtos pior-caso. A partir desta análise será também possível identificar os resíduos de substância ativa a procurar após higienização de um determinado equipamento. Para tal, foi necessário estabelecer e validar métodos de análise em HPLC (High Performance Liquid Chromatography) capazes de identificar e quantificar os resíduos das SAs em questão. A baixa frequência de produção de alguns dos produtos pior-caso impossibilitou o terminar dos estudos de validação de limpeza para o agitador de hélice e equipamentos de produção de cremes. O desenvolvimento destes procedimentos permitirá identificar potenciais riscos anteriormente desconhecidos, bem como a recolha e elaboração de documentos comprovativos da capacidade e adequabilidade dos procedimentos de limpeza aplicados.

Vitamin A, vitamin E, iron and zinc status in a cohort of HIV-infected mothers and their uninfected infants

Introduction We hypothesized that nutritional deficiency would be common in a cohort of postpartum, human immunodeficiency virus (HIV)-infected women and their infants. Methods Weight and height, as well as blood concentrations of retinol, α-tocopherol, ferritin, hemoglobin, and zinc, were measured in mothers after delivery and in their infants at birth and at 6-12 weeks and six months of age. Retinol and α-tocopherol levels were quantified by high performance liquid chromatography, and zinc levels were measured by atomic absorption spectrophotometry. The maternal body mass index during pregnancy was adjusted for gestational age (adjBMI). Results Among the 97 women 19.6% were underweight. Laboratory abnormalities were most frequently observed for the hemoglobin (46.4%), zinc (41.1%), retinol (12.5%) and ferritin (6.5%) levels. Five percent of the women had mean corpuscular hemoglobin concentrations < 31g/dL. The most common deficiency in the infants was α-tocopherol (81%) at birth; however, only 18.5% of infants had deficient levels at six months of age. Large percentages of infants had zinc (36.8%) and retinol (29.5%) deficiencies at birth; however, these percentages decreased to 17.5% and 18.5%, respectively, by six months of age. No associations between infant micronutrient deficiencies and either the maternal adjBMI category or maternal micronutrient deficiencies were found. Conclusions Micronutrient deficiencies were common in HIV-infected women and their infants. Micronutrient deficiencies were less prevalent in the infants at six months of age. Neither underweight women nor their infants at birth were at increased risk for micronutrient deficiencies.

Hyperhomocyst(e)inemia in chronic stable renal transplant patients

PURPOSE: Hyperhomocyst(e)inaemia is an important risk factor for atherosclerosis, which is currently a major cause of death in renal transplant patients. The aim of this study was to assess the influence of immunosuppressive therapy on homocyst(e)inemia in renal transplant recipients. METHODS: Total serum homocysteine (by high performance liquid chromatography), creatinine, lipid profile, folic acid (by radioimmunoassay-RIA) and vitamin B12 (by RIA) concentrations were measured in 3 groups. Group I patients (n=20) were under treatment with cyclosporine, azathioprine, and prednisone; group II (n=9) were under treatment with azathioprine and prednisone; and group III (n=7) were composed of renal graft donors for groups I and II. Creatinine, estimated creatinine clearance, cyclosporine trough level, lipid profile, folic acid, and vitamin B12 concentrations and clinical characteristics of patients were assessed with the aim of ascertaining determinants of hyperhomocyst(e)inemia. RESULTS: Patient ages were 48.8 ± 15.1 yr (group I), 43.3 ± 11.3 yr (group II); and 46.5 ± 14.8 yr (group III). Mean serum homocyst(e)ine (tHcy) concentrations were 18.07 ± 8.29 mmol/l in renal transplant recipients; 16.55 ± 5.6 mmol/l and 21.44 ± 12.1 mmol/l respectively for group I (with cyclosporine) and group II (without cyclosporine) (NS). In renal donors, tHcy was significantly lower (9.07 ± 3.06 mmol/l; group I + group II vs. group III, p<0.008). There was an unadjusted correlation (p<0.10) between age (r=0.427; p<0.005) body weight (r=0.412; p<0.05), serum creatinine (r=0.427; p<0.05), estimated creatinine clearance (r=0.316; p<0.10), and tHcy in renal recipients (group I +II). Independent regressors (r²=0.46) identified in the multiple regression model were age (coefficient= 0.253; p=0.009) and serum creatinine (coefficient=8.07; p=0.045). We found no cases of hyperhomocyst(e)inemia in the control group. In contrast, 38% of renal recipients had hyperhomocyst(e)inemia: 7 cases (35%) on cyclosporine and 4 (45%) without cyclosporine, based on serum normal levels. CONCLUSIONS: Renal transplant recipients frequently have hyperhomocyst(e)inemia. Hyperhomocyst(e)inemia in renal transplant patients is independent of the scheme of immunosuppression they are taking. The older the patients are and the higher are their serum creatinine levels, the more susceptible they are to hyperhomocyst(e)inemia following renal transplantation.

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 β-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.

Bioactive dihydroxyfuranonaphthoquinones from the bark of Tabebuia incana A.H. Gentry (Bignoniaceae) and HPLC analysis of commercial pau d'arco and certified T.incana bark infusions

Tabebuia incana A.H. Gentry (Bignoniaceae) is a tree from the Brazilian Amazon having medicinal uses and is one several Tabebuia spp. known as pau d'arco or palo de arco in this region. Fractionation of the bark ethanolic extract afforded a mixture of 5 and 8-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-diones (1 and 2, respectively) identified on the basis of nuclear magnetic resonance (NMR), infrared (IR) and mass (MS) spectra, whose in vitro antimalarial and antitumor activity have been shown previously. This is the first study on T. incana bark, and 2 are described in this species for the first time. Also, high performance liquid chromatography (HPLC) analysis of T. incana bark tea revealed the presence of the 1 + 2 mixture peak corresponding to a concentration in the range 10-6-10-5 M. The chromatograms of teas prepared from commercial pau d' arco and T. incana bark were also studied and the presence of the 1 + 2 peak has potential for quality control of commercial plant materials.

Combining analytical methodologies for the identification of Aspergillus section Flavi strains isolated from Brazilian agricultural commodities

The use of chemical analysis of microbial components, including proteins, became an important achievement in the 80’s of the last century to the microbial identification. This led a more objective microbial identification scheme, called chemotaxonomy, and the analytical tools used in the field are mainly 1D/2D gel electrophoresis, spectrophotometry, high-performance liquid chromatography, gas chromatography, and combined gas chromatography-mass spectrometry. The Edman degradation reaction was also applied to peptides sequence giving important insights to the microbial identification. The rapid development of these techniques, in association with knowledge generated by DNA sequencing and phylogeny based on rRNA gene and housekeeping genes sequences, boosted the microbial identification to an unparalleled scale. The recent results of mass spectrometry (MS), like Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF), for rapid and reliable microbial identification showed considerable promise. In addition, the technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with other biological techniques. At present, MALDI-TOF MS adds an additional step for polyphasic identification which is essential when there is a paucity of characters or high DNA homologies for delimiting very close related species. The full impact of this approach is now being appreciated when more diverse species are studied in detail and successfully identified. However, even with the best polyphasic system, identification of some taxa remains time-consuming and determining what represents a species remains subjective. The possibilities opened with new and even more robust mass spectrometers combined with sound and reliable databases allow not only the microbial identification based on the proteome fingerprinting but also include de novo specific proteins sequencing as additional step. These approaches are pushing the boundaries in the microbial identification field.

Proteins on microbial identification: past achievements, present state-of-the-art, and future challenges

The use of chemical analysis of microbial components, including proteins, became an important achievement in the 80’s of the last century to the microbial identification. This led a more objective microbial identification scheme, called chemotaxonomy, and the analytical tools used in the field are mainly 1D/2D gel electrophoresis, spectrophotometry, high-performance liquid chromatography, gas chromatography, and combined gas chromatography-mass spectrometry. The Edman degradation reaction was also applied to peptides sequence giving important insights to the microbial identification. The rapid development of these techniques, in association with knowledge generated by DNA sequencing and phylogeny based on rRNA gene and housekeeping genes sequences, boosted the microbial identification to an unparalleled scale. The recent results of mass spectrometry (MS), like Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF), for rapid and reliable microbial identification showed considerable promise. In addition, the technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with other biological techniques. At present, MALDI-TOF MS adds an additional step for polyphasic identification which is essential when there is a paucity of characters or high DNA homologies for delimiting very close related species. The full impact of this approach is now being appreciated when more diverse species are studied in detail and successfully identified. However, even with the best polyphasic system, identification of some taxa remains time-consuming and determining what represents a species remains subjective. The possibilities opened with new and even more robust mass spectrometers combined with sound and reliable databases allow not only the microbial identification based on the proteome fingerprinting but also include de novo specific proteins sequencing as additional step. These approaches are pushing the boundaries in the microbial identification field.

UV-visible scanning spectrophotometry and chemometric analysis as tools for carotenoids analysis in cassava genotypes (Manihot esculenta Crantz)

In this study, the metabolomics characterization focusing on the carotenoid composition of ten cassava (Manihot esculenta) genotypes cultivated in southern Brazil by UV-visible scanning spectrophotometry and reverse phase-high performance liquid chromatography was performed. Cassava roots rich in -carotene are an important staple food for populations with risk of vitamin A deficiency. Cassava genotypes with high pro-vitamin A activity have been identified as a strategy to reduce the prevalence of deficiency of this vitamin. The data set was used for the construction of a descriptive model by chemometric analysis. The genotypes of yellow-fleshed roots were clustered by the higher concentrations of cis--carotene and lutein. Inversely, cream-fleshed roots genotypes were grouped precisely due to their lower concentrations of these pigments, as samples rich in lycopene (redfleshed) differed among the studied genotypes. The analytical approach (UV-Vis, HPLC, and chemometrics) used showed to be efficient for understanding the chemodiversity of cassava genotypes, allowing to classify them according to important features for human health and nutrition.

Tocopherol profile of Brazil nut oil from different geographic areas of the Amazon region

The tocopherol content of Brazil nut oil from different Amazon regions (Manicoré-AM, Rio Preto da Eva-AM, São João da Baliza-RR, Caroebe-RR, Belém-PA, and Xapurí-AC) was investigated by normal-phase high-performance liquid chromatography. For all authentic oils, two isomers: α- and γ-tocopherols were observed (37.92-74.48 µg g-1, 106.88-171.80 µg g-1, respectively), and their levels were relatively constant among the oils having these geographic origins, which would enable to distinguish Brazil nut oil from other plant oils for authentication purposes. Commercial Brazil nut oils were also evaluated, and some of these oils demonstrated a tocopherol content that was very different from that of the authentic oils. Therefore, we suggest that the tocopherol profile of Brazil nut oil can be useful chemical marker for quality control and authentication.