Technical optimization of the synthesis of flavours

The goal of this thesis is the investigation and optimization of the synthesis of potential fragrances. This work is projected as collaboration between the University of Applied Sciences in Merseburg and the company Miltitz Aromatics GmbH in Bitterfeld‐Wolfen (Germany). Flavoured compounds can be synthesized in different ways and by various methods. In this work, methods like the phase transfer catalysis and the Cope‐rearrangement were investigated and applied, for getting a high yield and quantity of the desired substances and without any by‐products or side reactions. This involved the study of syntheses with different process parameters such as temperature, solvent, pressure and reaction time. The main focus was on Cope‐rearrangement, which is a common method in the synthesis of new potential fragrance compounds. The substances synthesized in this work have a hepta‐1,5‐diene‐structure and that is why they can easily undergo this [3,3]‐sigma tropic rearrangement. The lead compound of all research was 2,5‐dimethyl‐2‐vinyl‐4‐hexenenitrile (Neronil). Neronil is synthesized by an alkylation of 2‐methyl‐3‐butenenitrile with prenylchloride under basic conditions in a phase‐transfer system. In this work the yield of isolated Neronil is improved from about 35% to 46% by according to the execution conditions of the reaction. Additionally the amount of side product was decreased. This synthesized hexenenitrile involved not only the aforementioned 1,5‐diene‐structure, but also a cyano group, that makes this structure a suitable base for the synthesis of new potential fragrance compounds. It was observed that Neronil can be transferred into 2,5‐dimethyl‐2‐vinyl‐4‐hexenoic acid by a hydrolysis under basic conditions. After five hours the acid can be obtained with a yield of 96%. The following esterification is realized with isobutanol to produce 2,5‐dimethyl‐2‐vinyl‐4‐hexenoic acid isobutyl ester with quantitative conversion. It was observed that the Neronil and the corresponding ester can be converted into the corresponding Cope‐product, with a conversion of 30 % and 80%. Implementing the Cope‐rearrangement, the acid was heated and an unexpected decarboxylated product is formed. To achieve the best verification of reaction development and structure, scrupulous analyses were done using GC‐MS, 1H‐NMR and 13C‐ NMR.

Respiratory characteristics of Hoplostenum littorale (Siluriformes, Callichthyidae)

Hematological parameters, intraerythrocytic phosphates, hemoglobin, and whole blood Bohr effect of the South American armored catfish Hoplostenum littorale were studied during different seasons of the year. In addition, the degree of dependence on air breathing was determined for this species. The hematological parameters presented seasonal variations, which were not correlated to oxygen, temperature, and water level oscillations. Five anodic hemoglobin fractions were detected in starch gel electrophoresis. In addition to ATP, GTP and Fe-GTP being detected, 2,3-DPG was also detected in red blood cells of H. littorale. The latter is an intraerythrocytic phosphate characteristic to red blood cells of mammalians. The increased production of 2,3-DPG could be associated with decreasing Hb-O2 affinity and both features could be related to environmental temperature increase. Whole blood Bohr effect was influenced by water temperature. This study confirms H. littorale to be continuous and not obligate air breather, under all dissolved oxygen level conditions.

Síntese de moléculas contendo a unidade de piperazina como potenciais agentes antipsicóticos

Tese de Doutoramente em Ciências (área de especialização em Química).

In vitro and in vivo studies of temozolomide loading in zeolite structures as drug delivery systems for glioblastoma

Zeolites Y (faujasite) and MOR (mordonite) were used as hosts for temozolomide (TMZ), a current good-standard chemotherapeutic agent used in the treatment of glioblastoma brain tumors. TMZ was loaded into zeolites by liquid-phase adsorption at controlled pH. FTIR, 1H NMR, MS, SEM, UV/vis and chemical analysis demonstrated the successful loading of TMZ into zeolite hosts. The hydrolysis of TMZ in MTIC (TMZ metabolite) after the preparation of drug delivery systems (DDS) was observed in simulated body fluid. The effect of zeolites and DDS were evaluated on the viability of glioblastoma cell lines. Unloaded Y zeolite presented toxicity to cancer cells in contrast to MOR. In accordance, the best results in potentiation of the TMZ effect was obtained with MOR. We found that mordonite loaded with 0.026 mmol of TMZ was able to decrease the half maximal inhibitory concentrations (IC50) at least 3-fold in comparison to free temozolomide both in vitro and in vivo.

Synthesis of new peptide derivatives that self-assemble into nanostructured hydrogels for biomedical applications

Tese de Doutoramento em Ciências (área de especialização em Química)

Avaliação hemodinâmica e evolução clínica de crianças portadoras de cardiomiopatia dilatada grave candidatas a transplante cardíaco

OBJETIVO: Estudar o perfil dos parâmetros hemodinâmicos e a evolução clínica de crianças candidatas a transplante cardíaco, portadoras de cardiomiopatia grave. MÉTODOS: Foram 24 crianças, com idade entre 4 meses e 10 anos e 8 meses (média de 3,7±2,5 anos), no período de fevereiro/92 a maio/96, submetidas a estudo hemodinâmico e medidos os seguintes parâmetros: débito cardíaco, pressão média de artéria pulmonar (PMAP) e pressão capilar pulmonar. Foram calculados o índice de resistência vascular pulmonar (IRVP) e gradiente de pressão transpulmonar (GPT). RESULTADOS: Do ponto de vista evolutivo, 10 (41,6%) crianças foram transplantadas (grupo A), 5 (20,8%) aguardam o transplante (grupo B) e 9 (37,6%) faleceram (grupo C). Observou-se que a média das idades dos pacientes do grupo B foi significativamente menor que do grupo C. Dos dados hemodinâmicos, a PMAP, GTP e IRVP apresentaram médias significativamente menores no grupo A em relação ao grupo C. CONCLUSÃO: O perfil hemodinâmico de crianças candidatas ao transplante cardíaco mostrou-se compatível ao quadro clínico de insuficiência cardíaca grave. A idade foi o único fator que diferenciou o grupo B e C (p= 0,036). O IRVP, PMAP e o GTP foram fatores que diferenciaram de modo significativo o grupo A e o grupo C (p=0,010; p=0,044 e p=0,023, respectivamente). Quanto maior a idade no momento da indicação do transplante na criança, pior foi seu prognóstico.

New bio-strategies for ochratoxin A detoxification using lactic acid bacteria

The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Influence of chitosan coating on protein-based nanohydrogels properties and in vitro gastric digestibility

Chitosan coating was applied in Lactoferrin (Lf)-Glycomacropeptide (GMP) nanohydrogels by layer-by-layer coating process. A volume ratio of 0.1 of Lf-GMP nanohydrogels (0.2 mg.mL-1, at pH 5.0) to chitosan (1 mg.mL-1, at pH 3) demonstrated to be the optimal condition to obtain stable nanohydrogels with size of 230 ± 12 nm, a PdI of 0.22 ± 0.02 and a -potential of 30.0 ± 0.15 mV. Transmission electron microscopy (TEM) images showed that the application of chitosan coating in Lf-GMP did not affect the spherical shape of nanohydrogels and confirmed the low aggregation of nanohydrogels in solution. The analysis of chemical interactions between chitosan and Lf-GMP nanohydrogels were performed by Fourier transform infrared spectroscopy (FTIR) and by circular dichroism (CD) that revealed that a specific chemical interaction occurring between functional groups of protein-based nanohydrogels and active groups of the chitosan was established. The effect of chitosan coating on release mechanisms of Lf-GMP nanohydrogels at acid conditions (pH 2, 37 ºC) was evaluated by the encapsulation of a model compound (caffeine) in these systems. Linear Superposition Model was used to fit the experimental data and revealed that Fick and relaxation mechanisms are involved in caffeine release. It was also observed that the Fick contribution increase with the application of chitosan coating. In vitro gastric digestion was performed with Lf-GMP nanohydrogels and Lf-GMP nanohydrogels with chitosan coating and it was observed that the presence of chitosan improve the stability of Lf and GMP (proteins were hydrolysed at a slower rate and were present in solution by longer time). Native electrophoreses revealed that the nanohydrogels without coating remained intact in solution until 15 min and with chitosan coating remained intact until 60 min, during gastric digestion.

Conversion of Cn-unsaturated into Cn-2-saturated LCFA can occur uncoupled from methanogenesis in anaerobic bioreactors

Fat, oils, and grease present in complex wastewater can be readily converted to methane, but the energy potential of these compounds is not always recyclable, due to incomplete degradation of long chain fatty acids (LCFA) released during lipids hydrolysis. Oleate (C18:1) is generally the dominant LCFA in lipid-containing wastewater, and its conversion in anaerobic bioreactors results in palmitate (C16:0) accumulation. The reason why oleate is continuously converted to palmitate without further degradation via β-oxidation is still unknown. In this work, the influence of methanogenic activity in the initial conversion steps of unsaturated LCFA was studied in 10 bioreactors continuously operated with saturated or unsaturated C16- and C18-LCFA, in the presence or absence of the methanogenic inhibitor bromoethanesulfonate (BrES). Saturated Cn-2-LCFA accumulated both in the presence and absence of BrES during the degradation of unsaturated Cn-LCFA, and represented more than 50\% of total LCFA. In the presence of BrES further conversion of saturated intermediates did not proceed, not even when prolonged batch incubation was applied. As the initial steps of unsaturated LCFA degradation proceed uncoupled from methanogenesis, accumulation of saturated LCFA can be expected. Analysis of the active microbial communities suggests a role for facultative anaerobic bacteria in the initial steps of unsaturated LCFA biodegradation. Understanding this role is now imperative to optimize methane production from LCFA.

Persistent organic chemicals of emerging environmental concern

The use of chemicals and chemical derivatives in agriculture and industry has contributed to their accumulation and persistence in the environment. Persistent organic pollutants (POPs) are among the environmental pollutants of most concern since, when improperly handled and disposed, they can persist in the environment, bioaccumulate through the food web, and may create serious public health and environmental problems. Development of an effective degradation process has become an area of intense research. The physical/chemical methods employed, such as volatilization, evaporation, photooxidation, adsorption, or hydrolysis, are not always effective, are very expensive, and, sometimes, lead to generation/disposal of other contaminants. Biodegradation is one of the major mechanisms by which organic contaminants are transformed, immobilized, or mineralized in the environment. A clear understanding of the major processes that affect the interactions between organic contaminants, microorganisms, and environmental matrix is, thus, important for determining persistence of the compounds, for predicting in situ transformation rates, and for developing site remediation. Information on their risks and impact and occurrence in the different environmental matrices is also important, in order to attenuate their impact and apply the appropriate remediation process. This chapter provides information on the fate of pesticides and polycyclic aromatic hydrocarbons (PAHs), their impact, bioavailability, and biodegradation. © Springer Science+Business Media Dordrecht 2014.

Chemical functionalization in bionanoparticles

Dissertação de mestrado em Biofísica e Bionanossistemas

Engineering a commercial cold-adapted xylanase for improved low pH stability

Dissertação de mestrado em Genética Molecular

Olive pomace pretreatments to enhance its valorisation by solid-state fermentation

Dissertação de mestrado em Bioengenharia

Biotechnological production and application of fructooligosaccharides

Currently, prebiotics are all carbohydrates of relatively short chain length. An important group is the fructooligosaccharides, which are a special kind of prebiotics associated to their selective stimulation of the activity of certain groups of colonic bacteria that have a positive and beneficial effect on intestinal microbiota, reducing incidence of gastrointestinal infections, respiratory and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of fructooligosaccharides, such as solid-state fermentation utilizing various agroindustrial by-products. By optimizing the culture parameters, fructooligosaccharides yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of fructooligosaccharides. This paper is an overview on the results of recent studies on fructooligosacharides biosynthesis, physicochemical properties, sources, biotechnological production and applications.

Entwicklung einer Impulsmethode zur Ermittlung thermodynamischer und reaktionskinetischer Parameter in der heterogenen Katalyse

Heterogeneous catalysis, homogeneous catalysis, adsorption equilibrium, reaction kinetics, impulse method, hydrolysis of methyl formate, production of formic acid