Characterization of phenolic compounds and antioxidant properties of Glycyrrhiza glabra L. rhizomes and roots

The present work aims to characterize and quantify the phenolic composition and to evaluate the antioxidant activity of Glycyrrhiza glabra L. (commonly known as licorice) rhizomes and roots. The antioxidant potential of its methanol/water extract could be related with flavones (mainly apigenin derivatives), flavanones (mainly liquirintin derivatives), a methylated isoflavone and a chalcone, identified in the extract. Lipid peroxidation inhibition was the most pronounced antioxidant effect (EC50=0.24±0.01 µg/mL and 22.74±2.42 µg/mL in TBARS and -carotene/linoleate assays, respectively), followed by free radicals scavenging activity (EC50=111.54±6.04 µg/mL) and, finally, reducing power (EC50=128.63±0.21 µg/mL). In this sense, licorice extract could be used as a source of antioxidants for pharmaceutical, cosmetic and/or food industries.

The influence of kaolin application on key metabolic pathways associated with grape berry quality: a molecular and biochemical analysis

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Mechanism of extracellular silver nanoparticles synthesis by Stereum hirsutum and Fusarium oxysporum

The increasing interest for greener and biological methods of synthesis has led to the development of non-toxic and comparatively more bioactive nanoparticles. Unlike physical and chemical methods of nanoparticle synthesis, microbial synthesis in general and mycosynthesis in particular is cost-effective and environment-friendly. However, different aspects, such as the rate of synthesis, monodispersity and downstream processing, need to be improved. Many fungal-based mechanisms have been proposed for the formation of silver nanoparticles (AgNPs), mainly those involving the presence of nitrate reductase, which has been detected in filtered fungus cell used for AgNPs production. There is a general acceptance that nitrate reductase is the main responsible for the reduction of Ag ions for the formation of AgNPs. However, this generally accepted mechanism for fungal AgNPs production is not totally understood. In order to elucidate the molecules participating in the mechanistic formation of metal nanoparticles, the current study is focused on the enzymes and other organic compounds involved in the biosynthesis of AgNPs. The use of each free fungal mycelium of both Stereum hirsutum and Fusarium oxysporum will be assessed. In order to identify defective mutants on the nitrate reductase structural gene niaD, fungal cultures of S.hirsutum and F.oxysporum will be selected by chlorate resistance. In addition, in order to verify if each compound identified as key-molecule influenced on the production of nanoparticles, an in vitro assay using different nitrogen sources will be developed. Lately, fungal extracellular enzymes will be measured and an in vitro assay will be done. Finally, The nanoparticle formation and its characterization will be evaluated by UV-visible spectroscopy, electron microscopy (TEM), X-ray diffraction analysis (XRD), Fourier transforms infrared spectroscopy (FTIR), and LC-MS/MS.

Advances in microbial diagnosis: using mass spectrometry for proteomics and genomics applications

Since the last two decades mass spectrometry (MS) has been applied to analyse the chemical cellular components of microorganisms, providing rapid and discriminatory proteomic profiles for their species identification and, in some cases, subtyping. The application of MS for the microbial diagnosis is currently well-established. The remarkable reproducibility and objectivity of this method is based on the measurement of constantly expressed and highly abundant proteins, mainly important conservative ribosomal proteins, which are used as markers to generate a cellular fingerprint. Mass spectrometry based on matrix-assisted laser desorption ionization-time of flight (MALDI- TOF) technique has been an important tool for the microbial diagnostic. However, some technical limitation concerning both MALDI-TOF and its used protocols for sample preparation have fostered the research of new mass spectrometry systems (e.g. LC MS/MS). LC MS/MS is able to generate online mass spectra of specific ions with further online sequencing of these ions, which include both specific proteins and DNA fragments. In this work a set of data for yeasts and filamentous fungi diagnostic obtained through an international collaboration project involving partners from Argentina, Brazil, Chile and Portugal will be presented and discussed.

Use of a polyphasic approach including MALDI-TOF MS for identification of Aspergillus section Flavi strains isolated from food commodities in Brazil

Brazil is one the largest producers and exporters of food commodities in the world. The evaluation of fungi capable of spoilage and the production mycotoxins in these commodities is an important issue that can be of help in bioeconomic development. The present work aimed to identify fungi of the genus Aspergillus section Flavi isolated from different food commodities in Brazil. Thirty-five fungal isolates belonging to the section Flavi were identified and characterised. Different classic phenotypic and genotypic methodologies were used, as well as a novel approach based on proteomic profiles produced by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Type or reference strains for each taxonomic group were included in this study. Three isolates that presented discordant identification patterns were further analysed using the internal transcribed spacer (ITS) region and calmodulin gene sequences. The data obtained from the phenotypic and spectral analyses divide the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus, and Aspergillus tamarii. Final polyphasic fungal identification was achieved by joining data from molecular analyses, classical morphology, and biochemical and proteomic profiles generated by MALDI-TOF MS.

Efeito da porosidade nas propriedades físicas, mecânicas e tribológicas de mós abrasivas

Dissertação de mestrado integrado em Engenharia de Materiais

Polyphasic approach including MALDI-TOF MS/MS analysis for identification and characterisation of Fusarium verticillioides in Brazilian corn kernels

Fusarium verticillioides is considered one of the most important global sources of fumonisin contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG)5 and (GACA)4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol) and B2 (705.83 g/mol)

Biodegradação de Ochratoxin A por Pediococcus parvulus

Algumas bactérias do ácido láctico (BAL) são capazes de destoxificar micotoxinas através de processos de adsorção às suas paredes celulares ou através de processos de biotransformação em compostos menos tóxicos. Uma das micotoxinas mais importantes encontradas em produtos agrícolas é a ocratoxina A (OTA). A OTA é conhecida principalmente pela sua nefro e carcinogenicidade, estando classificada no Grupo 2B pelo IARC. O presente trabalho descreve a destoxificação de OTA por estirpes de Pediococcus parvulus que foram isoladas de vinhos do Douro. As estirpes foram identificadas e caracterizadas utilizando uma abordagem polifásica que utilizou métodos feno e genotípicos. Para identificar e caracterizar a sua capacidade para destoxificar a OTA, as estirpes foram cultivadas em meio MRS suplementado com esta micotoxina (1 µg/mL). A concentração de OTA, a temperatura de incubação e a concentração de inóculo foram os parâmetros cujo efeito na destoxificação foi avaliado. Verificou-se que a OTA foi degradada em OT pelas estirpes de P. parvulus em todas as condições testadas e que a estirpe tipo desta espécie não apresentou essa capacidade. Ademais, a OT foi confirmada por LC-MS/MS. A conversão de OTA em OT indica que a ligação amida presente na micotoxina foi hidrolisada por uma peptidase. Verificou-se também que a taxa de biodegradação da OTA depende do tamanho do inóculo e da temperatura de incubação. Às condições ótimas (10 9 CFU/mL e 30 ºC), 50% e 90% da OTA foi degradado em 6 e 19 h, respetivamente. Por outro lado, observou-se que as células mortas de P. parvulus adsorveram apenas 1,3% da OTA, o que exclui este mecanismo na eliminação da micotoxina pelas bactérias. A biodegradação de OTA por P. parvulus UTAD 473 foi também avaliada e observada em mostos de uvas. Experiências de vinificação foram também realizadas. Uma vez que algumas estirpes de P. parvulus têm propriedades probióticas relevantes, as estirpes isoladas de vinhos do Douro podem ser de particular interesse para aplicações em alimentos e rações de forma a neutralizar os efeitos tóxicos da OTA.

Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity

The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0-10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 hours. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (> 5 log) and more than 4 logs P. aeruginosa and Salmonella Typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine.

Determinação de psicofármacos em cabelo humano por técnicas cromatográficas hifenadas: um método alternativo não-invasivo

Dissertação de mestrado em Técnicas de Caracterização e Análise Química