Investigations on relational control of time perception

Tese de Doutoramento em Psicologia Básica

Plataforma de apoio à decisão nos cuidados de ginecologia e obstetrícia

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Informática Médica)

Evaluation of zearalenone in vitro removal by Saccharomyces cerevisiae strains isolated from bovine forage

Zearalenone (ZEN) is a mycotoxin that has relatively low acute toxicity. However, it is a potent oestrogen, interfering with the reproductive tract of animals. Among other effects, ZEN decreases animals fertility, and induces fibrosis in the uterus, breast cancer and endometrial carcinoma (Zinedine et al., 2007). Anti-mycotoxin additives (AMA) are defined as a group of products that, when added to animal feed, are capable of adsorbing, inactivating, or neutralizing mycotoxins in the gastrointestinal tract of animals. One example of these products are adsorbents based on yeast cell walls, a safe and beneficial animal feed additive (Abreu et al., 2008). When based on active cells, yeast based products also act as a probiotic, contributing to improve the general animal health because it stimulates their immune system and promotes the integrity of intestinal mucosa (Albino et al., 2006). Strains of Saccharomyces cerevisiae isolated from silage were tested for their ZEN removal capability. Their effect on - and b-zearalenol (-ZOL and b-ZOL) was also tested. Strains were grown on YPD separately supplemented with ZEN, -ZOL and b-ZOL, and their elimination from culture media was quantified over time by HPLC-FL.

O novo luxo: um novo olhar sobre o fenómeno

Dissertação de mestrado em Design e Marketing da Moda

Current situation on the occurrence of mycotoxins and toxigenic fungi in Portugal

Mycotoxins are secondary metabolites produced by filamentous fungi that are toxic for humans and animals in small amounts and that are found worldwide in a large number of agricultural commodities. They are usually ingested involuntarily, when contaminated plant products are consumed, and represent a great risk for public health. Therefore, governments throughout the world have imposed strict legal limits for their levels in food and feed products in order to reduce potential health risks for consumers. Despite of its ubiquity, the mycotoxin problem is mainly dependent on regional factors, such as the mycotoxigenic characteristics of the local mycoflora, the local climate conditions, and the local agricultural practices. For this reason, a constant vigilance from local governmental food safety agencies and from the local researcher community is needed. This communication will review the current situation on the occurrence of mycotoxigenic fungi in some Portuguese cultures, such as wine grapes, corn and dried fruits. Particular attention will be given to the incidence of mycotoxigenic Aspergillus strains in those cultures and to the levels of ochratoxin A, aflatoxins, cyclopiazonic acid and fumonisin B2 produced. Data will be discussed taking into account the geographical origin of the isolates and the particular climate conditions of each sampling region. An updated review on the levels of the main mycotoxins found in local products and in imported commodities will also be presented.

Simultaneous detection of cyclopiazonic acid and aflatoxin B1 by HPLC in methanol/water mobile phase

A simple procedure for the simultaneous detection of cyclopiazonic acid (CPA) and aflatoxin B1 from fungal extracts is presented, using a methanol and water mobile phase and fluorescence detection. This methodology has been tested with standard solutions of both mycotoxins CPA and Aflatoxin B1 and with methanolic extracts of Aspergillus section Flavi strains, previously characterized for their mycotoxin production profile. Previously available methodology required the use of two different chromatographic runs for these mycotoxins, with distinct columns and detectors (fluorescence detection with a post-column photochemical derivatization (PHRED) for aflatoxin B1 and UV detection for CPA). The proposed method detects both mycotoxins in a single run. Data from these assays will be presented and discussed.

The isolation of Aspergillus spp from harvested maize in three Portuguese regions

In Portugal, maize is the cereal that involves more agriculture explorations. Aspergillus spp., among other species, are usually associated with this cereal, during drying and storage, making this commodity susceptible to mycotoxins (such as aflatoxins, ochratoxins, and cyclopiazonic acid). The aim of this study was to evaluate the mycotoxigenic potential of isolated Aspergillus strains from these maize samples and correlate it with the sampling place, the weather conditions, and local practices during drying and storage. The samples were collected between November 2008 and April 2009 in maize association of producers facilities in Coimbra, Santarém and Portalegre. The isolated strains were divided in three distinct groups, Aspergillus section Flavi, Aspergillus section Nigri and others Aspergillus. The preliminary results show that there are differences between the incidence of these groups in the three sampling places, especially in Coimbra, probably due to a lower mean temperatures and higher humidity levels. These data will be presented and discussed.

Enzymes which detoxify ochratoxin A

Mycotoxins are fungal secondary metabolites found in some agricultural commodities which are toxic for humans and animals in small amounts. Mycotoxins are a global problem which can be partially controlled through prevention strategies that can be applied along the food and feed chain production. However, when mycotoxin formation can not be avoided and they come to be present in commodities some remediation strategies can also be used to reduce its levels on products, its bioavailability or its toxic effects. Among these remediation strategies, the biological methods are recently holding a relevant position, being widely studied in the last years. As a result, a great number of microorganisms that can degrade or detoxify several mycotoxins and the application of some of them were reported. Moreover, several enzymes which mediate these biological processes were identified, being by themselves studied in order to develop new biotechnological approaches to control the mycotoxin problem on commodities. The main enzymes known to detoxify ochratoxin A, their action and their present application in order to counteract the referred problem are reviewed and critically assessed.

Biosynthesis, detection and toxicology of ochratoxins - optimisation of production

Ochratoxin A (OTA) is a very well known mycotoxin found in several food commodities for which maximum limits are being discussed in EC in other to produce appropriate regulations. OTA is one of several ochratoxins produced by Aspergillus and Penicillium species. All the compounds in this group have a molecular structure very similar to OTA and some were already isolated from natural substrates. Several of these compounds such as ochratoxin , methyl and ethyl ester of ochratoxin A, 4-R and S-hydroxyochratoxin A, 10-hydroxyochratoxin A and ochratoxin A open lactone are commercially unavailable. However, they can be easily synthesized through OTA modification. With the main objective of its application on further research works, OTA production, isolation and purification has been optimised from an A. alliaceus strain grown on wheat medium. Synthesis and purification of some OTA derivatives has been achieved and an HPLC method for their detection was optimised. Data about their production by several species of Aspergillus will be presented. The toxicological properties of ochratoxins are still not very clear and a future EC safety limit for OTA will depend on e.g., a better clarification of its carcinogenity. Could OTA derivatives play a role here?

Bio-detoxification of mycotoxins by lactic acid bacteria from different food matrices

Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP- PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 g/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 m pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

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.

Desconformidade como alteração da vontade na venda de bens de consumo

Dissertação de mestrado em Direito dos Contratos e da Empresa

Estudo de estirpes probióticas como inoculantes de silagens para o controlo de micotoxinas

Dissertação de mestrado em Bioengenharia