Alternative technologies for producing sterile low acid food products

Four emerging high-energy non-thermal technologies may replace or augment heating for producing sterile low-acid food products. High pressure, high-voltage pulsed electric field, high-energy ultrasound and high-intensity pulsed light are all capable of reducing bacterial spore counts under certain conditions. However, only non-continuous high pressure treatments, at temperatures higher than ambient, are currently capable of completely inactivating spores and producing sterile food products. The first three technologies also reduce the resistance of spores to inactivation by heat.

Isolamento e caracterização de uma proteína tóxica produzida por um isolado açoriano de Bacillus weihenstephanensis

Dissertação de Mestrado, Biotecnologia em Controlo Biológico, 27 de Junho de 2013, Universidade dos Açores.

Occupational exposure to Aflatoxin B1 in swine production and possible contamination sources

Although the adverse health consequences of ingestion of food contaminated with aflatoxin B1 (AFB1) are known, relatively few studies are available on the adverse effects of exposure in occupational settings. Taking this into consideration, our study was developed aiming to elucidate the possible effects of occupational exposure to AFB1 in Portuguese swine production facilities using a specific biomarker to assess exposure to AFB1. In total, 28 workers participated in this study, providing blood samples, and a control group (n  = 30) was composed of subjects without any type of agricultural activity. Fungal contamination was also studied by conventional methods through air, surfaces, and new and used floor coverage. Twenty-one workers (75%) showed detectable levels of AFB1 with values ranging from <1 ng/ml to 8.94 ng/ml and with a mean value of 1.91 ± 1.68 ng/ml. In the control group, the AFB1 values were all below 1 ng/ml. Twelve different Aspergillus species were identified. Aspergillus versicolor presented the highest airborne spore counts (3210 CFU/m3) and was also detected in higher values in surfaces (>300 CFU/cm2). Data indicate that exposure to AFB1 occurs in swine barns, and this site serves as a contamination source in an occupational setting.

Species composition of arbuscular mycorrhizal fungi differ in semi-natural and intensively managed pastures in an isolated oceanic island (Terceira, Azores)

Copyright © Springer Science+Business Media Dordrecht 2014.

Estudo de atividades antimicrobianas em bactérias do género Bacillus isoladas nos Açores : otimização das condições de produção, identificação e caracterização de bacteriocinas

Dissertação de Mestrado, Ciências Biomédicas, 18 de Novembro de 2015, Universidade dos Açores.

Environmental impact caused by fungal and particle contamination of Portuguese swine

Social concerns for environmental impact on air, water and soil pollution have grown along with the accelerated growth of pig production. This study intends to characterize air contamination caused by fungi and particles in swine production, and, additionally, to conclude about their eventual environmental impact. Fiftysix air samples of 50 litters were collected through impaction method. Air sampling and particle matter concentration were performed in indoor and also outdoor premises. Simultaneously, temperature and relative humidity were monitored according to the International Standard ISO 7726 – 1998. Aspergillus versicolor presents the highest indoor spore counts (>2000 CFU/m3) and the highest overall prevalence (40.5%), followed by Scopulariopsis brevicaulis (17.0%) and Penicillium sp. (14.1%). All the swine farms showed indoor fungal species different from the ones identified outdoors and the most frequent genera were also different from the ones indoors. The distribution of particle size showed the same tendency in all swine farms (higher concentration values in PM5 and PM10 sizes). Through the ratio between the indoor and outdoor values, it was possible to conclude that CFU/m3 and particles presented an eventual impact in outdoor measurements.

Ocorrência e diversidade de leveduras no filoplano de plantas seleccionadas do Parque Natural da Serra da Arrábida

Dissertação apresentada para obtenção do Grau de Doutor em Biologia (especialidade Microbiologia), pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Cork stoppers industry: defining appropriate mould colonization

Aims: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. Methods and Results: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. Conclusions: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. Significance and Impact of the Study: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.

Cork stoppers industry: defining appropriate mould colonization

Aims: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. Methods and Results: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. Conclusions: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. Significance and Impact of the Study: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.

Novos circuitos regulatórios de controlo espacio-temporal do desenvolvimento em bacillus subtilis

RESUMO: A esporulação em Bacillus subtilis é controlada por uma cascata de factores sigma da polimerase do RNA. F e E controlam os estágios precoces do desenvolvimento no pré-esporo e na célula mãe, respectivamente. Numa fase intermédia da diferenciação, quando a célula mãe acaba por envolver o pré-esporo, F é substituído por G e E é substituído por K. Vários mecanismos asseguram que a actividade dos diferentes factores sigma seja confinada a uma janela temporal precisa na célula adequada. Neste estudo, investigámos a função de um factor anti-G, designado por CsfB. Mostramos que para além da sua função de inibição da actividade do factor G em células pré-divisionais, CsfB é também necessário na célula mãe num estágio tardio do desenvolvimento. Mostramos que a expressão de csfB é activada na célula mãe a partir de um promotor dependente de K. Contudo, demonstramos que CsfB interage directamente com E e não com K, e que CsfB é suficiente para inibir a actividade transcricional dependente de E em células vegetativas de B. subtilis. Propomos que CsfB contribui para reduzir o período dependente de E, na linha de expressão genética da célula mãe, desse modo reduzindo a sobreposição entre os regulões E e K e aumentado a fidelidade do processo de desenvolvimento. Uma segunda proteína, YabK, partilha semelhança estrutural com CsfB. YabK é produzida no pré-esporo sob o comando de F, e é necessária para a esporulação. YabK contribui para a transição F/G no programa genético do pré-esporo, porque uma mutação que torna F sensível a CsfB ultrapassa parcialmente a função de YabK na esporulação. No entanto, YabK e CsfB funcionam por mecanismos diferentes, uma vez que YabK não liga directamente a F.---------ABSTRACT: Gene expression during spore development in Bacillus subtilis is governed by a cascade of RNA polymerase sigma factors. F and E control the early stages of development in the forespore and in the mother cell, respectively. At an intermediate stage in the differentiation process, when the larger mother cell finishes engulfment of the smaller forespore, F is replaced by G and E is replaced by K. Several mechanisms ensure the proper timing of activation of the cell type-specific sigma factors. Here, we have investigated the funtion of an anti-sigma G factor, called CsfB. We show here that in addition to its role in inhibiting G in pre-divisional cells, CsfB is also required in the mother cell at a late stage in development. We show that the expression of csfB is activated in the mother cell from a K-specific promoter. However, we demonstrate that CsfB binds directly to E but not to K in a yeast two-hybrid assay, and that CsfB is sufficient to inhibit E-dependent transcriptional activity in vegetative cells of B. subtilis. We posit that CsfB contributes to shutting off the early, E-controlled period in the mother cell line of gene expression, thus reducing the overlap between deployment of the E and K regulons and increasing the fidelity of the developmental process. A second protein, YabK, shares structural similarity with CsfB. YabK is produced in the forespore under F control, and is required for efficient sporulation. YabK contributes to the transition from the F- to the G-dependent period of gene expression, because a mutation that renders F sensitive to CsfB partially bypasses the need for YabK. Yet, YabK and CsfB must function in the control of sigma factor activity by different mechanisms because YabK does not bind directly to F.

Microscopy- or DNA-based analyses: Which methodology gives a truer picture of stream-dwelling decomposer fungal diversity?

We assessed aquatic hyphomycete diversity in autumn and spring on oak leaves decomposing in five streams along a gradient of eutrophication in the Northwest of Portugal. Diversity was assessed through microscopy-based (identification by spore morphology) and DNA-based techniques (Denaturing Gradient Gel Electrophoresis and 454 pyrosequencing). Pyrosequencing revealed five times greater diversity than DGGE. About 21% of all aquatic hyphomycete species were exclusively detected by pyrosequencing and 26% exclusively by spore identification. In some streams, more than half of the recorded species would have remained undetected if we had relied only on spore identification. Nevertheless, in spring aquatic hyphomycete diversity was higher based on spore identification, probably because many species occurring in this season are not yet connected to ITS barcodes in genetic databases. Pyrosequencing was a powerful tool for revealing aquatic hyphomycete diversity on decomposing plant litter in streams and we strongly encourage researchers to continue the effort in barcoding fungal species.

Evaluation of bioactivities of a propolis sample (Gerês) of Portuguese origin

Dissertação de mestrado em Genética Molecular

Bacillus sphaericus mosquito pathogens in the aquatic environment

The fate of Bacillus sphaericus spores in the aquatic environment was investigated by suspending spores in dialysis bags in fresh and seawater. Spore viability was lost more rapidly in seawater. Neither B. sphaericus nor B. thuringiensis israelensis (B.t.i.) spores mixed with pond sediment appeared to attach to the sediment. However, rapid decrease in B.t.i. toxicity suggested attachment of parasporal bodies to sediment. B. sphaericus toxin settled more slowly and less completely. B. sphaericus spores fed to larvae of four aquatic invertebrates were mostly eliminated from the animal gut in less than one week. An exception was the cranefly (Tipula abdominalis) where spores persisted in the posterior gut for up to five weeks.

Effects of genetic alterations in arbuscular mycorrhizal fungi on plant growth and on their response to a change of host

Abstract :The majority of land plants form the symbiosis with arbuscular mycorrhizal fungi (AMF). The AM symbiosis has existed for hundreds of millions of years but little or no specificity seems to have co- evolved between the partners and only about 200 morphospecies of AMF are known. The fungi supply the plants most notably with phosphate in exchange for carbohydrates. The fungi improve plant growth, protect them against pathogens and herbivores and the symbiosis plays a key role in ecosystem productivity and plant diversity. The fungi are coenocytic, grow clonally and no sexual stage in their life cycle is known. For these reasons, they are presumed ancient asexuals. Evidence suggests that AMF contain populations of genetically different nucleotypes coexisting in a common cytoplasm. Consequently, the nucleotype content of new clonal offspring could potentially be altered by segregation of nuclei at spore formation and by genetic exchange between different AMF. Given the importance of AMF, it is surprising that remarkably little is known about the genetics and genomics of the fungi.The main goal of this thesis was to investigate the combined effects of plant species differences and of genetic exchange and segregation in AMF on the symbiosis. This work showed that single spore progeny can receive a different assortment of nucleotypes compared to their parent and compared to other single spore progeny. This is the first direct evidence that segregation occurs in AMF. We then showed that both genetic exchange and segregation can lead to new progeny that differentially alter plant growth compared to their parents. We also found that genetic exchange and segregation can lead to different development of the fungus during the establishment of the symbiosis. Finally, we found that a shift of host species can differentially alter the phenotypes and genotypes of AMF progeny obtained by genetic exchange and segregation compared to their parents.Overall, this study confirms the multigenomic state of the AMF Glomus intraradices because our findings are possible only if the fungus contains genetically different nuclei. We demonstrated the importance of the processes of genetic exchange and segregation to produce, in a very short time span, new progeny with novel symbiotic effects. Moreover, our results suggest that different host species could affect the fate of different nucleotypes following genetic exchange and segregation in AMF, and can potentially contribute to the maintenance of genetic diversity within AMF individuals. This work brings new insights into understanding how plants and fungi have coevolved and how the genetic diversity in AMF can be maintained. We recommend that the intra-ir1dividual AMF diversity and these processes should be considered in future research on this symbiosis.Résumé :La majorité des plantes terrestres forment des symbioses avec les champignons endomycorhiziens arbusculaires (CEA). Cette symbiose existe depuis plusieurs centaines de millions d'années mais peu ou pas de spécificité semble avoir co-évoluée entre les partenaires et seulement 200 morpho-espèces de CEA sont connues. Le champignon fournit surtout aux plantes du phosphate en échange de carbohydrates. Le champignon augmente la croissance des plantes, les protège contre des pathogènes et herbivores et la symbiose joue un rôle clé dans la productivité des écosystèmes et de la diversité des plantes. Les CEA sont coenocytiques, se reproduisent clonalement et aucune étape sexuée n'est connue dans leur cycle de vie. Pour ces raisons, ils sont présumés comme anciens asexués. Des preuves suggèrent que les CEA ont des populations de nucleotypes différents coexistant dans un cytoplasme commun. Par conséquent, le contenu en nucleotype des nouveaux descendants clonaux pourrait être altéré par la ségrégation des noyaux lors de la fonnation des spores et par l'échange génétique entre différents CEA. Etant donné l'importance des CEA, il est surprenant que si peu soit connu sur la génétique et la génomique du champignon.Le principal but de cette thèse a été d'étudier les effets combinés de différentes espèces de plantes et des mécanismes d'échange génétique et de ségrégation chez les CEA sur la symbiose. Ce travail a montré que chaque nouvelle spore produite pouvait recevoir un assortiment différent de noyaux comparé au parent ou comparé à d'autres nouvelles spores. Ceci est la première preuve directe que la ségrégation peut se produire chez les CEA. Nous avons ensuite montré qu'à la fois l'échange génétique et la ségrégation pouvaient mener à de nouveaux descendants qui altèrent différemment la croissance des plantes, comparé à leurs parents. Nous avons également trouvé que l'échange génétique et la ségrégation pouvaient entraîner des développements différents du champignon pendant l'établissement de la symbiose. Pour finir, nous avons trouvé qu'un changement d'espèce de l'hôte pouvait altérer différemment les phénotypes et génotypes des descendants issus d'échange génétique et de ségrégation, comparé à leurs parents.Globalement, cette étude confirme l'état multigénomique du CEA Glumus intraradices car nous résultats sont possibles seulement si le champignon possède des noyaux génétiquement différents. Nous avons démontrés l'importance des mécanismes d'échange génétique et de ségrégation pour produire en très peu de temps de nouveaux descendants ayant des effets symbiotiques nouveaux. De plus, nos résultats suggèrent que différentes espèces de plantes peuvent agir sur le devenir des nucleotypes après l'échange génétique et la ségrégation chez les CEA, et pourraient contribuer à la maintenance de la diversité génétique au sein d'un même CEA. Ce travail apporte des éléments nouveaux pour comprendre comment les plantes et les champignons ont coévolué et comment la diversité génétique chez les CEA peut être maintenue. Nous recommandons de considérer la diversité génétique intra-individuelle des CEA et ces mécanismes lors de futures recherches sur cette symbiose.

Segregation in a mycorrhizal fungus alters rice growth and symbiosis-specific gene transcription

Arbuscular mycorrhizal fungi (AMF) form symbioses with the majority of plants, improving plant nutrition and diversity. Evidence exists suggesting that AMF contain populations of genetically different nucleotypes coexisting in a common cytoplasm. This potentially has two important consequences for their genetics. First, by random distribution of nuclei at spore formation, new offspring of an AMF could receive different complements of nucleotypes compared to the parent or siblings-we consider this as segregation. Second, genetic exchange between AMF would allow the mixing of nuclei, altering nucleotype diversity in new spores. Because segregation was assumed not to occur and genetic exchange has only recently been demonstrated, no attempts have been made to test whether this affects the symbiosis with plants. Here, we show that segregation occurs in the AMF Glomus intraradices and can enhance the growth of rice up to five times, even though neither parental nor crossed AMF lines induced a positive growth response. This process also resulted in an alteration of symbiosis-specific gene transcription in rice. Our results demonstrate that manipulation of AMF genetics has important consequences for the symbiotic effects on plants and could be used to enhance the growth of globally important crops.