Abstract This thesis aimed at developing methods for tracking the environmental origins of microbial contaminants of the food chain. We worked on three targets: i) environmental mycobacteria ii) toxinogenic Bacillus species iii) post-harvest fungi in strawberry jam. Our aim was to develop methods for early detection of the above contaminants, which have the potential to endanger consumer health. We developed a novel method based on 16S rRNA hybridization for tracking the reservoirs of potentially pathogenic environmental mycobacteria in piggeries and soil. From 1010 to 1012 16S rRNA molecules of environmental mycobacteria were found per gram of peat, wood shavings and straw in piggeries with a high prevalence of infections. These beddings may thus be a source of mycobacteria for pigs. We found 1010–1011 of mycobacterial 16S rRNA molecules per gram of Finnish forest soil, indicating that the soil contained 107–109 mycobacteria per gram. These numbers exceed the previous cultivation-based estimates of mycobacterial content in Finnish soils. To elucidate the role of mastitis in the input of toxinogenic Bacillus into the dairy production chain, milks were sampled from mastitic cows. Twenty-three Bacillus isolates were screened for toxins using the sperm cell motility inhibition assay. Four of the six toxinogenic isolates found were identified as Bacillus pumilus and two as Bacillus licheniformis. The isolates produced toxic substances that were heat-stable (100 °C) and soluble in methanol, thus being of non-protein nature. The extracts prepared from the toxin-producing isolates disrupted the plasma membrane of exposed sperm cells at concentrations 1–15 μg ml-1 (B. pumilus) 20–30 μg ml-1 (B. licheniformis). The toxic action of the mastitis-associated B. licheniformis strains was similar to that of the lipopeptide lichenysin A. The genes for lichenysin synthetase were found in these strains by PCR. This study revealed that heat-stable toxin-producing strains of B. pumilus and B. licheniformis occur in milk of mastitic milking cows. They may enter the dairy production chain when milk of clinically healthy cows recovered from mastitis is sent to dairies. Many foodborne contaminant fungi are known to produce volatile organic compounds. We investigated the suitability of such metabolites as early indicators of fungal contamination of strawberry jam. We found that volatile organic compounds commonly produced by the contaminant fungi in strawberry jam were 2-pentanone, styrene, 3-methyl-1-butanol, 1,3-pentadiene and ethanol. The results indicate that these compounds could be used to detect fungal contamination of jam.

The basin of the river Piauitinga is located in south-central portion of the state of Sergipe, being responsible for water supply in some municipalities Sergipe, which did not avoid the areas around the springs, framed as environmental protection (as forestry code ) are almost in its entirety cleared. Thus, this study seeks to characterize the soils in their local environment to serve as a benchmark for future comparisons between areas of nascent and degraded in the process of recovery and assess physical attributes, chemical and microbiological soil around the springs of this river for more understanding the dynamics of semi-physical. The sources were classified as its recharge and about their conservation status. We selected the areas for revegetation of each source and these were grouped according to their position in the landscape. The environmental and pedological characteristics of the study sites was performed by observing the local landscape, the opening of micro-trenches and tradagens soil. The soils were described and classified morphologically. Slides were prepared for x-ray diffraction of clay. Could a group of springs according to their positions of landscape and local soil characteristics below (BA), with slope horizon A (EA), slope without horizon A (ES), with slope hydromorphic (EH) and foothills of gentle slope (SE). The attributes were analyzed in samples at depths of 0 to 0.1 m and 0.1 to 0.3 m. Were determined: texture, organic particulate matter, stability of macro aggregates, mean diameter of aggregates, the value of color, pH, organic C, Ca, Ca + Mg, Mg, Al, Na, K, P, H + Al, N, sum of bases, cation exchange capacity, base saturation, Al saturation of, microbial respiration, C and N microbial biomass and enzyme activity (FDA). The similarity and / or dissimilarity among sites (spatial) was investigated by multivariate analysis "non-metric multidimensional scaling (NMS), from the distance of Sorensen. Were determined by Pearson's correlation coefficients between the axes of the NMS ordination by and between the different attributes. The sites around the headwaters of the River High Piauitinga positions are distributed in erosicionais (breaking-of-focus), and download a single case of foot gentle slope of the top of coastal tablelands. Its main features are the strong hydromorphic (Gleissolos Cambisols and Haplic Gleissolos), and / or the low level of development (Cambisols and Haplic Plinthosols, both with much skeletal material - quartz and petroplinthite - many with eroded phase). The physical, chemical and biological studied by means of multivariate analysis allowed the construction of an environmental gradient, with decreasing quality of soil in the sequence: BA, EH, EA and ES. It is believed that it is possible redefinitions of strategies for the revegetation of the upper course of the spring basin, extrapolate soil quality in areas of springs of water bodies with similar characteristics and reflect on strategies for restoring degraded areas. / A bacia hidrográfica do rio Piauitinga está localizada na porção centro-sul do estado de Sergipe, sendo responsável pelo abastecimento de água de alguns municípios sergipanos, o que não evitou que as áreas do entorno de nascentes, enquadradas como de proteção ambiental (conforme código florestal), estejam quase em sua totalidade desmatadas. Assim, o presente estudo procura caracterizar os solos locais na sua ambiência para servir como um referencial em futuras comparações entre áreas de nascentes degradadas e em processo de recuperação e avaliar atributos físicos, químicos e microbiológicos do solo no entorno de nascentes deste rio, para maior entendimento da dinâmica do meio-físico. As nascentes foram classificadas quanto a sua recarga e quanto ao seu estado de conservação. Foram selecionadas as áreas de revegetação de cada nascente e estas foram agrupados de acordo com a sua posição na paisagem. A caracterização ambiental e pedológica dos sítios de estudo foi realizada pela observação da paisagem local, pela abertura de micro-trincheiras e por tradagens dos solos. Os solos foram descritos morfologicamente e classificados. Foram confeccionadas lâminas para a difração de raios-x da fração argila. Foi possível o agrupamento das nascentes de acordo com suas posições de paisagem e das características dos solos locais em baixada (BA), encosta com horizonte A (EA), encosta sem horizonte A (ES), encosta com hidromorfismo (EH) e sopé de encosta suave (SE). Os atributos foram analisados em amostras nas profundidades de 0 a 0,1m e 0,1 a 0,3m. Foram determinados: textura, matéria orgânica particulada, estabilidade de macro agregados, diâmetro médio de agregados, valor da cor, pH, C orgânico, Ca, Ca+Mg, Mg, Al, Na, K, P, H+Al, N, soma de bases, capacidade de troca catiônica, saturação de bases, saturação de Al, respiração microbiana, C e N da biomassa microbiana e atividade enzimática (FDA). A similaridade e/ou, dissimilaridade entre os sítios (ordenamento) foi averiguada pela análise multivariada non-metric multidimensional scaling (NMS), a partir da distância de Sorensen. Determinaram-se os coeficientes de correlação de Pearson entre os eixos da ordenação por NMS e entre os diferentes atributos. Os sítios do entorno das nascentes do alto curso do rio Piauitinga estão distribuídos em posições erosicionais (quebra-de-relevo), baixadas e um único caso de sopé de encosta suave de topo de tabuleiros costeiros. Suas principais características são o forte hidromorfismo (Gleissolos e Cambissolos Háplicos gleissólicos) e/ou, o baixo grau de desenvolvimento (Cambissolos Háplicos e Plintossolos, ambos com muito material esquelético quartzo e petroplintita muitos com fase erodida). As variáveis físicas, químicas e biológicas estudadas, por meio da análise multivariada, permitiram a construção de um gradiente ambiental, com a qualidade dos solos decrescente na seqüência: BA, EH, EA e ES. Acredita-se que seja possível redefinições de estratégias para a revegetação das nascentes do alto curso dessa bacia hidrográfica, extrapolar a qualidade do solo para áreas de nascentes de cursos d água com características semelhantes e refletir sobre estratégias para restauração de áreas degradadas.

A vida moderna tem gerado mudanças importantes nos hábitos alimentares em todo o mundo e observa-se um aumento da demanda por alimentos prontos para o consumo, tais como frutas e hortaliças minimamente processadas. As condições de embalagem e armazenamento destes produtos podem favorecer a multiplicação de bactérias psicrotróficas, destacando-se o patógeno Listeria monocytogenes. Para estudos de avaliação de riscos microbiológicos relativos a L. monocytogenes, dados quantitativos são fundamentais, mas, os métodos para enumeração disponíveis atualmente são trabalhosos e morosos. Há grande interesse no desenvolvimento de métodos rápidos para a determinação das populações de L. monocytogenes, o que pode reduzir significativamente o tempo de análise. Neste trabalho, foram analisadas 162 amostras de hortaliças minimamente processadas adquiridas no comércio varejista de Ribeirão Preto/SP, no período de setembro de 2007 a agosto de 2008. Foram realizados ensaios convencionais para enumeração de coliformes totais e termotolerantes, Escherichia coli e bactérias aeróbias psicrotróficas. Foi realizada a detecção de Listeria sp. por imunoensaio (Oxoid Listeria Rapid Test) e de Salmonella por método convencional. Alíquotas congeladas de amostras positivas para L. monocytogenes e Salmonella sp. foram também avaliadas quantitativamente empregando-se métodos convencionais. Para quantificação de L. monocytogenes foi utilizado o método da Reação de Polimerase em Cadeia (PCR) em tempo real, com primers de DNA baseados em 16S rRNA. Para a comparação do método de PCR em tempo real e método convencional de cultivo, foram inoculadas seis amostras de hortaliças minimamente processadas (alface, cheiro-verde, couve, almeirão, repolho e acelga) com três níveis de inoculação. Dentre as 162 amostras de hortaliças analisadas, L. monocytogenes foi detectada em uma amostra de couve e uma de cheiro-verde, com populações de 1,5 x 103 e 0,43 NMP/g, respectivamente. Salmonella sp. foi isolada de duas amostras de almeirão, com populações de 0,09 e 4,6 NMP/g. Contaminação por coliformes totais foi detectada em 132 (81,5%) amostras e por coliformes termotolerantes em 107 (66%) amostras. Escherichia coli foi confirmada em 86 (53,1%) amostras, enquanto que 158 (96,7%) amostras apresentaram população de bactérias psicrotróficas maior que 5 log UFC/g. Os resultados obtidos entre os dois métodos desenvolvidos apresentaram pouca variação e todos os resultados foram concordantes considerando-se o intervalo de confiança de 95% da técnica do NMP (SWANSON; PETRAN; HANLIN, 2001). A PCR em tempo real foi otimizada com a preparação comercial ABSOLUTETM QPCR SYBR® Green Mix (ABgene, Reino Unido) e os primers utilizados mostraram ser específicos para L. monocytogenes. As populações de microrganismos encontradas nestes alimentos indicaram a baixa qualidade microbiológica e evidenciaram a importância da implantação de programas para garantia da inocuidade de alimentos na cadeia produtiva das hortaliças minimamente processadas. A PCR em tempo real mostrou ser de fácil execução e diminuiu o tempo de obtenção de resultados para pouco mais que 48 horas em comparação com o tempo gasto (7 dias) quando as hortaliças foram analisadas por meio do método convencional de cultivo. / Modern lifestyles have deeply changed eating habits worldwide, with an increasing demand for ready-to-eat foods, including minimally processed fruits and leafy greens. Packaging and storage conditions of these products may favor the growth of psychrotrophic bacteria, such as the pathogen Listeria monocytogenes. For microbiological risk assessment, it is crucial to generate quantitative data on foodborne pathogens, but the enumeration methods currently available are labor and time consuming. There is great interest in the development of reliable and fast methods for enumeration of L. monocytogenes, in order to shorten the time needed to obtain quantitative results. In this study 162, samples of leafy vegetables minimally processed were acquired at retail market in Ribeirão Preto from September, 2007 to August 2008 and they were analysed by conventional enumerating methods for total and thermophilic coliforms, Escherichia coli and psychrotrophic aerobic bacteria. Presence or absence of Listeria spp. was evaluated by immunoassay (Oxoid Listeria Rapid test) and detection of Salmonella was performed by conventional methods. Frozen aliquots of Salmonella spp. and L. monocytogenes positive samples were quantitatively evaluated by conventional methods and L. monocytogenes was also enumerated by real-time Polymerase Chain Reaction (PCR) with DNA primers based on 16S rRNA. Real-time PCR method was applied to analyze six artificially contaminated vegetables and the results obtained with this methodology were similar to the ones obtained with conventional most probably number (MPN) technique with a confidence interval of 95% (SWANSON; PETRAN; HANLIN, 2001). Of the 162 samples analyzed, L. monocytogenes was detected in one sample of collard green and in one mixed bunch of parsley plus spring onion (1.5x103 and 0.43 MPN/g, respectively). Salmonella sp was detected in two samples of common chicory with populations of 0.09 and 4.6 MPN/g. One hundred and thirty two samples (81.5%) showed contamination by total coliforms and 107 (66%) by thermophilics coliforms. Escherichia coli was confirmed in 86 (53.1%) samples while 156 (96.7%) showed populations of psychrotrophic bacteria above of 5 log CFU/g. PCR was optimized with a commercial preparation ABSOLUTE TM QPCR SYBRÒ Green Mix (ABgene, UK) and the primers utilized were specific for L. monocytogenes. These results showed the low microbiologic quality of minimally processed vegetables samples studied and indicate an urge for implementation of quality programs from producer to processors of these ready-to-eat foods. Realtime In comparison with the conventional culture method, real-time PCR was more easily performed and the time required to obtain the results was reduced to ca. 48 hours, in comparison with 7 days for conventional method.

O pólen apícola, produto da aglutinação do pólen das flores com néctar e substâncias salivares das abelhas, tem sido promovido como suplemento da dieta humana por apresentar propriedades nutricionais e bioativas. Sessenta e duas amostras de pólen apícola desidratado foram avaliadas para as análises de compostos fenólicos, flavonoides, atividade antioxidante por três métodos (DPPH, sistema β-caroteno e ORAC), origem botânica, qualidade comercial (umidade, cinzas, lipídeos, proteínas e carboidratos) e sanitária (bolores e leveduras, aeróbios mesófilos, coliformes totais, E.coli, clostrídios sulfito redutores e S.aureus.), além da atividade antimicrobiana (Candida albicans, Escherichia coli, Staphylococcus aureus). Os valores obtidos para compostos fenólicos e flavonoides totais variaram de 12,60 a 84,22 mg GAE/g de pólen apícola (GAE: equivalentes em ácido gálico) e 1,90 a 36,85 mg de quercetina/g de pólen apícola respectivamente. O EC50, determinado pelo método do DPPH, variou de 0,35 a 13,42 mg pólen apícola/mL de extrato. Os extratos de pólen apícola apresentaram valores entre 52,58 e 98,37 % para o método do β-caroteno. Quando quantificada por ORAC, a atividade antioxidante medida ficou entre 132,98 e 575,85 µmol eq. trolox/g pólen apícola. Na avaliação da qualidade comercial foram obtidos os valores de 3,06% a 8,12% para umidade, de 1,94 a 4,61%, para cinzas, de 3,35 a 10,96% para lipídeos; de 17,73 a 34,73% para as proteínas, de 11,86 a 25,71% e de 2,77 a 15,87% para os açúcares frutose e glicose, respectivamente. Verificou-se que a presença do pólen apícola inibiu o crescimento de todos os microrganismos estudados. Candida albicans foi a mais resistente e o Staphylococcus epidermides foi o mais sensível. Observou-se 36 tipos polínicos diferentes, destacando-se: Cocos nucifera sp., Mimosa scabrella (Benth.), Mimosa caesalpiniaefolia sp., Eucalyptus sp., Myrcia sp., Asteraceae, Poaceae., Euphorbiaceae e Brassica que ocorreram com maior frequência entre as amostras estudadas. Foram observadas somente correlações moderadas e fracas entre os tipos polínicos presentes e os parâmetros avaliados para as amostras desidratadas de pólen apícola. / Bee pollen, a product of agglutination of flower pollen with nectar and bee salivary substances, has been promoted as a dietary supplement for human because of its nutritional and bioactive properties. Sixty-two samples of dehydrated bee pollen were analyzed for phenolics, flavonoids, antioxidant activity using three methods (DPPH, βcarotene and ORAC), botanical composition, commercial quality (moisture, ashes, lipids, proteins, carbohydrates), hygiene (aerobic mesophiles, yeasts and moulds, coliforms, Escherichia coli, Staphylococcus aureus and sulfite-reducing Clostridium), and antimicrobial activity (Candida albicans, Escherichia coli, Staphylococcus aureus). The obtained values for total phenolics ranged from 12.60 to 84.22 mg GAE/g bee pollen (GAE: gallic acid equivalents) while for total flavonoids ranged from 1.90 and 36.85 mg quercetin/g bee pollen. The EC50, determined by the DPPH method, ranged from 0.35 to 13.42 mg bee pollen/ml of extract. The bee pollen extracts showed values between 52.58 and 98.37% by β- carotene method. When measured by ORAC, antioxidant activity was between 132.98 and 575.85 µmols eq. trolox/g bee pollen. In the evaluation of commercial quality, the following results were achieved: 3.06% to 8.12% for moisture, 1.94 to 4.61% for ashes, 3.35 to 10.96% for lipids, 17.73 to 34.73% for proteins, from 11.86 to 25.71% and 2.77 to 15.87% for the carbohydrates glucose and fructose respectively. It was verified that the presence of the bee pollen inhibited the growth of all microorganisms studied. Candida albicans was the more resistant and Staphylococcus epidermides the more sensitive. Thirty-three pollen-types were identified mainly Cocos nucifera sp., Mimosa scabrella (Benth.) sp., Mimosa caesalpiniaefolia sp., Eucalyptus sp., Myrcia sp., Asteraceae sp., Poaceae sp., Euphorbiaceae sp. and Brassica sp. that occurred more frequently among the samples studied. It was observed only moderate and weak correlations between the pollen types present and the evaluated parameters for dehydrated samples of bee pollen

Recent studies have demonstrated that peanut and grape processing by-products may be richer sources of bioactive compounds as compared to their original raw material and feedstock; however, before their application as a source of nutraceuticals or in the prevention of lipid oxidation in food systems, certain technological challenges have to be addressed. This study discusses recent advances in the application of plant food processing by-products as sources of phenolic compounds with special emphasis on the profiling and screening of phenolics using high-performance liquid chromatography-mass spectrometry, their potential health benefits, and microbiologial safety. The major findings are summarized in chapters 2, 3, and 4. The first chapter deals with phenolics from grape by-products. In general, insoluble-bound phenolics were more effective in inhibiting copper-induced human LDL-cholesterol oxidation in vitro than free and esterified phenolics. Phenolic extracts from all fractions inhibited peroxyl radical-induced DNA strand breakage. The third chapter brings about the effects of gamma-irradiation on the microbial growth, phenolic composition, and antioxidant properties of peanut skin. Gamma-irradiation at 5.0 kGy decreased the microbiological count of the product. Total phenolic and proanthocyanidin contents, ABTS radical cation, DPPH radical, hydrogen peroxide, and hydroxyl radical scavenging capacities as well as the reducing power of the sample were increased upon gamma-irradiation in both the free and insoluble-bound phenolic fractions. The bioactivity of the free phenolics against in vitro human LDL-cholesterol oxidation and copper induced DNA strand breakage was improved upon gamma-irradiation. Phenolic compounds were positively or tentatively identified and their distribution was in the decreasing order of free > esterified > insoluble-bound forms. Procyanidin dimer A was increased in all phenolic fractions, whereas procyanidin dimer B decreased. Gamma-irradiation induced changes may be explained by molecular conversion, depolymerization, and cross-linking. In the fourth chapter, the ability of selected enzymes in improving the extraction of insoluble-bound phenolics from the starting material (experiment I) or the residues containing insoluble-bound phenolics (experiment II) were evaluated. Pronase and Viscozyme improved the extraction of insoluble-bound phenolics. Viscozyme released higher amounts of gallic acid, catechin, and prodelphinidin dimer A compared to Pronase treatment. Furthermore, p-coumaric and caffeic acids, as well as procyanidin dimer B, were extracted with Viscozyme but not with Pronase treatment. Solubility plays an important role in the bioavailability of phenolic compounds, hence this study may assist in better exploitation of phenolics from winemaking by-products as functional food ingredients or supplements. / Estudos recentes têm demonstrado que subprodutos da indústria processadora de amendoim e uva podem ser mais ricos em compostos bioativos em comparação às suas matérias-primas. No entanto, alguns desafios tecnológicos precisam ser enfrentados antes da sua aplicação como fonte de compostos nutracêuticos ou na prevenção da oxidação lipídica em sistemas alimentares. Este estudo discute os recentes avanços na aplicação de subprodutos da indústria processadora de amendoim e uva como fontes de compostos fenólicos. Especial ênfase foi dada a sua caracterização por cromatografia líquida acoplada à espectrometria de massas, aos potenciais benefícios à saúde e à segurança microbiológica. As principais conclusões estão apresentadas nos capítulos 2, 3 e 4. O primeiro capítulo trata de compostos bioativos de subprodutos da indústria de suco de uva e da produção vinícola. A fração da qual foram extraídos os compostos fenólicos ligados à parede celular foi predominante. Em geral, esta fração também foi a mais eficaz na inibição da oxidação do LDL - colesterol in vitro quando comparada à fração que continha os fenólicos livres e os esterificados. Os compostos fenólicos de todas as frações inibiram o dano oxidativo ao DNA induzido por radicais peroxila. O terceiro capítulo fala sobre os efeitos da irradiação gama sobre a carga microbiana, a composição fenólica e as propriedades antioxidantes da película de amendoim. A irradiação gama (5,0 kGy) diminuiu a contagem microbiana do produto. Os compostos fenólicos totais, o teor de proantocianidinas e a capacidade dos extratos em neutralizar radicais como o ABTS, DPPH e espécies reativas de oxigênio como o peróxido de hidrogênio e radicais hidroxila, assim como o poder redutor da amostra, aumentaram devido à irradiação gama em ambas as frações (contendo fenólicos livres e ligados à parede celular). A bioatividade dos compostos fenólicos livres contra a oxidação do LDL-colesterol in vitro e contra os danos oxidativos ao DNA aumentou com a irradiação gama. Os compostos fenólicos foram positivamente ou tentativamente identificados, distribuindo-se entre: fenólicos livres > esterificados > ligados. Houve aumento na concentração de dímeros de procianidina A em todas as frações, enquanto a concentração de dímeros de procianidina B diminuiu. Essas alterações podem ser explicadas pela conversão molecular, despolimerização e formação de ligações cruzadas. No quarto e último capítulo, enzimas selecionadas foram aplicadas à matéria-prima inicial (experimento I) ou nos resíduos contendo apenas compostos fenólicos insolúveis (experimento II). Pronase e Viscozyme aumentaram a extração de compostos fenólicos insolúveis (ligados à parede celular). Viscozyme liberou maiores quantidades de ácido gálico, catequina e dímero de prodelfinidina A em comparação ao tratamento com Pronase. Além disso, os ácidos p-cumárico e ácido caféico, bem como o dímero de procianidina B, foram extraídos com Viscozyme, mas não com Pronase. A solubilidade desempenha um papel importante na biodisponibilidade de compostos fenólicos. Desta forma, o terceiro estudo oferece uma alternativa para a exploração de compostos fenólicos de subprodutos da indústria vinícola como ingredientes alimentares com propriedades funcionais ou suplementos alimentares.

Escherichia coli enteropatogênica atípica (aEPEC) faz parte de um grupo de patógenos capazes de formar um tipo de lesão característica em cultura de tecidos epiteliais, denominada attaching and effacing (A/E). Os genes que são necessários para produção da lesão A/E estão localizados em uma ilha de patogenicidade denominada região LEE (locus of enterocyte effacement). A transcrição de genes da região LEE está sujeita a regulação por vários fatores, entre eles quorum sensing, termo utilizado para designar um mecanismo de regulação gênica dependente da concentração celular. Esse mecanismo é usado por bactérias Gram-positivas e Gram-negativas e em ambos os casos envolve a produção e detecção de moléculas sinalizadoras extracelulares, denominadas autoindutores. Até o momento, pelo menos quatro sistemas de quorum sensing foram descritos, entre eles o sistema de autoindutor AI-3 encontrado em bactérias Gram-positivas e Gram-negativas. Diversos mecanismos celulares, entre eles a expressão de fatores de virulência em amostras de EPEC e EHEC, são regulados por esse fenômeno. O principal objetivo deste estudo foi verificar se existe uma possível regulação por quorum sensing na interação in vitro de uma amostra de E. coli da microbiota intestinal com amostras de aEPEC. Após a confirmação da produção de AI-3 por amostras de E.coli da microbiota intestinal foram realizados ensaios de adesão e quantificação utilizando meio pré-condicionado com esta amostra, epinefrina e bloqueadores que confirmaram que os padrões de adesão de aEPEC obtidos em menor tempo são devidos a presença de AI-3 no meio pré-condicionado, indicando a participação de quorum sensing nessa interação. Além disso, foi observado um fenômeno citotóxico nas células que não é produzido pelo AI-3. / Atypical Enteropathogenic Escherichia coli (aEPEC) are part of a group of pathogens capable of forming a type of lesion characteristic of epithelial tissues in culture, called attaching and effacing (A/E). The genes that are required for production of A/E lesion are located in a pathogenicity island called LEE region (locus of enterocyte effacement). The transcription of LEE genes in the region is subject to regulation by various factors, including quorum sensing, a term used to describe a mechanism of gene regulation dependent on cell concentration. This mechanism is used by Gram-positive and Gram-negative and in both cases involves the production and detection of extracellular signaling molecules, called autoinducers. So far, four systems of quorum sensing have been described, including the system of autoinducers AI-3 found in Gram-positive and Gram-negative bacteria. Several cellular mechanisms, including expression of virulence factors in EPEC and EHEC are regulated by this phenomenon. The main objective of this study was to determine whether there is a possible regulation by quorum sensing in the in vitro interaction of a strains of E. coli of the intestinal microbiota with strains aEPEC. After confirming the production of AI-3 in E. coli of the intestinal microbiota were performed adhesion assays and quantification using means preconditioned with this strains, epinephrine, and blockers who confirmed that patterns of adherence of aEPEC obtained in less time are due to the presence of AI-3 in the preconditioned means, indicating the involvement of quorum sensing in this interaction. Furthermore, we observed a phenomenon that cytotoxic cells is not produced by AI-3.

This thesis deals with the Air to Ground Heat Exchangers (AGHEx) and their effects on the hygienic quality of the supplied air and the microbial microclimate in the interior of buildings. The theoretical part focuses on current findings and knowledge in the field of warm air heating, ventilation of the low-energy and energy passive houses and ground heat exchanger for the ventilation systems to family houses. The next chapter deals with the quality of the indoor environment and the influence on HVAC systems on the building microclimate. The experimental part of the thesis presents the results of energy simulations of operation of air to ground heat exchanger, obtained using the simplified model, and the data from long-term monitoring of experimental AGHEx built at FME BUT. Furthermore, the results of microbiological research of several already operating air to ground heat exchangers are evaluated. The research was carried out using two different sampling methods: the method using swabs taken from the pipe wall, and the sedimentation (gravimetric) method. The conclusion part mentions the practical experiences of users and knowledge of the author relating to the design, operation and use of air to ground heat exchangers.

Orientador: Antonio Batista Filho / Banca: Carlos Frederico Wilcken / Banca: Edson Luiz Furtado / Banca: José Eduardo Marcondes de Almeida / Resumo: Os objetivos deste trabalho foram avaliar diferentes meios de cultura na produção, semi-sólida e líquida, de propágulos de fungos de M. anisopliae e B. bassiana, bem como a tolerância desses propágulos a ação da radiação ultravioleta e da temperatura. Os experimentos foram conduzidos no Laboratório de Controle Biológico do Instituto Biológico, em Campinas, SP. Foram realizados, para M. anisopliae e B. bassiana, 6 repetições para cada um dos 17 tratamentos: amido de milho, arroz integral, arroz parboilizado, arroz tipo 1 (testemunha), arroz tipo 2, aveia em flocos, canjiquinha, farelo de trigo, farinha de mandioca crua, farinha de milho amarela, farinha de trigo especial, fubá, milho em grãos, polvilho azedo, soja em grãos, trigo moído e turfa. A viabilidade foi feita em placas de Petri plásticas contendo BDA. Para os ensaios com radiação ultravioleta e temperatura, utilizou-se a mesma metodologia para a viabilidade, mas cada tratamento sendo exposto à radiação por 0, 25 e 50 segundos e, em diferentes temperaturas: 20, 25, 30 e 35oC. Inoculou-se, em torre de Potter, 2 mL da suspensão de fungo de cada tratamento em lagartas de Diatraea saccharalis. Para a concentração os melhores tratamentos de M. anisopliae e B. bassiana foram: arroz parboilizado tipo 1, arroz tipo 1, arroz tipo 2, farinha de milho amarela, fubá e trigo moído. A viabilidade de todos os tratamentos foi superior a 94,00%; quanto maior o tempo de exposição ao ultravioleta menor foi o número de conídios férteis. À temperatura de 35oC ocorreu perda significativa da viabilidade de conídios e todos os tratamentos se mostraram virulentos. Para a 2 produção líquida de blastosporos de M. anisopliae e B. bassiana, foram avaliados 12 tratamentos, com 6 repetições cada, compostos pelas combinações entre as concentrações de Carbono (C), na forma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The goals of this work were to evaluate different medias (semi-solid and liquid) as for the production of M. anisopliae and B. Bassiana propagules, and also the tolerance of these propagules to ultraviolet radiation and temperature. The experiments were carried out at the Biological Control Laboratory of the Instituto Biológico, at Campinas, São Paulo, Brazil. For both M. anisopliae and B. bassiana, 6 repetitions were performed for each one of the 17 treatments: corn stearch, full rice, parboiled rice, type 1 rice, type 2 rice, oat flakes, canjiquinha, wheat flour, raw cassava flour, yellow corn flour, special wheat flour, corn flour, corn in grains, cassava stearch, soy in grains, crushed wheat and turf. The viability analysis was done in plastic plates containing BDA. For each one of the bioassays in ultraviolet and temperature exposition, the same methodology was used for viability analysis, but each treatment was exposed to the UV radiation during 0, 25 and 50 seconds, and at different temperatures: 20, 25, 30 and 35oC. Using the Potter tower, 2 mL of fungus suspension, from each tratment, were inoculated into the Diatraea saccharalis caterpillars. Regarding the sporulation, the best M. anisopliae and B. bassiana treatments were: parboiled rice, type 1 rice, type 2 rice, yellow corn flour, corn flour and crushed wheat. The viability of all the treatments was superior to 94,00%. Also, as longer was the duration of the exposition to the UV, as smaller was the number of fertile conidia. At 35oC, a significant loss of conidia viability was observed, and all the treatments have shown some level of virulence. For the M. anisopliae and B. bassiana blastospores production over liquid media, 12 treatments were 4 evaluated, with 6 repetitions each, composed by the combinations of carbon (C), presented as D-glicose anidra (40% Carbon) and... (Complete abstract click electronic access below) / Doutor

Tiger nuts (Cyperus esculentus L) are recognized as a high potential, alternative source of food nutrients. However, there is limited scientific literature on the technological possibilities for developing value-added foods, such as fermented products from tiger nut milk. Therefore, strategies for producing and improving the properties of fermented tiger nut milk were investigated for generating lactose-free, nutritious yogurt-like products with acceptable sensory properties and a prolonged shelf life quality. A wet-milling procedure was standardized for extracting tiger nut milk from tiger nuts, and the effects of the extraction process on nutrient distribution, colour properties and colloidal stability of the milk were analyzed. Next, tiger nut milk was enriched with proteins and/or hydrocolloids and the impact of the additives on the physical properties of the milk were determined. Enriched tiger nut milk was fermented by using classical yogurt cultures and the obtained products were analyzed for the microbiological, physico-chemical and sensory characteristics. Additionally, effects of enriching tiger nut milk with microbial transglutaminase cross-linked proteins on the microbiological and physico-chemical properties were evaluated. Higher wet-milling intensity improved the nutrient composition, colloidal stability and colour of the milk. Enrichment of tiger nut milk with milk proteins and xanthan gum enhanced the viscosity and stability, and after fermentation, led to homogenous gel-like products with superior microbiological, physico-chemical and different sensory properties compared to the fermented plain tiger nut milk. Microbial transglutaminase cross-linked proteins improved the physical characteristics of the fermented product, especially during storage. This product would be relevant in many developing countries with high prevalence of lactose intolerance, limited access to nutritious food but show a high distribution of tiger nut vegetation.:1. Introduction and aim 1 2. Literature review 4 2.1 Tiger nut, origin, nutritional value and food use 4 2.2 Tiger nut milk, preparation and nutrient composition 7 2.3 Colloidal characteristics of tiger nut milk 9 2.4 Factors accounting for the dispersion stability of tiger nut milk 10 2.5 Enhancing tiger nut milk stability 12 2.6 Properties of fermented tiger nut milk 17 2.7 Microbial transglutaminase and properties of fermented tiger nut milk 18 3. Methodology 21 3.1 Extraction and characterisation of tiger nut milk 21 3.1.1 Sample collection and preparation 21 3.1.2 Tiger nut milk extraction 21 3.1.3 Nutrient analysis of tiger nuts 22 3.1.4 Analysis of tiger nut products 23 3.1.5 Particle size distribution 24 3.1.6 Colloidal stability 25 3.1.7 Colour measurement 25 3.2 Stabilisation of tiger nut milk dispersion 26 3.2.1 Tiger nut milk preparation 26 3.2.2 Preparation of tiger nut milk enrichments 26 3.2.3 Gravitational stability of enriched tiger nut milk 27 3.2.4 Accelerated gravitational stability of enriched tiger nut milk 28 3.2.5 Viscosity of TNM mixtures 29 3.3 Extraction and characterisation of globular tiger nut proteins 29 3.3.1 Protein extraction and fractionation 29 3.3.2 Molecular mass of globular tiger nut proteins 31 3.3.3 Denaturation temperature of globular tiger nut proteins 32 3.3.4 Isoelectric point of globular tiger nut protein 33 3.4 Properties of fermented tiger nut milk enriched with proteins 34 3.4.1 Materials and Reagents 34 3.4.2 Preparation of plain and enriched tiger nut milk 34 3.4.3 Fermentation of plain and enriched tiger nut milk 35 3.4.4 Viable counts of starter cultures in fermented tiger nut milk systems 36 3.4.5 Chemical analysis of unfermented and fermented tiger nut milk 36 3.4.6 Physical analysis of fermented tiger nut milk products 37 3.4.7 Sensory analysis of fermented tiger nut milk products 38 3.5 Microbial transglutaminase and fermented tiger nut milk property 38 3.5.1 Preparation of plain and enriched tiger nut milk 38 3.5.2 Fermentation of plain and enriched tiger nut milk 39 3.5.3 Analysis of the enzymatically cross-linked proteins 39 3.5.4 Viable counts 40 3.5.5 pH and titratable acidity 40 3.5.6 Syneresis and viscosity 41 3.5.7 Colour of fermented tiger nut products 41 3.6 Statistical analysis 41 4. Results and discussion 43 4.1 Extraction and characteristics of tiger nut milk 43 4.1.1 Material recovery, mass transfer and yield of tiger nut solids 43 4.1.2 Nutrient composition of tiger nut products 45 4.1.3 Physical properties of tiger nut milk 48 4.1.3.1 Particle size distribution of extracted tiger nut milk 48 4.1.3.2 Colloidal stability of tiger nut milk 49 4.1.3.3 Colour stability of tiger nut milk 51 4.2 Stabilisation of tiger nut milk 53 4.2.1 Effects of enrichments on the stability of tiger nut milk 53 4.2.2 Effects of pH and temperature on the stability of enriched TNM 56 4.2.3 Effects of enrichments on the rheology of tiger nut milk 58 4.3 Tiger nut protein extraction and characterisation 60 4.3.1 Protein extraction and fractionation 60 4.3.2 Molecular mass of tiger nut protein 62 4.3.3 Thermal denaturation of tiger nut protein 63 4.3.4 Isoelectric point of tiger nut proteins 66 4.4 Properties of fermented tiger nut milk enriched with proteins 67 4.4.1 Acidification and gel formation during fermentation 67 4.4.2 Microbiological properties of fermented enriched tiger nut milk 70 4.4.3 Physico-chemical properties of fermented enriched tiger nut milk 71 4.4.4 Sensory properties of fermented tiger nut milk products 76 4.5 Microbial transglutaminase and fermented tiger nut milk property 77 4.5.1 Effects on tiger nut milk fermentation 77 4.5.2 Microbiological properties during storage of fermented product 81 4.5.3 Physico-chemical properties during storage of fermented product 83 4.5.4 Effects on colour of fermented tiger nut product 86 5. Conclusions and outlook 88 Bibliography 90 List of figures 111 List of tables 115 List of Publications 116 Poster and presentations 116 / Erdmandeln (Cyperus esculentus L) haben ein hohes Potential als alternative Quelle Lebensmittelinhaltsstoffen. Allerdings gibt es nur in begrenztem Ausmaß Literatur über technologische Möglichkeiten zur Entwicklung von Mehrwert-Lebensmitteln wie fermentierter Erdmandelmilch. Daher wurden Strategien zur Herstellung und Verbesserung der Eigenschaften von fermentierter Erdmandelmilch zur Erzeugung laktosefreier joghurtähnlicher Produkte mit akzeptablen sensorischen Eigenschaften untersucht. Für die Extraktion der Erdmandelmilch wurde ein Nassmahlverfahren standardisiert und der Einfluss des Verfahrens auf die Nährstoffverteilung, die Farbeigenschaften und die kolloidale Stabilität der Milch analysiert. Als nächstes wurde Erdmandelmilch mit Proteinen und/oder Hydrokolloiden angereichert, und der Einfluss der Additive auf die physikalischen Eigenschaften des Extrakts bestimmt. Angereicherte Erdmandelmilch wurde mit klassischen Joghurtkulturen fermentiert, und die mikrobiologischen, physikalisch-chemischen und sensorischen Eigenschaften der Produkte wurden untersucht. Zusätzlich wurden Effekte der Anreicherung von Erdmandelmilch mit enzymatisch vernetzten Proteinen auf die mikrobiologischen und physikalisch-chemischen Eigenschaften bewertet. Eine höhere Nassmahlintensität verbesserte die Nährstoffzusammensetzung, die kolloidale Stabilität und die Farbe der Milch. Die Anreicherung erhöhte die Viskosität und Stabilität und führte nach der Fermentation zu homogenen gelartigen Produkten mit verbesserten mikrobiologischen, physikalisch-chemischen und sensorischen Eigenschaften im Vergleich zur fermentierten Erdmandelmilch. Mikrobielle Transglutaminase-vernetzte Proteine verbesserten die physikalischen Eigenschaften des fermentierten Produkts, insbesondere während der Lagerung. Dieses Produkt wäre in vielen Entwicklungsländern mit hoher Prävalenz von Laktoseintoleranz und begrenztem Zugang zu nahrhaften Lebensmitteln als Alternative von Interesse.:1. Introduction and aim 1 2. Literature review 4 2.1 Tiger nut, origin, nutritional value and food use 4 2.2 Tiger nut milk, preparation and nutrient composition 7 2.3 Colloidal characteristics of tiger nut milk 9 2.4 Factors accounting for the dispersion stability of tiger nut milk 10 2.5 Enhancing tiger nut milk stability 12 2.6 Properties of fermented tiger nut milk 17 2.7 Microbial transglutaminase and properties of fermented tiger nut milk 18 3. Methodology 21 3.1 Extraction and characterisation of tiger nut milk 21 3.1.1 Sample collection and preparation 21 3.1.2 Tiger nut milk extraction 21 3.1.3 Nutrient analysis of tiger nuts 22 3.1.4 Analysis of tiger nut products 23 3.1.5 Particle size distribution 24 3.1.6 Colloidal stability 25 3.1.7 Colour measurement 25 3.2 Stabilisation of tiger nut milk dispersion 26 3.2.1 Tiger nut milk preparation 26 3.2.2 Preparation of tiger nut milk enrichments 26 3.2.3 Gravitational stability of enriched tiger nut milk 27 3.2.4 Accelerated gravitational stability of enriched tiger nut milk 28 3.2.5 Viscosity of TNM mixtures 29 3.3 Extraction and characterisation of globular tiger nut proteins 29 3.3.1 Protein extraction and fractionation 29 3.3.2 Molecular mass of globular tiger nut proteins 31 3.3.3 Denaturation temperature of globular tiger nut proteins 32 3.3.4 Isoelectric point of globular tiger nut protein 33 3.4 Properties of fermented tiger nut milk enriched with proteins 34 3.4.1 Materials and Reagents 34 3.4.2 Preparation of plain and enriched tiger nut milk 34 3.4.3 Fermentation of plain and enriched tiger nut milk 35 3.4.4 Viable counts of starter cultures in fermented tiger nut milk systems 36 3.4.5 Chemical analysis of unfermented and fermented tiger nut milk 36 3.4.6 Physical analysis of fermented tiger nut milk products 37 3.4.7 Sensory analysis of fermented tiger nut milk products 38 3.5 Microbial transglutaminase and fermented tiger nut milk property 38 3.5.1 Preparation of plain and enriched tiger nut milk 38 3.5.2 Fermentation of plain and enriched tiger nut milk 39 3.5.3 Analysis of the enzymatically cross-linked proteins 39 3.5.4 Viable counts 40 3.5.5 pH and titratable acidity 40 3.5.6 Syneresis and viscosity 41 3.5.7 Colour of fermented tiger nut products 41 3.6 Statistical analysis 41 4. Results and discussion 43 4.1 Extraction and characteristics of tiger nut milk 43 4.1.1 Material recovery, mass transfer and yield of tiger nut solids 43 4.1.2 Nutrient composition of tiger nut products 45 4.1.3 Physical properties of tiger nut milk 48 4.1.3.1 Particle size distribution of extracted tiger nut milk 48 4.1.3.2 Colloidal stability of tiger nut milk 49 4.1.3.3 Colour stability of tiger nut milk 51 4.2 Stabilisation of tiger nut milk 53 4.2.1 Effects of enrichments on the stability of tiger nut milk 53 4.2.2 Effects of pH and temperature on the stability of enriched TNM 56 4.2.3 Effects of enrichments on the rheology of tiger nut milk 58 4.3 Tiger nut protein extraction and characterisation 60 4.3.1 Protein extraction and fractionation 60 4.3.2 Molecular mass of tiger nut protein 62 4.3.3 Thermal denaturation of tiger nut protein 63 4.3.4 Isoelectric point of tiger nut proteins 66 4.4 Properties of fermented tiger nut milk enriched with proteins 67 4.4.1 Acidification and gel formation during fermentation 67 4.4.2 Microbiological properties of fermented enriched tiger nut milk 70 4.4.3 Physico-chemical properties of fermented enriched tiger nut milk 71 4.4.4 Sensory properties of fermented tiger nut milk products 76 4.5 Microbial transglutaminase and fermented tiger nut milk property 77 4.5.1 Effects on tiger nut milk fermentation 77 4.5.2 Microbiological properties during storage of fermented product 81 4.5.3 Physico-chemical properties during storage of fermented product 83 4.5.4 Effects on colour of fermented tiger nut product 86 5. Conclusions and outlook 88 Bibliography 90 List of figures 111 List of tables 115 List of Publications 116 Poster and presentations 116

This research investigates interacting biological, chemical, and physical factors affecting the efficiency of microbiological wastewater treatment at the W. J. Smith Wood- Preserving Company in Denison, Texas. The treatment process consisted of collecting exhaust boiler water containing unidentified boiler treatment compounds, steam condensate contaminated with preservatives and wood extracts, plant process waters, and rainfall runoff from plant grounds. With a 5-minute residence time, wastewater was passed over 2 oxidation towers in series, each containing approximately 47,000 square feet of surface area. Suspended solids were removed from the wastewater before discharge. Various amino acids such as serine, aspartate, cysteine, phenylalanine, alanine, proline, glycine, histidine, and tyrosine significantly stimulated phenol degradation in the laboratory. The plant wastewater contained approximately 0.1 mg/l of several of the stimulatory amino acids. It was assumed that these concentrations provided maximal stimulation in the field situation. The plant wastewater also contained sufficient nitrogen to permit the organisms to degrade up to 100 mg phenol/1 of water examined. Amino acids in the wastewater probably serve as a source of microbial nutrition. Toxicity of the wastewater to fish was not caused by the presence of phenol, phenol degradation products, or traces of pentachlorophenol. The wastewater was rendered non-toxic by diluting with between 4 to 9 volumes of stream water. Toxicity could also be removed by chemical coagulation followed by activated carbon adsorption. As a result of biological treatment, the plant now discharges the treated wastewater into the municipal sewage treatment facility.