Bacillus cereus est une bactérie largement disséminée dans la nature, contaminant ainsi les aliments en contact avec le sol. En France, cette bactérie est considérée comme le quatrième agent de toxi infection alimentaire collective. Pour être pathogène, B. cereus doit être capable de se multiplier lors des différentes étapes de transformation et notamment au cours de la réfrigération. Le but de cette étude a été d'étudier les mécanismes moléculaires de la réponse adaptative au froid et en particulier le rôle des hélicases à ARN de B. cereus ATCC 14579. Le gène cshA, codant pour une hélicase à ARN putative, a été identifié par une approche de mutagénèse aléatoire, comme jouant un important dans l’adaptation au froid de B. cereus. La souche ATCC 14579 possède 5 gènes codant pour des hélicases à ARN, cshA à cshE qui sont tous fortement surexprimés à 10°C par rapport à 37°C et quel que soit le stade de croissance considéré. La délétion simple des gènes cshA, cshB et cshC conduit à l’apparition de phénotypes cryosensibles, se traduisant par une incapacité d'adaptation au froid par rapport à la souche sauvage, associée à une modification de la morphologie cellulaire. De plus, CshA, CshB et CshC possèdent chacune un domaine de température où leur action est prépondérante. Elles semblent également être impliquées dans l’adaptation au stress oxydant et au stress basique, alors que CshD et E n’ont pas de rôle dans l’adaptation aux stress testés. Nous avons montré que CshA est indispensable à basse température, pour permettre le maintien de la stabilité des ribosomes avec lesquels elle interagit directement, mais aussi pour réguler la dégradation des ARNr. L’identification des partenaires protéiques interagissant avec CshA suggérent qu'elle puisse être également impliquée dans un complexe de dégradation des ARN / Bacillus cereus is a widespread bacteria, thus contaminating all raw materials in contact with soil. In France, B. cereus is considered as the fourth causative agent of foodborne illness. To be pathogenic, B. cereus should multiply during the various stages of food processing and particularly during preservation at low temperature. The aim of this study was to study molecular mechanisms of the adaptive response at low temperature and more precisely the involvement of the B. cereus ATCC 14579 RNA helicases. The cshA gene encoding a putative RNA helicase was identified by a random mutagenesis approach, as playing a major role in cold adaptation of B. cereus. The ATCC 14579 strain possesses 5 genes encoding putative RNA helicases, cshA to cshE, which were all strongly overexpressed at 10°C versus 37°C, whatever the growth stage. The simple deletion of cshA, cshB, and cshC lead to a cold-sensitive phenotype, resulting in an inability to adapt at 10 °C compared to the wild type strain, associated to a huge modification of cell morphology. In addition, CshA, CshB and CshC have a temperature range where their action is decisive. The role of these three RNA helicases also appears to be important in adaptation to oxidative and basic stresses while CshD and E did not appear to be involved in the adaptation to the tested stresses. The RNA helicase CshA has the most important role in adaptation to cold. We demonstrated that CshA is essential at low temperature to allow the maintenance of ribosome stability. CshA interacts directly with ribosomes, and also regulate rRNA degradation. The identification of protein partners that interact with CshA suggests that it could be involve in a complex of RNA decay

Lorsqu'un biofilm se développe dans des conditions statiques, deux populations, sessiles et planctoniques, coexistent et peuvent échanger des cellules. Cependant, l'immigration de cellules planctoniques dans un biofilm n'a, jusqu'à présent, fait l'objet que de peu d'études dans le cas d'un biofilm monoespèce. Chez B. cereus, un pathogène alimentaire, notre équipe a récemment montré que des bactéries planctoniques mobiles peuvent pénétrer en profondeur à l'intérieur d'un biofilm formé en immersion. L'objectif du présent travail était, en partant de ces données, de déterminer le rôle du recrutement dans le développement du biofilm formé en interface air-liquide, et de caractériser ce processus d’un point de vue physiologique et moléculaire. Nous avons montré que la population planctonique est massivement recrutée par le biofilm, mais que, dans nos conditions expérimentales, ce recrutement ne contribue que de façon marginale à la croissance du biofilm. Nous avons mis au point deux dispositifs expérimentaux (en interface air-liquide et en immersion) permettant de quantifier le recrutement, ce qui nous a permis de cribler une banque de mutants, obtenue par mutagenèse aléatoire, pour sélectionner des clones inaptes à être recrutés. Le criblage de 1700 clones a abouti à la sélection d'un gène: Bthur002_62720. La délétion de ce gène par échange allélique affecte fortement la capacité du mutant à être recruté, et la complémentation rétablit le phénotype sauvage. Ce gène code pour une protéine probablement localisée dans l'enveloppe bactérienne. Il est porté par un plasmide, pCT8513, et pourrait être un élément mobile dont l'acquisition augmenterait fortement la capacité de la bactérie réceptrice à être recrutée par un biofilm. Enfin, nous avons mis en évidence le rôle du locus eps dans le recrutement des cellules planctoniques par un biofilm formé en interface air-liquide. Ce locus est homologue du locus epsA-O de Bacillus subtilis, requis chez cette espèce pour la production des exopolysaccharides de la matrice du biofilm. Chez B. cereus, nous avons montré que le locus eps est impliqué dans la formation d’une gaine d’exopolysaccharides faiblement liée à la paroi bactérienne. Cette couche d'exopolysaccharides contribue, avec d'autres exopolysaccharides d'origine inconnue, à la formation de la matrice du biofilm, et joue un rôle important dans l'adhésion de la bactérie sur des surfaces inertes et vivantes. En favorisant l'adhésion de la bactérie sur des surfaces vivantes, le locus eps pourrait faciliter son intégration dans le biofilm. Il pourrait également être impliqué dans le pouvoir pathogène de la bactérie. / When biofilm is developing in static conditions, cell exchanges between sessile and planktonic coexisting population can emerge. Up to now, very few are known about the implication of planktonic cells integration in monospecies biofilm development. in B. cereus, a foodborne pathogen, our team have shown that motility is a key factor for biofilm development and for the deep penetration of motile planktonic bacteria inside a biofilm formed in immersed condition. Based on these data, the purpose of the present work was to determine the role of recruitment in the development of biofilm in air-liquid interface and to characterize this phenomenon physiologically and in a molecular aspect. We showed that a massive planktonic population is integrated in the developing biofilm, however, in our experimental conditions this recruitment contributes only marginally to the biofilm growth. We have developed two recruitment systems (in air-liquid interface and in immersion condition), to quantify recruitment, which has allowed us to screen a library of mutants obtained by random mutagenesis in order to select clones unable to be recruited by a preformed biofilm. Screening of 1700 clones resulted in the selection of a gene: Bthur002_62720. The deletion of this gene by allelic exchange strongly affects the ability of the mutant to be recruited, and complementation restored the wild type phenotype. This gene encodes a protein probably localized in the bacterial envelope. It is carried by a plasmid, pCT8513, and could be a mobile element whose acquisition would greatly increase the ability of the recipient bacterium to be recruited by a biofilm. Finally, we have highlighted the role of the eps locus in the recruitment of planktonic cells in a biofilm formed at air-liquid interface. This locus is homologous to Bacillus subtilis epsA-O locus, required in this species for the production of exopolysaccharides of the biofilm matrix. In B. cereus, we have shown that the eps locus is involved in the formation of an exopolysaccharides sheath weakly bound to the bacterial cell wall. This exopolysaccharides layer contributes with other exopolysaccharides of unknown origin, to the formation of the biofilm matrix, and plays an important role in the adhesion of bacteria on inanimate and living surfaces.By promoting bacterial adhesion on living surfaces; the eps locus could help bacteria integration into the biofilm. It could also be involved in the pathogenicity of bacteria.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-06-02T10:37:53Z No. of bitstreams: 1 texto completo.pdf: 807554 bytes, checksum: 658a487e853c7bd48eaec0404d9f045c (MD5) / Made available in DSpace on 2016-06-02T10:37:53Z (GMT). No. of bitstreams: 1 texto completo.pdf: 807554 bytes, checksum: 658a487e853c7bd48eaec0404d9f045c (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O leite e os produtos lácteos podem se contaminar com Bacillus cereus, um micro-organismo que dependendo do número, da estirpe e das condições de processamento e comercialização, além de produzir lipases e proteases causadoras de off-flavor nos produtos, está associado a duas síndromes distintas que atingem os humanos: uma de natureza diarréica causada pela toxinas não hemolítica nhe, hemolítica hbl e citotoxina cytk e outra emética, causada pela toxina denominada de cereulide ces. Avaliou-se a presença de B. cereus no leite pasteurizado, leite em pó, leite UHT, queijo ricota e sobremesas lácteas, bebidas lácteas e leite achocolatado comercializados supermercados da microrregião de Viçosa, Minas Gerais. nos Também, determinou-se a presença de fatores de virulência de B. cereus nos isolados e avaliou-se a produção de enterotoxina diarreica. A ocorrência de B. cereus em diferentes produtos lácteos foi avaliada utilizando métodos quantitavivo e qualitativo. Das 129 amostras analisadas, foi observada a presença de B. cereus em 69 (53,4%) considerando ambos os métodos. As contagens do micro-organismo variaram de 1,30 log UFC/mL no leite pasteurizado até 5,32 log UFC/g na ricota. A presença do B. cereus foi detectada no leite UHT apenas no teste qualitativo. Os 69 isolados que apresentaram caraterísticas fenotípicas de B. cereus foram confirmados como sendo dessa espécie pela técnica de PCR para o gene 16S, e foram classificados como pertencentes a três estirpes diferentes (B. cereus KAVK4, B. cereus SVK1 e B. cereus ATCC 4342), sendo predominante em todos os produtos analisados a estirpe B. cereus KAVK4. Dos 69 isolados analisados para a presença de genes produtores de enterotoxinas, 68 (98 %) apresentaram o gene nhe. Este gene foi expresso nos 68 isolados com a produção de pelo menos 6 ng/mL da nhe, limite mínimo de detecção desta toxina na metodologia utilizada. Dos 69 isolados confirmados como B. cereus, 40 (57 %) apresentam a amplificação do gene hbl, em 30 (75 %) destes constatou-se a produção de pelo menos 20 ng/mL da toxina hbl, limite de detecção do kit utilizado. A presença do gene cytk foi verificada nos 69 isolados analisados. A ocorrência de pelo menos um gene produtor de enterotoxina foi constatada em todos isolados, o que indicaria um alto potencial patogênico das estirpes presentes nos produtos lácteos. / Milk and dairy products may be contaminated with Bacillus cereus, a micro- organism that depending on the number, of the strain and of the processing and marketing conditions can produce lipases and proteases that cause off-flavor in dairy product and it is associated with two distinct syndromes that affect humans: one diarrhea caused by toxins, not hemolytic nhe, hemolytic hbl and cytotoxin k cytk and another emetic caused by toxin, called cereulide. The aim of this study was to evaluate the presence of B. cereus in milk products marketed in the supermarkets of Viçosa city, Minas Gerais, Brazil. Also, it was determined the presence of virulence factors of B. cereus isolates and it was evaluated the diarrheal enterotoxin production. The occurrence of B. cereus in different dairy products was evaluated using quantitavive and qualitative methods. Of the 129 samples analyzed, it was observed the presence of B. cereus in 69 (53.4%) considering both methods. The microorganism counts vary from 1.30 log UFC/ml in milk pasteurized up to 5.32 log UFC/g in the ricotta. The presence of B. cereus was detected in the UHT milk only in qualitative testing. The 69 isolates with phenotypic characteristics of B. cereus were confirmed as this species by PCR for gene 16S, and they were classified as belonging to three different strains (B. cereus KAVK4, B. cereus SVK1 and B. cereus ATCC 4342), being predominant in all the products analyzed strain B. cereus KAVK4. From the 69 isolates analyzed for the presence of enterotoxin producing genes, 68 (98%) had the nhe gene. This gene was expressed in the 68 isolates with the production of at least 6 ng / ml of nhe, minimum detection limit of the methodology of this toxin. From the 69 isolates confirmed as B. cereus, 40 (57%) showed the amplification of the gene hbl, 30 (75%) of them it was found containing at least 20 ng / mL of the hbl toxin, according kit detection limit .The presence of cytk gene was found in 69 isolates analyzed. The occurrence of at least one enterotoxin producing gene was found in all isolates, which would indicate a high potential pathogenic strains present in dairy products.

Orientador: Arnaldo Yoshiteru Kuaye / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T22:35:32Z (GMT). No. of bitstreams: 1 Esper_LucianaMariaRamires_M.pdf: 799385 bytes, checksum: 87dd89627de1603ab2c21bcb0f84f8aa (MD5) Previous issue date: 2006 / Resumo: A ricota é um tipo de queijo fresco, de origem italiana, obtido pela precipitação das proteínas do soro do queijo, por acidificação associada ao calor, cuja produção aumenta a cada ano, justificado em parte pela procura por alimentos mais saudáveis e de baixo valor calórico. O teor de umidade, em geral de 70%, caracteriza a ricota como sendo um alimento de muito alta umidade, o que a torna bastante susceptível à contaminação microbiana, podendo ocasionar doenças de origem alimentar, mesmo sendo submetida à refrigeração. O objetivo desse trabalho foi o de avaliar a qualidade microbiológica e parâmetros físicoquímicos de amostras de ricotas comercializadas no município de Campinas-S.P. A conformidade das informações nutricionais declaradas nos rótulos, com o estabelecido pela RDC nº 360/2003 da ANVISA foi avaliada. Para qualidade microbiológica foi utilizada como referência a Resolução da Diretoria Colegiada nº 12/2001 da Agência Nacional de Vigilância Sanitária, além de pesquisas complementares como a contagem de bolores e leveduras, Bacillus cereus mesófilos e psicrotróficos e sua capacidade de produção de toxinas, a presença de enterotoxinas estafilocócicas na ricota e a produção destas por cepas isoladas de estafilococos, e os fatores de virulência de Listeria monocytogenes isoladas das amostras.Os resultados das análises físico-químicas demonstraram grande variabilidade em todos os parâmetros avaliados entre as amostras e marcas. Particularmente em relação à gordura, segundo a Portaria nº 146/96 do Ministério da Agricultura do Abastecimento e da Reforma Agrária, 8,89% das amostras seriam classificados como queijo magro, 42,22% queijo semi- gordo, 40,00% queijo gordo e 8,89% como queijo extra- gordo. Na maioria dos rótulos as informações nutricionais se apresentavam em desacordo com a legislação (> ±20% de tolerância) sendo 60% em relação à proteína, 60% em relação ao valor energético total e 66,7% em relação à gordura. Estes resultados enfatizam a necessidade do estabelecimento de padrões de identidade para melhor controle da qualidade do produto e segurança do consumidor. Os resultados das análises microbiológicas demonstraram que 46,7% das amostras estavam em desacordo com o padrão estabelecido pela RDC nº 12/2001.O número de amostras acima do permitido pela legislação em relação a coliformes termotolerantes foi de 46,7%, estafilococos coagulase positiva, 2,2% e Listeria monocytogenes, 6,7% . Não foi isolada Salmonella em nenhuma das amostras. Além dos critérios microbiológicos exigidos pela legislação uma avaliação complementar mostrou que 51,1% da amostras estavam contaminadas por B.cereus, sendo que 28,9% com contagens na faixa de 104 a 106 UFC/g; 47,5% contaminadas por bolores e 97,5% por leveduras ambas com elevado nível de contaminação. Embora não tenha sido detectada a presença de toxinas estafilocócicas nas ricotas, 23,64% dos isolados de estafilococos eram produtores de toxinas. Destes, 69,23% eram estafilococos coagulase negativa e 30,77% estafilococos coagulase positiva, evidenciando a importância dos estafilococos coagulase negativa. Quanto ao potencial enterotoxigênico de B. cereus, 85,7% (36/45) dos isolados analisados, foram positivos para o Kit BDE-VIA. Foram identificados 11 perfis toxigênicos na pesquisa de genes codificadores de enteroroxinas pela técnica de PCR, atestando o alto potencial enterotoxigênico dos isolados de B.cereus. Na avaliação da patogenecidade dos isolados de Listeria monocytogenes, 100% apresentaram o gene actA do tipo 4 e hly do tipo 1, classificados portanto, como linhagem do tipo I, esta linhagem encontrada na maior parte dos surtos e casos de listeriose em humanos. O presente trabalho revela que a ricota deveria merecer maior atenção por parte da comunidade científica, setor produtivo e órgãos de vigilância sanitária visando a melhoria da qualidade e conseqüente segurança do consumidor tendo em vista o seu consumo crescente e utilização em dietas especiais / Abstract: Ricotta is a soft white cheese, of Italian origin, obtained from the precipitation of proteins from the whey of cheese through heating associated with acidification. The production of ricotta cheese increases every year, thanks to the widespread search for healthier foods with low caloric value. Ricotta cheese is characterized by its high moisture content (70% in general), which makes it susceptible to microbiological contamination and therefore able to cause food poisoning, even when stored under refrigeration. This work evaluates the microbiological quality and some physical-chemical parameters (pH, titratable acidity, moisture, protein, fat, salt and ash) of commercial samples of ricotta cheese at the local market of Campinas city, in the state of Sao Paulo, Brazil. We have first evaluated the compliance of the nutritional information in labels with that established by the applicable regulation RDC nº 360/2003 of ANVISA and the variation between values declared in the labels and those obtained through laboratorial analyses. In order to determine microbiological quality, the standard RDC nº12/2001 from ANVISA was used as reference, as well as complementary research, including the counting of mold, yeast, mesophilic and psycrotrophic Bacillus cereus and their potential enterotoxin production capacity. In addtion, the presence of staphylococcal enterotoxin in ricotta and the production of enterotoxin by isolated strains of staphylococci, and also the virulence factors of Listeria monocytogenes strains isolated from the samples. The physical-chemical analyses resulted in great variability among the samples of ricotta cheese for all the parameters evaluated. Particularly regarding fat, according to regulation Portaria nº146/96 of MAARA, 8.89% of the samples would be considered fat-free cheese, 42.22% low-fat cheese, 40.00% high-fat cheese and 8.89% as extra high-fat cheese. In most of the labels the nutritional information failed to comply with the regulation (over ±20% tolerance): 60% regarding protein content, 60% regarding total energetic value and 66.7% regarding fat content. Such results stress the need for identity standards to improve quality control of the product and consumer safety. According to the results of the microbiological analyses, 46.7% of the samples failed to meet the standards established by the RDC nº 12/2001 regulation. A great deal of the samples had microbiological content above the levels allowed by the regulation: 46,7%, for thermotolerant coliforms, 2.2% for coagulase-positive staphylococci, and 6,7% for Listeria monocytogenes. Salmonella was not isolated in any of the samples. Besides the microbiological criteria required by the regulation, a complementary evaluation showed that 51.1% of the samples were contaminated by B. cereus, being that 28.9% with countings in the band 104 to 106 UFC/g; also, 47.5% of the samples were contaminated by mold and 97.5% by yeast, both at high contamination levels. Although the presence of staphylococcal enterotoxins was not detected in the ricotta analyzed, 23.64% of the staphylococci isolated strains were toxin producers. Out of these, 69,23 % were coagulase-negative staphylococci and only 30.77% were coagulase-positive staphylococci, what demonstrates the importance of the coagulase-negative staphylococci strains. As for the enterotoxigenic potential of B. cereus, 85.7% (36/45) of the isolated B. cereus strains analyzed were positive for the BDE-VIA Kit. PCR technique was applied to enteroroxin code genes and identified 11 toxigenic profiles, what demonstrates the high enterotoxigenic potential of the isolated B. cereus strains. In the assessment of the pathogenic potential of isolated L. monocytogenes strains, 100% presented the genes actA type 4 and hly type 1, therefore lineage I was found in most of the outbreaks and isolated cases of listeriosis in human beings. This work points out that ricotta cheese should be given greater attention by the scientific community, the productive sector and the food safety agencies aiming at the improvement of the quality of the product and therefore the security of the consumer, inasmuch as ricotta cheese has been increasingly consumed and used in special diets / Mestrado / Mestre em Tecnologia de Alimentos

Orientadores: Arnaldo Yoshiteru Kuaye, Marcelo Palma Sircili / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T20:40:16Z (GMT). No. of bitstreams: 1 Esper_LucianaMariaRamires_D.pdf: 999342 bytes, checksum: 3f9fabfdcb18d73bafce5fd0ce00935e (MD5) Previous issue date: 2010 / Resumo: A contaminação de fórmulas infantis por Enterobacter sakazakii (Cronobacter spp.) e Bacillus cereus pode ter como origem o contato do alimento com biofilmes formados em ambientes, utensílios e equipamentos empregados na sua produção ou posterior reconstituição nos locais de distribuição. A grande preocupação em relação a estas bactérias é a presença das mesmas em fórmulas infantis, produtos estes utilizados como fonte de alimentação para lactentes de forma exclusiva ou em combinação com outros alimentos. A formação de biofilmes, assim como outros mecanismos celulares como por exemplo a produção de bacteriocinas e fatores de virulência, podem ser modulados pelo processo de comunicação célula-célula ou quorum sensing - mecanismo de sinalização célula-célula mediada pelo acúmulo de uma classe ou mais de moléculas sinalizadoras produzidas pela célula e excretadas para o meio externo. Por sua vez, a quebra deste sistema, pela degradação das moléculas sinalizadoras de comunicação, denomina-se quorum quenching. Neste trabalho objetivou-se, primeiramente, a avaliação da dinâmica de formação de biofilmes mono e multi-espécies de E. sakazakii (Cronobacter spp.) e B. cereus em superfície de aço inoxidável utilizando-se como meios de cultivos fórmula Infantil (FI) e caldo Luria Bertani (LB) e a eficácia de soluções de ácido peracético e de hipoclorito de sódio na inativação desses biofilmes. Outro objetivo principal foi pesquisar a ocorrência dos sistemas quorum sensing e quorum quenching em E. sakazakii (Cronobacter spp.) e B. cereus e a possível influência das moléculas sinalizadoras na sensibilidade destas bactérias aos antimicrobianos. A formação de biofilmes ocorreu de forma mais intensa ao utilizar-se a fórmula infantil, quando comparado com o meio de cultivo LB, fato este relevante por ser a fórmula infantil o mais conhecido e importante veículo de transmissão de E. sakazakii (Cronobacter spp.). Tanto em cultivo mono-espécie quanto em cultivo misto, o nível de contagem de E. sakazakii (Cronobacter spp.) superou o de B. cereus e para ambos o maior desenvolvimento ocorreu em cultivos mono-espécie. Em todos os biofilmes de E. sakazakii (Cronobacter spp.) e B. cereus isoladamente e em cultura mista, produzidos em caldo LB e independente do tempo de formação, as contagens foram reduzidas a níveis inferiores a 1 logUFC/cm2 quando em contato com soluções de ácido peracético ([500mg/L]) e hipoclorito de sódio ([100mg/L]) por 15 minutos. No entanto, para os biofilmes produzidos em fórmula infantil ocorreram algumas situações, em que as contagens de microrganismos após o contato com as soluções de sanitizantes, foram superiores a 1 logUFC/cm2, evidenciando assim a ineficácia do procedimento de sanitização para alguns biofilmes formados a 5 dias ou mais e a necessidade da higienização das superfícies ser realizada o mais próximo do término do processamento ou reconstituição destes alimentos. Evidenciou-se a existência dos sistemas quorum sensing e quorum quenching, através dos testes de atividades biológicas das culturas, extratos e suas frações. Os testes com os biossensores revelaram-se positivos para a produção de homoserinas lactonas em cepa de E. sakazakii (Cronobacter spp.) e negativos para a espécie B. cereus. Em cultivo misto de E. sakazakii (Cronobacter spp.) e B. cereus observou-se a redução e/ou a não detecção das homoserinas lactonas fato este possivelmente associado ao fenômeno quorum quenching. A possível presença de moléculas sinalizadoras AI- 2 e AI-3 foi evidenciada, sendo confirmada a presença de moléculas sinalizadoras AI-1. A caracterização do AI-1 realizada por cromatografia gasosa acoplada à espectrometria de massa (CG-EM) revelou a capacidade da cepa de E. sakazakii (Cronobacter spp.) em produzir três compostos, N-heptanoil-HSL, N-dodecanoil- HSL e N-tetradecanoil-HSL, substâncias estas ainda não reportadas na literatura para o microrganismo em estudo. Os autoindutores sintéticos C7-HSL, C12-HSL e C14-HSL adicionados a cultura de E. sakazakii (Cronobacter spp.) e B. cereus não exerceram efeito sobre a sensibilidade aos antimicrobianos, sugerindo que estas moléculas não estariam envolvidas em mecanismos de resistência a estes antimicrobianos. Em resumo este trabalho representa um importante passo no estudo da formação de biofilmes e dos sistemas quorum sensing e quenching para as bactérias em questão, cujos conhecimentos são de grande interesse na segurança dos alimentos / Abstract: The contamination of infant formulas by Enterobacter sakazakii (Cronobacter spp.) and Bacillus cereus can occur due to contact with biofilms formed in the environment and on equipment used in their production and reconstitution. There is concern about these bacteria due to their presence in foods used as a source of nutrition for infants. Biofilm formation as a wide spectrum of important processes is reported to be regulated by quorum sensing, including, for example, antibiotic production and virulence. Cell-to-cell communication or bacterial quorum sensing is a signaling mechanism that refers to the ability of bacteria to respond to chemical molecules called autoinducers, in response to cell density. Degradation of the signaling molecule prevents it from accumulating in sufficient amounts, leading to disruption of the communication system, known as quorum quenching. The aim of this study was first to evaluate the formation of mono and multi-species biofilms of Enterobacter sakazakii (Cronobacter spp.) and B. cereus on stainless steel surfaces using infant formula (FI) and Luria Bertani (LB) broth as the culture media, and the efficacy of sodium hypochlorite and peracetic acid in inactivating these biofilms. The other objective was to investigate the involvement of Enterobacter sakazakii (Cronobacter spp.) and Bacillus cereus in quorum sensing and quorum quenching systems and the possible influence of the signaling molecules on the sensibility of these bacteria to antimicrobials. The formation of biofilms was greater when using the infant formula than the Luria Bertani broth, this fact being important since the infant formula is considered to be an important vehicle in the transmission of Enterobacter sakazakii (Cronobacter spp.) to infants. In both cases, the growth of the mono species was greater than in the multi species culture. In all the biofilms formed in the Luria Bertani broth, independent of the time of formation, the counts were reduced to less than 1 log CFU/cm2 when in contact with sodium hypochlorite ([100mg/L]) or peracetic acid ([500mg/L]). However for the biofilms produced in the infant formula, many situations occurred, with counts more than1 log CFU/cm2 in some situations after the contact with sanitizers highlighting the need for na efficient sanitization of the surfaces as soon as the processing or reconstitution of these foods has finished. The experiments with biosensors provided evidence of the occurrence of quorum sensing and quorum quenching systems using bioassays with the cultures and extracts of the microorganisms under examination. These bioassays were positive for the production of homoserine lactone by E. sakazakii (Cronobacter spp.) but negative for B.cereus. In the mixed culture a reduction and/or non-detection of the homoserine lactone was observed, a fact that could be associated with quorum quenching. A possible presence of the signaling molecules AI-2 and AI-3 was evident and the presence of AI-1 was confirmed. Gas chromatography-mass spectrometry (GC-MS) analyses allowed for a chemical identification of the production of N-heptanoyl-HSL, N-dodecanoyl- HSL and N-tetradecanoyl-HSLby the E. sakazakii (Cronobacter spp.), a fact not previously reported in the literature. The addition of synthetic signaling molecules (N-heptanoyl-HSL, N-dodecanoyl-HSL and N-tetradecanoyl-HSL) had no significant effect on the sensibility of the E. sakazakii (Cronobacter spp.) and B. cereus strains to antimicrobials. In summary this study represents an important step in biofilm's formation, quorum sensing and quorum quenching in these bacteria that are of great interest in food safety / Doutorado / Doutor em Tecnologia de Alimentos

Orientadores: Arnaldo Yoshiteru Kuaye, Dirce Yorika Kabuki / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T05:00:36Z (GMT). No. of bitstreams: 1 Fernandes_MegdaSilva_D.pdf: 2553051 bytes, checksum: ee968bf858cc0b427d8f6b79c37338b7 (MD5) Previous issue date: 2014 / Resumo: Enterococcus faecium e Enterococcus faecalis são espécies de patógenos oportunistas que infectam principalmente imunocomprometidos. Estas espécies são encontradas em produtos lácteos e possuem capacidade de formar biofilme em superfícies que contatam com os alimentos. A sua remoção é muito dependente dos procedimentos de higienização. Os Enterococcus spp. utilizam o sistema de comunicação célula-célula (quorum sensing) para a formação de biofilmes. A formação de biofilme mono e multiespécie, a eficácia dos procedimentos de higienização no controle destes biofilmes e a produção de moléculas sinalizadoras de quorum sensing por cepas de E. faecalis, E. faecium, Bacillus cereus e Listeria monocytogenes foram avaliadas. Os ensaios foram realizados com cupons de aço inoxidável e variando-se a temperatura (7, 25 e 39 °C) e o tempo (0, 1, 2, 4, 6 e 8 dias). Após 1 e 8 dias de contato nas temperaturas de 25 e 39 °C, os cupons foram submetidos a diferentes processos de higienização. Os sanitizantes testados foram: hipoclorito de sódio (0,2%), ácido peracético (0,2%), quaternário de amônio (3,0%) e biguanida (1,0%). A detecção das moléculas sinalizadoras de quorum sensing AI-2 foi realizada através da avaliação do gene luxS e de ensaio biológico de bioluminescência. Nenhum dos micro-organismos avaliados foi capaz de formar biofilmes a 7 ?C. Enterococcus sp. foram capazes de formar biofilmes, com contagens acima de 8 log ufc/cm2 para as temperaturas de 25 e 39 °C após 8 dias de contato. Em cultivo multiespécie, a temperatura 25 °C favoreceu o desenvolvimento do biofilme de L. monocytogenes (contagens acima de 6 log ufc/cm2). Por sua vez, a 39 °C observou-se o efeito negativo no desenvolvimento do biofilme de L. monocytogenes em cultivo misto, com redução significativa nas contagens ao longo do tempo (valores abaixo de 0,4 log ufc/cm2). As contagens de B. cereus, para ambas as temperaturas em diferentes tempos de exposição situaram-se abaixo de 4,1 log ufc/cm2. Em contrapartida, a contagem de esporos de B. cereus evoluiu ao longo do tempo, atingindo contagens em torno de 4,6 log ufc/cm2. A limpeza com tensoativo aniônico complementada por outra etapa (limpeza ácida, limpeza ácida + sanitização ou sanitização) foi capaz de remover os biofilmes mono e multiespécie em todas as condições testadas. O ácido peracético foi o sanitizante mais eficiente e a biguanida o menos eficiente. Todas as cepas de Enterococcus spp. e B. cereus apresentaram o gene luxS e induziram o fenômeno de bioluminescência em Vibrio harveyi BB170, indicando a presença de autoindutores AI-2 / Abstract: Enterococcus faecium and Enteroccus faecalis are opportunistic pathogens species that infect mainly immunocompromised individuals. These species are found in dairy products and are capable of forming biofilms on surfaces that contact with food. Their removal is highly dependent on the cleaning procedures. It is known that enterococci use the cell-cell communication (quorum sensing) to biofilm formation. The formation of mono- and multi-species biofilm, the effectiveness of sanitization procedures to control these biofilms and the production of signaling molecules of quorum sensing (AI-2) by strains of E. faecalis, E. faecium, Bacillus cereus and Listeria monocytogenes were evaluated in this work. The biofilms were grown on stainless steel coupons at various incubation temperatures (7, 25 and 39 °C) and times (0, 1, 2, 4, 6 and 8 days). After 1 and 8 days of contact at 25 and 39 °C, the coupons were subjected to different sanitation procedures: anionic tensioactive cleaning, acid-anionic tensioactive cleaning, sanitization, anionic tensioactive cleaning + sanitization, acidic- anionic tensioactive cleaning + sanitization and chlorinated alkaline cleaning. The sanitizers tested were: sodium hypochlorite (0.2%), peracetic acid (0.2%), quaternary ammonium (3%), and biguanide (1%). The detection of AI-2 molecules was performed by evaluating the luxS gene and biological bioluminescence assay. None of the microorganisms evaluated was able to form biofilms at 7 °C. Enterococcus sp. were able to form biofilms, with counts above 8 log CFU/cm2 for the temperatures of 25 and 39 °C after 8 days of contact. In multi-species culture, the temperature of 25 °C favored the development of L. monocytogenes biofilms (counts above 6 log CFU/cm2). On the other hand, at 39 °C it was observed a negative effect in the development of L. monocytogenes biofilms in mixed culture, with a significant reduction in counts over time (values below 0.4 log CFU/cm2). The counts of B. cereus, for both temperatures at different exposure times were below 4.1 log CFU/cm2. In contrast, the spore counts of B. cereus evolved over time, reaching scores of around 4.6 log CFU/cm2. The anionic tensioactive cleaning complemented by an aditional step (acid cleaning, acid cleaning + sanitization or sanitization) was able to remove mono- and multi-species biofilms in all tested conditions. The peracetic acid was the most effective sanitizer and the less efficient was biguanide. All strains of Enterococcus spp. and B. cereus showed the luxS gene and induced the phenomenon of bioluminescence in Vibrio harveyi BB170, indicating the presence of AI-2 autoinducers / Doutorado / Tecnologia de Alimentos / Doutora em Tecnologia de Alimentos

Bacillus cereus est un pathogène opportuniste à l'origine de deux types de toxi-infections alimentaires classées en syndrome émétique ou diarrhéique. Le syndrome diarrhéique résulte de la production d'entérotoxines (Hbl, Nhe et CytK) au niveau de l'intestin grêle de l'hôte, caractérisé par une atmosphère anaérobie et un faible potentiel d'oxydo-réduction (POR). La capacité de B. cereus à se développer et à produire des entérotoxines dans ces conditions est sous le contrôle de deux systèmes qui agissent, en partie, indépendamment du régulateur pléiotrope connu, PlcR (Phospholipase C Regulator). Il s'agit du système à deux composants ResDE et de la protéine Fnr (Fumarate Nitrate Reductase). Le but de cette étude a été de caractériser d'un point de vue fonctionnel l'implication du régulateur Fnr et du système ResDE dans la toxinogenèse de B. cereus. Les résultats ont montré que la régulation de la transcription de hbl et nhe était sous le contrôle direct et indirect de Fnr et de ResD. En aérobiose, la fixation de Fnr (forme Apo) sur les régions promotrices des gènes de structure des entérotoxines (pnhe et phbl) et des gènes de régulation (presDE, pfnr et pplcR) dépend des conditions redox. L'affinité de ResD pour pnhe, phbl, presDE, pfnr et pplcR dépend des séquences de ces régions promotrices et son affinité pour les régions promotrices presDE et pfnr dépend de son état de phosphorylation. ResD et ApoFnr sont capables de se fixer simultanément sur les régions promotrices étudiées et sont également capables d'interagir physiquement en l'absence d'ADN. Nous avons proposé un modèle de régulation de la toxinogenèse dans lequel ResDE et Fnr pourraient agir en synergie. Enfin des expériences de double hybride ont permis de mettre en évidence que la protéine PlcR pourrait interagir in vivo avec les régulateurs ResD et Fnr. La régulation de la toxinogenèse impliquerait donc la formation d'un complexe multi-moléculaire / Bacillus cereus is an opportunistic pathogen responsible of two types of food-borne diseases, classified as emetic and diarrhoeal syndromes. The diarrhoeal syndrome results from the production of enterotoxins (Hbl, Nhe and CytK) in the host small intestine, which constitutes a high reducing anoxic environment. The ability of B. cereus to produce enterotoxins and grow well in such environment is controlled by two global regulators that may function independently of the pleiotropic virulence regulator PlcR (Phospholipase C Regulator). These two regulators are the two-component system ResDE and the redox regulator Fnr (Fumarate Nitrate Reductase). The aim of this study was to establish the role of Fnr and ResDE in the virulence regulatory pathway of B. cereus. The results showed that transcriptional regulation of hbl and nhe was directly and indirectly controlled by Fnr and ResD. In aerobiosis, Fnr interaction (apo form) with the promoter regions of the enterotoxin structural genes (pnhe and phbl) and the enterotoxin regulator genes (presDE, pfnr and pplcR) depends on its oligomeric state. DNA binding affinity of ResD for pnhe, phbl, presDE, pfnr and pplcR depends on the promoter sequences and affinity for presDE and pfnr depends on its phosphorylation state. ResD and Fnr were found to physically interact and simultaneously bind their target DNAs. We proposed a model for regulation of enterotoxin genes expression in which ResD and Fnr could act synergically. Finally, yeast two-hybride experiments showed that PlcR could physically interact in vivo with Fnr and ResD. Enterotoxin genes expression of B. cereus could thus be controlled through a mechanism including a ternary complex

L’effet de la température et de la composition du milieu en nutriments sur les propriétés des spores (résistance et germination) de B. cereus a été largement étudié contrairement à l'effet de l'anaérobiose. Or, les cellules végétatives de B. cereus peuvent se retrouver dans une grande variété de milieux naturels avec un faible niveau d'oxygène (intestin, sol, lignes de traitement des aliments…) où la sporulation peut avoir lieu. Les spores produites dans ces conditions anaérobies pourraient donc avoir des propriétés particulières. Dans ce travail, un panel de 18 souches de B. cereus appartenant aux groupes phylogénétiques de II à VII a été étudié pour sa capacité à sporuler en anaérobiose dans un milieu de sporulation approprié que nous avons développé (MODS). En anaérobiose, la capacité de sporulation a été plus faible et plus hétérogène qu’en aérobiose. La souche AH187 a produit le niveau de spores le plus important en anaérobiose, elle a donc été choisie pour étudier les propriétés de ces spores. Les spores produites en anaérobiose étaient plus résistantes à la chaleur humide entre 90°C et 100°C, à 1M de NaOH, 1M d'acide nitreux et à la lumière pulsée. Aucune différence dans la résistance à 5 % de peroxyde d'hydrogène ou à 0.25 mM de formaldéhyde, ni aux UV-C, n'a été observée entre les deux conditions. En présence de L- alanine, les spores produites en anaérobiose germaient plus efficacement que celles produites en aérobiose tandis qu’aucune différence dans la germination n’a été observée en présence d'inosine. Aucune différence dans la taille des spores produites dans les deux conditions n’a été observée par microscopie électronique à transmission. Toutefois, les spores obtenues dans des conditions anaérobies avaient un exosporium endommagé ou dans certains cas un exosporium complètement détaché, contrairement aux spores produites dans des conditions aérobies. Afin de comprendre les différences dans la capacité de sporulation de B.cereus entre les 2 conditions, des PCR en temps réel (RT-PCR) ont été utilisées pour étudier l'expression des gènes de l'initiation de la sporulation spo0A, spo0B, spo0F, KinA et kinB. Les cinétiques d'expressions des gènes spo0A, spo0B, spo0F et KinA avaient la même tendance. Ils étaient caractérisés par une expression plus élevée en anaérobiose par rapport à l’aérobiose au début et à la fin de la phase exponentielle de croissance. En outre, l'expression du gène kinB était caractérisée par une augmentation en anaérobiose par rapport à l’aérobiose pour atteindre un pic entre 4 h (milieu de phase exponentielle) et 6 h (début de phase stationnaire) de croissance. Les gènes spo0A, spo0B, spo0F, KinA et kinB sont exprimés de manière différentielle entre l’aérobiose et l’anaérobiose. Ces données pourraient aider à comprendre la différence de capacité de sporulation de B. cereus entre la condition aérobie et anaérobie / The effect of temperature and nutrient composition of the medium on B. cereus spore properties (resistance and germination) has been extensively studied unlike to the effect of anaerobiosis. Nevertheless, B. cereus vegetative cells can be found in a large variety of natural environments with low oxygen level (intestine, soil, food processing line) where sporulation take place. Spores produced in these anaerobic environments could have particular properties. In this work, a panel of B. cereus strains belonging to phylogenetic groups II to VII was studied for their capacity to sporulate in anaerobiosis in an appropriate sporulation medium we developed (MODS). In anaerobiosis, sporulation ability was lower and more heterogeneous than in aerobiosis. The B. cereus AH187 strain produced the highest level of spores in anaerobiosis, it was therefore chosen to study spore properties. Spores produced in anaerobiosis were more resistant to wet heat from 90°C to 100 °C, 1M NaOH, 1M nitrous acid and pulsed light. No difference in resistance to 5 % hydrogen peroxide or 0.25 mM formaldehyde or UV-C was observed between these two conditions. In the presence of L-alanine, spores produced in anaerobiosis germinated more efficiently than spore produced in aerobiosis. No difference in germination was observed with inosine. No difference in the spores size produced in the two conditions was observed by transmission electron microscopy. However, spores obtained under anaerobic conditions had a damaged exosporium, or in some cases a completely detached exosporium, unlike spores produced under aerobic conditions. To understand differences in sporulation ability between both conditions, Real-time reverse transcription-PCR was used to study the expression the expression of sporulation initiation genes spo0A, spo0B, spo0F, kinA and kinB. The kinetics of gene expression spo0A, spo0B, spo0F and kinA had the same trend. They were characterized by a higher expression in anaerobiosis compared to aerobiosis at the beginning and the end of exponential growth phase. Furthermore, kinB gene expression was characterized by an increase in anaerobiosis compared to aerobiosis to achieve a peak between 4 (middle exponential phase) and 6 (early stationary phase) hours of growth. The spo0A, spo0B, spo0F, kinA and kinB genes are differentially expressed between aerobiosis and anaerobiosis. These data may help to understand the difference in B. cereus sporulation capacity between aerobic and anaerobic condition

Bacillus cereus est une bactérie à coloration de Gram positive capable de s'adapter à un grand nombre de stress tels que les bas pH ou les bas potentiels d'oxydo-réduction (Eh). Si certains des mécanismes d'adaptation de B. cereus à chacun de ces stress sont connus, les intéractions entre ces deux facteurs sur la physiologie bactérienne n'ont jamais été étudiés. Nous nous sommes donc intéressés à l'effet des variations de Eh sur la résistance au stress acide de B. cereus, aux adaptations métaboliques de B. cereus à pH acide et à l'intéraction de cette bactérie avec son environnement redox. Les résultats obtenus ont démontré, dans un premier temps, que la survie de B. cereus au choc acide était légèrement augmentée sous atmosphères à Eh réducteurs par rapport aux atmosphères à Eh oxydants. Nous avons également observé une réorientation majeure du métabolisme de B. cereus exposé à pH acide depuis la fermentation des acides mixtes vers la fermentation butanediolique. Cette réorientation joue vraisemblablement un rôle important dans la résistance au stress acide de B. cereus. Dans un second temps, nous avons étudié les intéractions de B. cereus avec son environnement redox et nous avons montré l'importance des groupements thiols exofaciaux dans l'activité réductrice de cette bactérie. L'ensemble de ces résultats permettent de mieux comprendre la physiologie de B. cereus confronté à un stress acide et aux variations de Eh et ouvre la voie à de nombreuses pistes de recherches novatrices. / Bacillus cereus is a Gram positive bacterium able to adapt and survive to numerous stress, including acid stress or oxydo-reduction potential (Eh) variations. Some adaptations are documeted for each of these two stress. However, the interaction between Eh and pH on B. cereus physiology was never studied. Here, we focus on the impact of Eh variation on the acid resistance of B. cereus, on the metabolic adaptation of these bacteria under low pH and on the interaction of bacterial cells with their redox environement. Results obtained demonstrate that the acid survival of B. cereus was slighlty higher under reductive Eh than under oxdative Eh. Concerning acid adaptations, we observed a major metabolic adjustement for cells cultivated at low pH with an important shift from mixed acid fermentation to butanediolic fermentation. Finally, we demonstrate the importance of exofacials thiols groups in the reductive abilities of B. cereus. All these conclusions will help to better understand the response of B. cereus exposed to acid stress and Eh.

Orientadores: Arnaldo Yoshiteru Kuaye, Raquel Monteiro Cordeiro de Azevedo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T01:42:08Z (GMT). No. of bitstreams: 1 Soares_CelinaMara_D.pdf: 3203111 bytes, checksum: 0aa846a0bcf7bb5eb0b55f95d9a7f9ff (MD5) Previous issue date: 2005 / Resumo: Amostras ambientais e de alimentos foram coletadas em dois restaurantes institucionais da cidade de Campinas/SP para a investigação das fontes de contaminação por Bacillus cereus e caracterização do perfil enterotoxigênico dos isolados. A pesquisa foi realizada em três etapas: i) análise da contaminação ambiental geral; ii) diagnóstico da contaminação durante processamentos de pratos cárneos e; iii) estudo do comportamento do microrganismo em substrato cárneo mantido a 10 e 30°C. A avaliação da contaminação ambiental geral e durante os processamentos revelou a presença do microrganismo em 80 (74,8%) das 107 amostras de ar ambiente (2,0 a 38,4 UFC/m³) e em 58 (46,4%) das 125 amostras de superfícies de bancadas e de equipamentos analisadas. O microrganismo também foi identificado em amostras de carne processada (10² UFC/g a 2,4x10³ UFC/g) e de condimentos e temperos (1,0x10 a 5,0x10² UFC/g) coletadas durante os processamentos. A produção de enterotoxinas foi investigada através da técnica da reação da polimerase em cadeia (PCR) para os genes hblA, hblD e hblC (codificadores da "hemolisina BL" - HBL) e para os genes nheA, nheB e nheC (codificadores da "enterotoxina não hemolítica" - NHE). Do total de 124 isolados, 117 (94,3%) foram positivos para ao menos um dos genes pesquisados; 18 (14,5%) foram positivos para os três genes codificadores da HBL; 25 (20,2%) foram positivos para os três genes codificadores da NHE; e 7 (5,6%) foram positivos para todos os genes. Um teste imunoenzimático de detecção da NHE (kit BDE-VIA: Bacillus Diarrhoeal Enterotoxin Visual Immunoassay; Tecra) também foi utilizado. Os resultados obtidos com o BDE-VIA revelaram que 89 (71,8%) dos 124 isolados são produtores de NHE. B. cereus apresentou rápido crescimento em substrato cárneo mantido a 30°C, com tempo de geração entre 28,8 e 36,0 min, indicando que a carne exposta a temperaturas abusivas é propícia ao desenvolvimento do microrganismo. A 10°C, o tempo de geração oscilou entre 10,16 e 28,38 h. A presença de cepas potencialmente enterotoxigênicas observadas em amostras ambientais atestam a importância do ambiente como fonte de contaminação pelo microrganismo. Os resultados da pesquisa permitem concluir que, tanto o controle do microrganismo no ambiente de serviços de alimentação como o adequado processamento dos alimentos, são fundamentais para a redução do risco de doenças de origem alimentar associadas a B. cereus / Abstract: Environmental and food samples were collected in two food services in the city of Campinas/SP for investigation of Bacillus cereus contamination sources and characterization of strains toxin profiles. The research has been made in three steps: i) analysis of general environmental contamination; ii) diagnosis of contamination during processing cooked meat; and iii) study of microorganism behavior in meat substrate stored at inappropriate temperatures. The results indicated the presence of microorganism in 80 (74.8%) of the 107 environmental air samples (2.0 to 38.4 CFU/m³) and in 58 (46.4%) of the 125 samples of equipments and benches surfaces. The microorganism also was identified in cooked meat samples (10² CFU/g to 2.4x10³ CFU/g) and in spices samples (1.0x10 CFU/g to 5.0x10² CFU/g), collected during the processing. The potential of enterotoxin production was investigated using polymerase chain reaction (PCR) methods for genes hblA, hblD e hblC (encoding hemolysin BL) and for genes nheA, nheB and nheC (encoding non-hemolytic enterotoxin - NHE). From all the 124 strains, 117 (94.3%) were positive for at least one gene; 16 (12.9%) were positive for the three HBL encoding genes; 25 (20.2%) were positive for the three NHE encoding genes; and 7 (5.6%) were positive for all genes. The Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDE-VIA; Tecra) also was used for NHE detection. The results obtained with BDE-VIA revealed that 89 (71.8%) from the 124 strains are NHE producers. B. cereus cells showed fast growth in meat substrate stored at 30°C with generation time between 28.8 and 36.0 min, indicating that meat stored at inadequate temperatures is propitious to the microorganism development. At 10°C, the generation time varied between 10.16 and 28.38 h. The presence of potentially enterotoxigenic strains in the environmental samples confirm the importance of environment as a source of microorganism contamination. The results allow to conclude that either the control of microorganism in food services environment either the proper food processing are fundamental to reduce the risks of foodborne diseases associated to B. cereus / Doutorado / Tecnologia de Alimentos / Doutor em Tecnologia de Alimentos