Orientadores: José Luiz Pereira, Maristela Nascimento / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T16:21:16Z (GMT). No. of bitstreams: 1 Silva_IvoneFranciscada_M.pdf: 1262965 bytes, checksum: 1fe0878a62acfd4e2e373d7b14691653 (MD5) Previous issue date: 2011 / Resumo: Os microrganismos são extremamente importantes no processamento do cacau, sob dois aspectos. O primeiro é o aspecto tecnológico, porque desempenham um papel fundamental no desenvolvimento do sabor do chocolate. O segundo é o aspecto de saúde pública, porque podem transmitir doenças de origem alimentar. O chocolate já foi incriminado em vários surtos de salmonelose e, em alguns deles, a contaminação por Salmonella originou-se dos ingredientes de cacau. O objetivo do presente estudo foi verificar a ocorrência de enterobactérias, incluindo Salmonella em cada etapa do processamento do cacau até a obtenção do chocolate além de determinar características fisico-químicas como pH e atividade de água. Foram analisadas 364 amostras, obtidas de produtores e processadores de cacau da Bahia e comerciantes de chocolate do Estado de São Paulo, sendo 119 amostras de cacau durante o pré-processamento nas fazendas produtoras; 150 amostras de derivados processados de cacau e 65 amostras comerciais de chocolate. As polpas de cacau apresentaram resultados microbiológicos e físico-químicos de acordo com a legislação vigente. As sementes que chegam do campo, antes de entrarem na fermentação, não apresentaram contaminação por E. coli, o que indica que a matéria prima é uma fonte menos provável de entrada de enteropatógenos, dentre eles Salmonella, no pré-processamento do cacau. Na etapa de fermentação não houve o isolamento de Salmonella, nem foi observada correlação entre a presença ou contagem de E. coli e o tempo de fermentação. Todas as fazendas produtoras apresentaram contaminação por E. coli. Durante a etapa de secagem das amêndoas não foi detectada presença de Salmonella, já a porcentagem de amostras contaminadas por E. coli aumentou em todas as fazendas (40% de amostras contaminadas) com relação a etapa anterior. Nas amêndoas fermentadas e secas foi detectada a presença de Salmonellaem uma amostra, além de ter sido verificado um aumento na porcentagem de amostras contaminadas por E. coli em relação a etapa anterior. Quanto cacau processado, nas amostras de Nibs analisadas houve o isolamento de bactérias do grupo enterobactérias e coliformes totais em 17 e 20% das amostras, mesmo após terem sido submetidos a um processo de torração com temperaturas acima de 110°C. Para os demais produtos foi verificada contaminação de até 7% das amostras analisadas. Em relação ao chocolate as amostras analisadas apresentaram resultados microbiológicos dentro dos padrões estabelecidos pela legislação. Os resultados obtidos nos levam a concluir que: o pré-processamento do cacau é uma etapa onde ocorre a introdução e multiplicação de microrganismos indicadores e patogênicos como a Salmonella, sendo que as etapas de secagem e armazenamento se mostraram as mais críticas; a etapa de processamento também oferece risco de introdução e/ou sobrevivência de microrganismos patogênicos como Salmonella / Abstract: Microorganisms are extremely important in the processing of cocoa for many reasons. First of all is the technological aspect because some microorganims play a key role in the improvement of chocolate flavor. The other aspect is on public health, mainly on the transmission of food-borne diseases. Chocolate has already in several outbreaks of salmonellosis, in some of them Salmonella source was cocoa ingredient. Therefore, the aim of this study was to investigate the occurrence of been Enterobacteriaceae, including Salmonella, throughout cocoa and chocolate chain incremented. Furthermore, physico-chemical properties such as pH and water activity were determined. The amount of 364 samples, obtained from manufacturers of Bahia state and cocoa traders from São Paulo state, were analyzed, including 119 samples of cocoa during the pre-processing on farms, 150 samples of cocoa processed products and 65 samples of commercial chocolate. The results indicated that the microbiological and physical-chemical analyses of the cocoa pulp were according to law. Cocoa sucs before the fermentation process are not. source of Salmonella and E.coli. In the fermentation stage, Salmonella was not isolated, but all farms showed contamination by E. coli, without correlation between the counting of pathogens and fermentation time. In the drying step, Salmonella was not detected as opposed to E. coli counting, that increased 40% in all farms with respect to the previous step. In the storage only one sample showed Salmonella. In Nibs samples were isolated Enterobacteriaceae and total coliforms in respectively 17% and 20% of samples, despite of roasting processing with temperatures above 110ºC. For other products the contamination of 7%. Commercial chocolate samples showed results seconding to the standards established by law.The results lead us to conclude that the pre-processing of cocoa is the stage where there is the introduction and multiplication of indicators microorganisms and Salmonella. The drying and storage were the most critical steps. The processing stages, also offer risk of introduction and survival of pathogens such as Salmonella / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

Orientador: Raphael Lucio Andreatti Filho / Coorientador: Adriano Sakai Okamoto / Banca: Alessandre Hataka / Banca: Ana Angelita Sampaio / Resumo: A microbiota intestinal das aves exerce papel fundamental no desenvolvimento de seu potencial produtivo por garantir o bom funcionamento do trato gastroentérico e do sistema imunológico a ele intimamente ligado. Por isso diversos aditivos já foram utilizados para favorecer o desenvolvimento de uma microbiota saudável, entretanto, ainda não temos informações sobre o perfil desejável para ela. Sendo assim, nos propusemos a utilizar a Eletroforese em Gel com Gradiente de Temperatura (TGGE), para avaliar a microbiota do ceco de aves alimentadas com dietas acrescidas de halquinol, ácido caprílico e Lactobacillus, e posteriormente desafiadas com Salmonella Enteritidis (SE). Aos 10, 20, 30 e 42 dias de vida foram quantificadas as imunoglobulinas IgA, IgM, IgY e interleucinas 17 e 8, avaliada a colonização cecal de SE (UFC/mL), observação histopatológica de tonsila cecal, e esses resulatados relacionados com a microbiota cecal avaliada pelo método TGGE. O tratamento com ácido caprílico foi igualmente eficaz ao halquinol na diminuição da colonização por SE, seguidos do grupo tratado com Lactobacillus. As concentrações de Il-8 e IgM demonstraram relação com a maturação e desenvolvimento imune local e sistêmico das aves, porém, não identificamos relação com o aumento da complexidade da microbiota, entre os diferentes trtamentos e nos grupos desafiados. No presente estudo também n"ao foi possível reproduzir a técnica de TGGE / Abstract: The intestinal microbiota of birds plays a fundamental role in the development of their production potential to ensure the proper functioning of a gastrointestinal tract and immune system closely linked to it. So many additives have been used to encourage the growth of a healthy microbiota, however, we still have no information about the desired profile for her. So we set out to use the Gel Electrophoresis with Temperature Gradient (TGGE) to assess the cecal microbiota of birds fed diets plus halquinol, caprylic acid and Lactobacillus, and subsequently challenged with Salmonella Enteritidis (SE). At 10, 20, 30 and 42 days old were quantified immunoglobulins IgA, IgM, IgY and interleukins 17:08 assessed cecal colonization IF (CFU / ml), cecal tonsil histopathological observation, and those related to resulatados cecal microbiota evaluated by TGGE method. The treatment with caprylic acid was equally effective in reducing the halquinol colonization by IF, followed by the group treated with Lactobacillus. IL-8 and IgM concentrations shown maturation and compared with the local and systemic immune development of the poultry, however, we do not identify relationship with the increased complexity of the microbiota between different trtamentos and challenged groups. In this study it was also not possible to reproduce the TGGE / Mestre

De acordo com a Food and Agriculture Organization of the United Nations (FAO, 2013), o consumo mundial de carne de frango tem aumentado de maneira significativa nas últimas décadas. Por outro lado, a preocupação dos produtores de alimentos, com a inocuidade de seus produtos também tem aumentado na mesma proporção. Durante as etapas da operação de abate de maneira geral, as contaminações cruzadas são as principais causas de disseminação de microrganismos patogênicos nos produtos obtidos e causadores de gastroenterites no consumidor. Espécies da enterobactéria Salmonella se enquadram como um dos maiores riscos desse tipo de doença devido a sua associação com os inúmeros surtos ocorridos a nível mundial, após o consumo desses produtos. Algumas enterobactérias possuem um gene blaKPC, que codifica a enzima carbapenemase, que confere resistência a antibióticos carbapenêmicos, agravando mais a situação. Alguns fatores de virulência encontrados nesse gênero de bactéria podem ainda estar associados a capacidade de adesão e formação de biofilmes em superfícies inertes, dificultando operações de higienização nas linhas de processamento. Assim sendo, a presente pesquisa objetivou a verificação da capacidade de estirpes de Salmonella spp isoladas de aviário e linha de abate de frangos de um frigorífico no estado do Rio Grande do Sul produzirem biofilmes e apresentarem resistência a antibióticos carbapenêmicos. Na avaliação da produção de biofilme, foi empregada a técnica de microplacas de polietileno e produção de cápsula segundo Stepanovic et al. (2004) e Rodrigues et al. (2006), respectivamente.Foram pesquisados os fatores de virulência de salmonelas, representados pelos genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH,e spvC, utilizando o método de Reação de cadeia de Polimerase (PCR), descrita por Borges et al. (2013). As estirpes foram submetidas ao teste de resistência a antibióticos carbapenêmicos pelo método de disco difusão com carbapenêmicos segundo Clinical and Laboratory Standards Institute-CLSI (2010) e pesquisa do gene de resistência a carbapenêmicos blaKCP, pela técnica de PCR, segundo NAAS et al. (2007). Obteve-se quatro perfis genéticos das estirpes de Salmonella spp.: perfil 1: genes ifpA, agfA, invA e avrA; perfil2: genes ifpA, agfA, sefA, invA e avrA; perfil 3: genes invA e avrA; perfil4: genes ifpA,agfA e invA. Observou-se a resistência das estirpes somente ao antibiótico imipenen. Entre as 36 estirpes de Salmonella spp. isoladas, todas foram consideradas produtoras de biofilme in vitro, sendo que 69% destas, apresentaram-se como fortes produtoras, 25% como moderadas, e apenas 6% como fracas produtoras. O método Agar Vermelho Congo não se mostrou eficiente para teste presuntivo de produção de biofilme para estirpes de Salmonella spp. Não foi evidenciado o gene blaKPC nas estirpes de Salmonella spp. isoladas na presente pesquisa. / According to Food and Agriculture Organization of the United Nations (FAO, 2013), the world consumption of meat of chicken has been higher significantly in the last decades. On the other hand, the concern of the food producers with the safety of their products also has increased in the same proportion. During the steps of the slaughter operation in general, cross contamination are the main causes of pathogenic microorganisms dissemination in the obtained products and the causes of gastroenteritis in the consumer. Species of the Enterobacter Salmonella fall as the highest risks of this kind of disease, due to their association with countless outbreaks which occurred worldwide, after the consumption of these products. Some enterobacter have a blaKPC gene, which codes for the carbapenemase enzyme that confers resistance to carbapenems antibiotics, aggravating the situation. Some virulence factors found in this bacteria genes can also be associated to the ability of adherence and the formation of biofilms in inert surfaces, making it difficult the operations of sanitation in the processing lines. Thus, the present research aimed to verify the capacity of Salmonella spp. strains isolated from aviary and slaughter line of chicken in a fridge of Rio Grande do Sul state to produce biofilms and be resistant to carbapenems antibiotics. In the evaluation of biofilm production, it was used the polyethylene microplates and capsule production technique according to Stepanovic et al. (2004) and Rodrigues et al. (2006), respectively. The virulence factors of salmonella were researched, represented by the genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, e spvC, using the Polymerase Chain Reaction (PCR) method described by Borges et al. (2013). The lineages were submitted to the carbapenems antibiotic resistence test by the disk diffusion method with carbapenems according to the Clinical and Laboratory Standards Institute-CLSI (2010), and the research of the resistance to carbapenems gene blaKCP, by the strains Salmonella spp.: profile 1: genes ifpA, agfA, invA and avrA.; profile 2: genes ifpA, agfA, sefA, invA and avrA; profile 3: genes invA and avrA; profile 4: genes ifpA, agfA and invA. It was observed the resistance of the strains only to the imipenen antibiotic. Among the other 36 cultures of Salmonella spp. isolated, all were considered to produce biofilm in vitro, of which 69% were strong producers, 25% moderate, and only 6% were low producers. The method Congo Red Agar was not efficient to the presumptive test of biofilm production for the Salmonella spp. strains. It was not evidenced the gene blaKPC in Salmonella spp. strains isolates in this research.

Publicación a texto completo no autorizada por el autor / Identifica molecularmente los serotipos de cepas sospechosas de Salmonella spp., aisladas mediante protocolos microbiológicos estandarizados, a partir de 272 muestras pareadas de hisopados rectales y vaginales de reproductoras primerizas clínicamente sanas de un criadero del distrito de Pachacamac. El ADN de los aislados sospechosos, evaluados hasta pruebas bioquímicas, es extraído y analizado mediante la técnica de PCR múltiple, para detectar la presencia de los genes inv A, prot6E y fliC específicos para Salmonella spp., Salmonella Enteritidis y Salmonella Typhimurium, respectivamente; encontrándose en 12 cepas aisladas que amplifican al gen inv A; de éstas, 10 (83.3%) amplifican para el gen fliC y ningún aislado amplifica el gen prot6E. Estos resultados confirman que Salmonella Typhimurium es el patógeno predominante en cobayos reproductoras al primer parto en esta crianza comercial. / Tesis

O-Acetylserine Sulfhydrylase (OASS) is a pyridoxal phosphate enzyme that catalyzes the reaction of O-acetyl-Lserine with sulfide to give L-cysteine. OASS is present as two isoforms, designated -A and -B. The kinetic mechanism of OASS-A is well known and there is also much known concerning the acid-base chemistry of the enzyme. However, little is known concerning the location of the rate determining steps, the sequencing of chemical steps that occur at the active site, or the nature of the rate determining transition states. The studies performed to help elucidate these aspects of the OASS-A mechanism included determination of the thermodynamics of both half reactions, along with studies utilizing substrate analogs of OAS halting the reaction at specific points along the reaction pathway allowing the identification of reaction intermediates. The free energy change of the first half reaction was shown to be -5.7 Kcal/mole while the second half reaction was shown to be, for all intents and purposes, irreversible. Intermediates along the reaction pathway that have been previously identified include the internal Schiff base and the a-aminoacrylate. The external Schiff base was identified using the analogs cysteine, alanine, and glycine while the geminal diamine was identified using the analog serine. Formation of the external aldimine was shown to be pH dependent with a pK of 8.1 ± 0.3 most likely representing a general base that accepts a proton from the a-amine of cysteine to facilitate a nucleophilic attack on C4r of the PLP imine. Formation of the geminal diamine was also shown to be pH dependent with two pK values having an average value of 8.1. One of the groups most likely represents the general base which accepts a proton from the a-amine of cysteine while the second group likely interacts with the amino acid side chain to orientate the amino acid into the correct configuration.

O-Acetyl-L-serine sulfhydrylase-A (OASS-A) forms acetate and L-cysteine from O-acetyl-L-serine (OAS) and sulfide. One molecule of the cofactor pyridoxal 5'- phosphate (PLP) is bound in each holoenzyme protomer.

La infección por Salmonella spp., es una de las causas más importantes de gastroenteritis en seres humanos a nivel mundial, constituyendo un problema de salud pública. El proceso de beneficio favorece la diseminación de microorganismos y pueden aumentar la frecuencia de Salmonella en el producto final, teniendo como posibles fuentes de contaminación el agua de lavado de las canales o carcasas, ruptura de vísceras durante el eviscerado o condiciones higiénicas deficientes del establecimiento. En el presente estudio se busco determinar la presencia de Salmonella para ello se analizaron 170 muestras de superficie corporal entre carcasas y canales y 170 muestras de hisopado cloacal. Estas muestras fueron adquiridas de 17 centros de beneficio clandestino de Lima Metropolitana. Para la superficie corporal se utilizó el método de enjuague descrito por el USDA/FSIS (2008). Se utilizó la metodología tradicional para el aislamiento de Salmonella. En los centros de beneficio donde no se realizaba eviscerado el porcentaje de muestras de superficie corporal de carcasas que resultaron positivas a Salmonella spp. fue de 21.3% y de 28.8% para hisopado cloacal; mientras que en los centros de beneficio donde se realizaba eviscerado el 25.6% de muestras de superficie corporal de canales estaban contaminadas y se obtuvo un 35.6% de muestras de hisopado cloacal positivas. El grado de concordancia de los resultados para ambos métodos de toma de muestra fue insignificante (k=0.074, k=0.146). En conclusión los resultados de este estudio muestran que Salmonella spp. está presente en los centros de beneficio clandestino de Lima Metropolitana. -- Palabras clave: Salmonella, canales, beneficio, clandestino, hisopado cloacal, superficie corporal / -- Salmonella spp., is one of the most important causes of gastroenteritis in humans worldwide, constituting a public health problem. The slaughtering process favors the spread of microorganisms and may increase the frequency of Salmonella in the final product. The possible sources of contamination are the wash water of the carcasses, the rupture of the viscera during evisceration and the poor hygiene of the establishment. In this study we sought to determine the presence of Salmonella. For this purpose 170 samples of the body surface of carcasses (among eviscerated and non eviscerated) and 170 cloacal swabs were analyzed. These samples were acquired from 17 slaughterhouses in Metropolitan Lima. For the body surface, we used the method of rinsing described by USDA / FSIS (2008). We used the traditional method for the isolation of Salmonella. In slaughterhouses where the evisceration was not carried out the percentage of samples of body surface carcasses tested positive for Salmonella spp., was 21.3%; and 28.8% for cloacal swabs. While in slaughterhouses where the evisceration was carried out, 25.6% of body surface samples were contaminated and 35.6% of the cloacal swabs were positive. The degree of concordance of the results for both sampling methods was insignificant (k = 0.074, k = 0.146). In conclusion the results of this study show that Salmonella spp. is present in the slaughterhouses of Metropolitan Lima. -- Keywords: Salmonella, chicken carcasses, cloacal swabs, body surface, slaughterhouse / Tesis

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A chemically defined medium for Salmonella growth was developed and optimised using supplements of amino acids, nucleosides, vitamins and different carbon sources. The medium developed was compared to commercially available pre-enrichment media BPW and Salmosyst. Growth of Salmonella was significantly higher in BPW and Salmosyst than the medium developed. The amino acid and nucleoside supplement was directly compared with peptone. The results show peptone to be nutritionally superior and promoting better growth of Salmonella. Three different ELISA assays were used to detect Salmonella growing in four different media. The ELISA assay sensitivity was determined and a degree of media interference with the immunoassays was established. Salmonella culture viability was investigated using three different procedures: differential culturing on selective and non-selective media; fluorescence microscopy with BACLIGHT stained cells and flow cytometry analysis of BACLIGHT and BEP stained cells. Flow cytometry was found to be the most consistent, sensitive and rapid procedure for cell viability measurement. Clusters of viable cells unable to grow on solid media and therefore remaining undetectable by cultural methods were identified using flow cytometry. Severely heat injured Salmonella was used to determine media recoverability. The results indicate that media which contain peptone recover injured Salmonella better than chemically defined or other media. Detection of Salmonella was performed using PCR assay after sample pre-enrichment. The amplification of Salmonella DNA extracted using a crude method resulted in an assay sensitivity of 20 Salmonella cells in pure cultures. The specificity of the oligonucleotide primers employed in the PCR assay was confirmed. Non-salmonella organisms present in high numbers interfered with PCR detection of Salmonella. Food components also interfered with PCR amplification and reduced the assay sensitivity. Interference by food components and non-salmonella DNA was eliminated by the use of a 24 hour pre-enrichment followed by a 3 hour secondary enrichment, a rapid DNA extraction and template preparation. Using this system it was possible to detect 3 Salmonella cells per gram of food in the presence of 106 non-salmonella cells within 28 hours.

No description available.