This thesis deals with the isolation of probiotic DNA from meat products and its assesment by PCR methods. In this thesis is developed homogenization of samples of sausages with kopist, preparation of sausage cells lysates and isolation of DNA by using of magnetic microparticles. The DNA was isolated from sausage lysates by using magnetic microparticles. Isolated DNA was further amplified in genus and spesies-specific PCR methods. In tested products was proven presence of DNA of domain Bacteria, type Lactobacillus and Bifidobacterium. In one product was proven presence of species Lactobacillus acidophilus and Bifidobacterium animalis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to evaluate the effects of partial replacement of pork fat for emulsion containing canola oil in Italian type sausage. Three treatments were developed, including: control (100% pork fat, no fat replacement), T1 (15% fat was replaced by emulsion) and T2 (30% of the fat emulsion was replaced by emulsion). The parameters of pH, water activity, instrumental color, TBA and weight loss of the salami and the quantification of lactic acid bacteria, Staphylococcus coagulase negative, total coliforms, fecal coliforms, Salmonella sp. and Staphylococcus coagulase positive were studied during the processing. After, the fermented sausages were stored for 90 days, being evaluated the same parameters analyzed during processing, with the exception of water activity and weight loss. Furthermore, were analyzed sensory properties, chemical composition and fatty acid profiles of salami. After 28 days of processing, it was found that the incorporation of the emulsion with canola oil did not affect the attributes of pH, water activity and color of the salami, but resulted in a greater amount of TBARS in T2. Higher moisture content and lower weight loss were observed in treatments with the presence of canola oil. On the other hand, during the 90 days of storage was observed that the addition of oil resulted in lower pH values in salami and changes in color of the products, when compared with control. Concerning the oxidative stability, the T1 had the lowest level among the treatments, with values of 1.87 mg MA / kg sample, while the T2 had TBARS values above 4.00 mg MA/kg and the control sample 2,83 mg MA /kg sample. The microbiological analysis performed during the processing and the storage periods showed that the presence of the canola oil did not affect the development of lactic bacteria, in contrast to Staphylococcus coagulase negative that were higher than control. The microbiological safety of the sausage was maintained independent of treatment, since the acidification by fermentation ensured the elimination of total coliforms and non-detection of fecal coliforms, Salmonella sp. and Staphylococcus coagulase positive. There was also that the treatments with addition of canola oil pre-emulsified did not differ in the percentage of protein, but showed differences in fat content compared with control. Moreover, we observed the reduction in the composition of saturated fatty acids and an increase of polyunsaturated fatty acids, and changes in the ratio of polyunsaturated /saturated, which contribute to improve the nutritional profile of the salami. Furthermore, the incorporation of the canola oil emulsion did not alter the sensory properties of the products. Therefore, the substitution of pork fat by canola oil pre-emulsified is a viable alternative for diversification of products, and the substitution of 15% fat resulted in products with better nutritional quality and showed values similar to salami with incorporation of 30% of the canola oil emulsion. / Este trabalho teve por objetivo avaliar os efeitos da substituição parcial da gordura suína por emulsão contendo óleo de canola em salames tipo Italiano. Foram elaborados três tratamentos, entre eles: controle (100% de gordura suína), T1 (15% do toucinho foi substituído pela emulsão) e T2 (30% do toucinho foi substituído pela emulsão). Durante o período de processamento, foram avaliados os parâmetros de pH, atividade de água, cor, TBARS e quebra de peso das peças, além das quantificações de bactérias lácticas, Staphylococcus coagulase negativa, coliformes totais, coliformes fecais, Salmonella sp. e Staphylococcus coagulase positiva. Após o término da fabricação, os embutidos fermentados foram armazenados durante 90 dias em temperatura ambiente, sendo analisados os mesmos parâmetros verificados durante o processamento, com exceção da atividade de água e perda de peso. Além disso, foram analisadas as propriedades sensoriais, a composição química e o perfil de ácidos graxos dos embutidos prontos. Ao final de 28 dias de processamento, verificou-se que a incorporação da emulsão não afetou os atributos de pH, atividade de água e cor dos embutidos, mas resultou em maior valor de TBARS no T2. Além disso, maior umidade e menor perda de peso foram verificadas nos tratamentos com a presença do óleo de canola. Por outro lado, durante os 90 dias de armazenamento, foi observado que a adição do óleo resultou em menores valores de pH nos salames e alterações nos parâmetros que avaliam a coloração dos produtos, quando comparados com o controle. Quanto a estabilidade oxidativa, o T1 apresentou o menor nível entre os tratamentos, com valores de 1,87 mg MA/Kg amostra, enquanto que o T2 teve valores de TBARS acima de 4,00 mg MA/kg amostra e o controle 2,83 mg MA/kg amostra. As análises microbiológicas realizadas durante o período de processamento e estocagem dos embutidos indicaram que a presença do óleo de canola não interferiu no desenvolvimento das bactérias lácticas, ao contrário do Staphylococcus coagulase negativa, que apresentou valor maior que o controle. A segurança microbiológica dos salames foi mantida, independente do tratamento, já que a acidificação do meio garantiu a eliminação dos coliformes totais e a não detecção de coliformes fecais, Salmonella sp. e Staphylococcus coagulase positiva. Verificou-se, também, que os tratamentos com adição de óleo de canola pré-emulsionado não diferiram estatisticamente na porcentagem de proteína, mas apresentaram diferença no teor de gordura, quando comparado com o controle. Foi observada a redução na composição de ácidos graxos saturados e a elevação dos ácidos poli-insaturados, além de alterações na razão de poli-insaturados/saturados, as quais contribuem para a melhoria do perfil nutricional dos embutidos. Além disso, a incorporação da emulsão com óleo de canola não alterou as propriedades sensoriais dos produtos. Dessa forma, a substituição de gordura suína por emulsão com óleo de canola em salame é uma alternativa viável para a diversificação de produtos, sendo que a substituição de 15% da gordura resultou em produtos com melhor qualidade e apresentou valores nutricionais semelhantes ao embutido elaborado com a incorporação de 30% de emulsão com óleo de canola.

Los embutidos fermentados ligeramente acidificados son un grupo de productos tradicionales mediterráneos, caracterizados por un pH superior a 5,3.Para un control eficiente de la seguridad microbiológica de los embutidos se necesitan técnicas rápidas para la identificación y recuento de los microorganismos patógenos a estudiar. En el presente trabajo, se desarrolló una técnica para la enumeración de L. monocytogenes que combinó el método del número más probable y la identificación mediante PCR específica. Para la detección de Salmonella spp. y L. monocytogenes se desarrolló un sistema de PCR-multiplex que permitió la identificación de ambos patógenos de forma simultánea en una sola reacción.El estudio de la calidad microbiológica de los embutidos fermentados ligeramente acidificados se completó con la caracterización de las comunidades microbianas más importantes en estos productos. Se identificaron a nivel de especie los aislados de bacterias del ácido láctico (BAL), de enterococos y de cocos gram-positivos catalasa-positivos (CGC+). Posteriormente se realizó una tipificación molecular de los mismos mediante RAPD y análisis del perfil plasmídico y se estudiaron las principales características de interés higiénico-sanitario y tecnológico de las cepas.Mediante PCR se identificó Lactobacillus sakei como la especie predominante (74%), seguida por Lactobacillus curvatus (21,2%). La actividad aminoácido-descarboxilasa se asoció a la especie L. curvatus (el 66% de los aislados presentaron esta actividad).La identificación de los enterococos se realizó mediante PCR-multiplex y por secuenciación del gen sodA. Enterococcus faecium fue la especie de enterococos predominante (51,9%) seguida por Enterococcus faecalis (14,2%).Todas las cepas de E. faecalis presentaron genes asociados a factores de virulencia. E. faecalis presentó mayor resistencia a antibióticos que el resto de las especies de enterococos estudiadas. Tan sólo una cepa de E. faecium presentó el genotipo vanA (que confiere resistencia de alto nivel a la vancomicina).La identificación de los aislados de CGC+ (mediante PCR específica y amplificación de la región intergénica 16S-23S ARNr) demostró que Staphylococcus xylosus es la especie predominante en los embutidos fermentados ligeramente acidificados (80,8%).La amina biógena más común en los CGC+ fue la feniletilamina, producida por un 10,8% de aislados. Un pequeño porcentaje de aislados fueron mecA+ (4,6%), presentando además resistencia a múltiples antibióticos. El potencial enterotoxigénico de las cepas de CGC+ fue muy reducido (3,3% de los aislados), detectándose únicamente el gen entC.El estudio pormenorizado de las comunidades bacterianas de interés permitió la selección de 2 cepas de L. sakei y 2 cepas de S. xylosus con características tecnológicas e higiénico-sanitarias óptimas. Para evaluar su efectividad como cultivos iniciadores se elaboraron dos tipos de embutidos ligeramente ácidos, chorizo y fuet, inoculados con microorganismos patógenos (Salmonella spp., L. monocytogenes y S. aureus). El uso de cultivos iniciadores permitió el control de L. monocytogenes, Enterobacteriaceae y Enterococcus así como del contenido en aminas biógenas. Los recuentos de Salmonella spp. disminuyeron de forma significante durante la maduración de los embutidos, independientemente del uso de cultivos iniciadores. El uso del tratamiento de alta presión (400 MPa) en los embutidos madurados consiguió la ausencia de Salmonella spp. en los lotes tratados. / Low-acid fermented meat products (final pH, 5.3 to 6.2) are a group of traditional Mediterranean products with a great diversity within the regions.To control the microbial quality of this kind of sausages is necessary to use rapid methods able to produce results quickly and reliably. In this study, a highly sensitive PCR-based method to detect and quantify L. monocytogenes in fermented sausages was developed. This method combined the high sensitivity of the most-probable-number method with a L. monocytogenes specific PCR assay. Also a multiplex-PCR based method for the simultaneous identification of L. monocytogenes and Salmonella spp. was designed. The study of the microbial quality of the slightly fermented sausages was followed by the characterization of the microbial communities of this kind of products. Lactic acid bacteria (LAB), enterococci and Gram-positive catalase-positive cocci (GCC+) were identified at species level. RAPD-PCR and plasmid profiling were used to evaluate the genetic diversity within species and to identify identical isolates of the same strain. Safety and hygienic properties were also studied in order to characterize the isolates in detail. With this aim, bacteriocin production, biogenic amine production and antibiotic susceptibility were determined. By species-specific PCR, Lactobacillus sakei was identified as the predominant LAB (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). The production of biogenic amines was mainly related to the species L. curvatus (66% of the isolates were biogenic amine-producers).Species-specific PCR and partial sequencing of sodA gene were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species (51.9%) followed by Enterococcus faecalis (14,2%). All the E. faecalis strains carried virulence genes. E. faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides).Species-specific PCR and amplification of the 16S-23S rDNA intergenic region were used to identify GCC+ population. Staphylococcus xylosus was the predominant species (80.8%) in this kind of sausages.Tyramine was the most intense biogenic amine produced, although by only 4.6% of the GCC+ isolates. Phenylethylamine was more frequently detected (10.8% of isolates) but at lower levels. A low percentage of isolates (4.6%) showed mecA genes displaying also resistance to multiple antibiotics. Only 3.3% of isolates showed staphylococcal enterotoxins genes, all identified as entC gene.The study of the safety and technological properties of the isolates allowed to select 2 strains of L. sakei and 2 strains of S. xylosus on the basis of their technological and safety characteristics. To evaluate their suitability as starter cultures two types of low acid fermented sausages, fuet and chorizo, were manufactured. Batters were inoculated with L. monocytogenes, S. enterica and S. aureus. Starter cultures were able to control the growth of L. monocytogenes, Enterobacteriaceae, Enterococcus and the biogenic amine content. Salmonella spp counts decreased significantly during ripening independently of the use of starter culture and product.High hydrostatic pressure treatment was necessary to assure absence of Salmonella spp. in final products.