[pt] Uma sonda fotoluminescente do tipo turn-on foi avaliada para a detecção de putrescina usando pontos quânticos de grafeno (GQDs). Diferentes nanopartículas de carbono fotoluminescentes foram preparadas usando a abordagem bottom-up, usando ácido cítrico como precursor, sozinho ou misturado com outros compostos contendo heteroátomos (N e/ou S) visando funcionalização da nanoestrutura. Observou- se que as nanopartículas preparadas com ácido cítrico e tioureia (GQDs-TU) apresentaram melhor perfil aumento da fotoluminescência (resposta analítica) na presença dessa amina biogênica. As condições experimentais foram ajustadas para o melhor perfil de resposta e para obter os parâmetros analíticos de mérito. No ensaio em batelada, a curva analítica normalizada (concentração L-L0 versus PUT) foi linear (R2 = 0,9498) até 90 mg L1 . O limite de quantificação (LOQ) foi de 15,1 mg L-1 e o limite de detecção (LOD) foi de 4,5 mg L-1 . Além disso, a estratégia proposta para a determinação indireta de putrescina foi adaptada para um sistema de análise por injeção em fluxo (FIA) a fim de aumentar a frequência analítica, diminuir os resíduos e automatizar a quantificação do analito. Após a otimização do ensaio, com a faixa linear cobrindo o intervalo até 50 mg L-1 (R2 = 0,9980), os valores de LOD e LOQ foram 3,0 mg L1 e 9,9 mg L-1 , respectivamente. / [en] A photoluminescent turn-on probe was evaluated for putrescine detection using graphene quantum dots (GQDs). Different photoluminescent carbon nanoparticles were prepared using a bottom-up approach, employing citric acid as a precursor, either alone or mixed with other compounds containing heteroatoms (N and/or S) for nanostructure functionalization. It was observed that the nanoparticles prepared with citric acid and thiourea (GQDs-TU) exhibited a more pronounced increase in photoluminescence (analytical response) when exposed to this biogenic amine. Experimental conditions were fine-tuned to optimize the response profile and obtain the analytical parameters of merit. In the batch assay, the normalized analytical curve (L-L0 concentration versus PUT) was linear (R2 = 0,9498) up to 90 mg L-1 . The limit of quantification (LOQ) was 15,1 mg L-1 and the limit of detection (LOD) was 4,5 g L-1 . Furthermore, the proposed strategy for the indirect determination of putrescine was adapted for a flow injection analysis (FIA) system to increase the analytical frequency, reduce waste, and automate analyte quantification. After optimization of the assay, with the linear range covering the range up to 50 mg L-1 (R2 = 0,9980), the LOD and LOQ values were 3,0 mg L-1 e 9,93 mg L-1 , respectively.

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.