Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-17T20:36:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_VivianePedrosoFausto.pdf: 2096881 bytes, checksum: c58d1ae11d22b250146714c3cf084b16 (MD5) / Made available in DSpace on 2018-08-17T20:36:49Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_VivianePedrosoFausto.pdf: 2096881 bytes, checksum: c58d1ae11d22b250146714c3cf084b16 (MD5) Previous issue date: 2017-04-28 / Listeria monocytogenes is a food-borne bacterium that causes an infection called listeriosis, Although rare, this infection can be lethal for immunocompromised, pregnant, and stillborn. The objective of this work was to develop, characterize, evaluate the stability, anti-Listeria monocytogenes activity, cytotoxicity and genotoxicity of nanostructures (nanocapsules and nanoemulsion) containing Geranium essential oil (OEG), as well as verify synergistic effects with the essential oils of Cinnamon, Lemongrass And Thyme. The characterization of the essential oils was performed by GCMS. The nanocapsules (NC) were prepared by the interfacial deposition method of the preformed polymer while Geranium nanoemulsion (NEG) was prepared by homogenization using an Ultra-Turrax T18. The analysis of the stability indicators was performed by the evaluation of particle size, polydispersity index, zeta potential and pH for 60 days. The bactericidal effect of the different concentrations of the essential oils, pure or in combination and the synergism were determined by the serial microdilution technique, the cytotoxicity was determined by the MTT assays and determination of the LDH in VERO cells and the genotoxicity was evaluated by the GEMO test. Results: The NCGs produced had mean nanometer diameters of (232.6 nm), polydispersity indices below 0.053, pH 3.5 and zeta potentials of about -20.7 mV. The study demonstrated that the stability of the formulations is temperature dependent, remaining more stable at refrigeration temperatures (4 °C) or at stable temperatures (25 ° C). The NEG had mean nanometer diameters of (136.2.6 nm), polydispersity indices below 0.039, pH 3.4 and zeta potentials of about -8.1 mV. The MICs obtained for Geranium, Cinnamon, Campi-lemon and Thyme oils were 10975 mg / ml, 0.05 mg / ml, 0.18 mg / ml and 0.35 mg / ml, respectively. The CIM of NEG was 0.0625 mg / ml and the nanocapsules did not demonstrate antimicrobial activity in the test performed. It was possible to observe at least one point of synergism between the OE of geraniums and the tested EOs.the nanostructures did not show synergistic effect with OE. In the MTT assay OEG and NEG showed increased cell viability at concentrations of 10, 100 and 1000 μg / μl as compared to the control, the LDH assay shows that both OEG and NEG at concentrations of 0,1, 1, 10, 100 and 1000 μg / μl were not cytotoxic. In the GEMO test the OEG had a genoprotective effect on lymphocytes at concentrations of 10, 100 and 1000 μg / μl. The study demonstrated that both OG and NEG do not present tocicity 7 and genotoxicity but present very high MIC values to be used in food, and the synergism and nanostructuring were a good alternative to reduce the amount of oil required for an antimicrobial response. / A Listeria monocytogenes é um microrganismo contaminante de alimentos causador da listeriose, uma infecção que embora rara pode ser letal para indivíduos imunodeficientes, gestantes e neonatos. O objetivo deste trabalho foi desenvolver, nanoestruturas contendo óleo essencial de Gerânio (OEG), caracterizar, avaliar a atividade contra L. monocytogenes, a cito e genotoxicidade, bem como verificar efeitos sinérgicos com os óleos essenciais de Canela, Capim-limão e Tomilho. A caracterização dos óleos essenciais foi realizada por GC-MS. As nanocápsulas (NC) foram preparadas pelo método de deposição interfacial do polímero pré-formado enquanto a nanoemulsão de Gerânio (NEG) foi preparada por homogeneização utilizando um T18 Ultra-Turrax. A análise dos indicadores de estabilidade foi realizada pela avaliação do tamanho de partícula, índice de polidispersão, potencial zeta e pH por 60 dias. O efeito bactericida das diferentes concentrações dos óleos essenciais e o sinergismo foram determinados pela técnica de microdiluição seriada, a citotoxicidade foi determinada pelos ensaios de MTT e LDH e a genotoxicidade foi avaliada pelo teste GEMO. Resultados: As NCG produzidas apresentaram diâmetros nanométricos médios de (232,6 nm), índices de polidispersão abaixo de 0,053, pH de 3,5 e potenciais zeta de cerca de -20,7 mV. O estudo demonstrou que a estabilidade das formulações são mais estáveis em temperatura ambiente (25 ºC). A NEG apresentou diâmetros nanométricos médio de (136,2 nm), índices de polidispersão abaixo de 0,039, pH de 3,4 e potenciais zeta de cerca de -8,1 mV. As CIM obtidas para os óleos de Gerânio, Canela, Campi-limão e Tomilho foram de 10975 mg/ml, 0,05 mg/ml, 0,18 mg/ml e 0,35 mg/ml respectivamente. A CIM da NEG foi de 0,0625 mg/ml e as nanocápsulas não demonstraram atividade antimicrobiana no teste realizado. Foi possível observar pelo menos um ponto de sinergismo entre o OEG os OE testados. As nanoestruturas não apresentaram efeito sinérgico com os OE. No ensaio de MTT o OEG e a NEG apresentaram um aumento da viabilidade celular nas concentrações de 10, 100 e 1000 μg/μl quando comparado com o controle. O ensaio de LDH mostrou que tanto o OEG quanto a NEG nas concentrações de 0,1, 1, 10, 100 e 1000 μg/μl não foram citotóxicas. No teste GEMO o OEG apresentou um efeito genoprotetor sobre os linfócitos nas concentrações de 10, 100 e 1000 μg/μl. O presente estudo demonstrou que tanto o OEG quanto a NEG não apresentaram toxicidade e genotoxicidade porém apresentam valores de CIM muito altos para poder serem utilizados em alimentos, podendo ser o sinergismo e a nanoestruturação uma boa alternativa para reduzir a quantidade de óleo necessária para uma resposta antimicrobiana.

Laticínios são facilmente susceptíveis à contaminação por L. monocytogenes, que pode vir a formar biofilmes nos equipamentos e utensílios e se tornar reservatório de uma recontaminação de produtos lácteos pasteurizados. O objetivo do trabalho foi analisar a formação de biofilmes por L. monocytogenes em diferentes condições encontradas em laticínios como temperatura, tempo e presença de E. coli, assim como avaliar a eficiência do processo de limpeza utilizado, Clean in Place (CIP), frente a esses biofilmes formados. Para isso, foi utilizado um modelo experimental com cupons de aço inoxidável da mesma especificação do pasteurizador de leite. Os cupons foram imersos em um béquer contendo leite UHT integral e TSB-YE, contaminados artificialmente com uma suspensão de L. monocytogenes. Os cupons permaneceram durante um período de dez horas sob agitação constante a temperatura de 5 e 35ºC, visando a aderência das cepas na superfície, seguida de incubação em diferentes tempos (18 e 114 horas) e temperaturas (5 e 35ºC) para a formação do biofilme, seguidos do processo CIP. As populações bacterianas dos biofilmes foram avaliadas por amostragem por suabe e por microscopia eletrônica de varredura (MEV). A formação de biofilme por L. monocytogenes foi significativamente influenciada pela temperatura, sendo que a de 5°C prolongou sua sobrevivência sobre aço inoxidável, provavelmente pela formação de polímeros extracelulares, produzidos em maior número a esta temperatura. Portanto a refrigeração não deve ser usada como única forma de prevenção da contaminação dos alimentos. O substrato e a presença de E. coli não influenciaram na formação de biofilmes pela L. monocytogenes. Em todas as condições estudadas, o processo de limpeza CIP foi eficiente para remover as células de L. monocytogenes a níveis não detectados pelo método suabe ou tornando essas células inviáveis. / Dairy industries are easily susceptible to contamination by L. monocytogenes, which could form biofilms in equipments and utensils and become a source of recontamination of pasteurized dairy products. The objective of this work was to analyze the formation of biofilms by L. monocytogenes at different conditions found in dairy industries such as temperature, time and presence of E. coli, as well as to evaluate the efficiency of the cleaning process used called Clean in Place (CIP) against the biofilms formed. To do so, an experimental model with stainless steel coupons of the same specification as the milk pasteurizer was used. The coupons were immersed in a glass flask containing whole UHT milk and Tryptic Soy BOH + 0.6% Yeast Extract (TSB-YE), artificially contaminated with a suspension of L. monocytogenes. The coupons remained for a period of ten hours under constant agitation at temperature of 5 and 35oC, aiming at the adherence of the strains on the surface, followed by incubation at different times (18 and 114 hours) and temperatures (5 and 35oC) for the formation of the biofilm, followed by CIP process. The bacterial populations of biofilms were evaluated by sampling through swab and scanning electron microscopy (SEM). The formation of biofilms by L. monocytogenes was significantly influenced by temperature, and temperature of 5°C prolonged their survival on stainless steel, probably due to the formation of extracellular polymers, produced in greater numbers at this temperature. Therefore, refrigeration should not be used as the only way to prevent food contamination. The substrate and the presence of E. coli did not influence the formation of biofilms by L. monocytogenes. In all conditions studied, the CIP cleaning process was efficient to remove L. monocytogenes cells at levels not detected through the swab method or making these cells unviable.

Submitted in fulfilment of the requirements for the degree of Master of Applied Science in Food Science and Technology, Durban University of Technology, 2015. / Listeria monocytogenes have been implicated as a public health concern worldwide. The study explored the survival of non-adapted, heat adapted and chlorine adapted L. monocytogenes on tomatoes; as well as the survival of non-adapted, heat adapted and chlorine adapted biofilms after exposure to sodium dodecyl sulphate (SDS), levulinc acid, sodium hypochlorite solution. Contact time of 1, 3 and 5 minutes was used. The survival of L. monocytogenes was monitored at 0, 24, 48 and 72 hours. The sanitizers were used individually or combined as follows; 1% sodium dodecyl sulphate individually; 0.5% levulinic acid individually; 200 ppm sodium hypochlorite solution individually and 0.5% levulinic acid/0.05% sodium dodecyl sulphate in combination (mixture). The samples were kept at 4 °C throughout the period of assessment. The effect of these sanitizers on pH, total soluble solids (TSS) and titratable acidity (TA) was also determined. Furthermore, the attachment of L. monocytogenes on tomatoes was investigated using a scanning electron microscope. Highest log reduction of non-adapted L. monocytogenes were observed on tomatoes treated with 1% SDS and least log reduction was achieved when tomatoes were treated with sodium hypochlorite solution. Though the log reduction achieved by 0.5% levulinic acid was higher that sodium hypochlorite solution, it was lower than log reduction achieved when 0.05% SDS / 0.5% levulinic acid mixture was used for all contact times. Using non-adapted L. monocytogenes, SDS was able to destroy all L. monocytogenes at 1, 3 and 5 minutes contact time. The trend was the same when heat adapted and chlorine adapted L. monocytogenes were used. There was no significant log reduction observed with biofilms. More favourable results were observed as contact time was increased from 1 to 5 minutes. Though there was a decrease in surviving bacteria from 1 to 3 minutes contact time, this decrease was not significant. The study investigated if exposure to sanitizer has an effect on pH, titratable acidity (TA) and total soluble solids (TSS) of the tomatoes. It was revealed that levulinic acid and mixture can have detrimental effect on pH, TA and TSS of tomatoes. The TA and TSS of samples treated with levulinic acid and mixture varied significantly (P ≤ 0.05) compared to the control sample. Although the TA and TSS of samples treated with SDS and sodium hypochlorite solution were different from the control, the differences were not significant. As much as sanitizers have the potential to reduce the bacterial population in fresh produce they may not completely destroy pathogens. Chlorine based sanitizers such as sodium hypochlorite though frequently used in the fresh produce industry, are not the best sanitizer to be used against food borne pathogens. Other sanitizers such as SDS used alone or in combination with another sanitizer can achieve better results than the widely used sodium hypochlorite solution as observed in this study. Stress adapted pathogens become less responsive to sanitizers during subsequent treatments. Through this research, it was established that biofilms are resistant to sanitizers. Though application of sanitizers in fresh produce is cheaper and simpler to apply, there is need to monitor varying concentrations of sanitizers, contact time and minimise contact with sub-surfaces as this could lead to sensory quality losses.

In this project the factors influencing the stability and antimicrobial activity of allyl isothiocyanate (AITC) against Campylobacter jejuni, Salmonella or Listeria monocytogenes as well as factors that enhance sinigrin (glucosinolate in Oriental mustard) hydrolysis by these pathogens were investigated. The minimum inhibitory concentration (MIC) of AITC against 5 strains of each of Salmonella or L. monocytogenes, ranged from 60-100 ppm at 37 ºC. This was reduced to 10-40 ppm at 21 ºC and a further reduction to 5-10 ppm against strains of L. monocytogenes was observed at 4 ºC. This was attributed to greater stability of AITC as temperature was decreased. C. jejuni strains were more susceptible to AITC with MICs of 0.63-1.25 ppm and 2.5-5 ppm at 37 and 42 ºC, respectively. AITC was more inhibitory at ≤ 21 ºC against Salmonella with acidic pH or against L. monocytogenes with neutral pH. C. jejuni, Salmonella and L. monocytogenes strains and mixtures had the ability to degrade sinigrin to form inhibitory concentrations of AITC, and sinigrin hydrolysis was significantly enhanced by higher incubation temperature (21 ºC > 10 ºC > 4 ºC), the presence of 10 mM ferric or ferrous irons, and the presence of < 0.25% glucose. This project also investigated the antimicrobial activity of AITC or Oriental mustard extract alone or combined with ethylenediamine tetraacetic acid (EDTA), malic acid and acetic acid in edible antimicrobial coatings against C. jejuni and Salmonella on fresh, refrigerated, vacuum-packed chicken breasts or L. monocytogenes on refrigerated, cured roast chicken. Malic acid improved the antimicrobial activity of Oriental mustard extract against L. monocytogenes, while EDTA improved its activity against Salmonella. Incorporation of 25 to 50 µl/g AITC or 100 to 250 mg/g Oriental mustard extract in 0.5%κ-carrageenan/2%chitosan coatings, prepared using 1.5% malic or acetic acid, reduced L. monocytogenes on cooked, cured, vacuum-packed chicken slices 4.2 to > 7.0 log10 CFU/g, compared to uncoated chicken by 70 d at 4 ºC. In addition, 0.2%κ-carrageenan/2%chitosan coatings (prepared using a 1% acetic acid solution) containing 250 mg/g mustard extract or 50 µl/g AITC reduced Salmonella numbers on vacuum-packed chicken breasts 3.0 log10 CFU/g by 21 d at 4 ºC. Further, 0.2%κ-carrageenan/2%chitosan coatings containing 50 or 100 µl/g AITC reduced numbers of C. jejuni on fresh, vacuum-packed chicken breasts > 5.0 log10 CFU/g (C. jejuni cells were not detected) after 5 d storage at 4 ºC, while coatings containing 200 to 300 mg/g Oriental mustard extract or 25 µl/g AITC reduced C. jejuni numbers by 3.6 to 4.6 log10 CFU/g. Numbers of lactic acid and aerobic bacteria on poultry meat products were significantly reduced by the coatings. It is clear that κ-carrageenan/chitosan coatings containing either AITC, mustard extract alone or combined with EDTA, malic or acetic acid significantly reduced C. jejuni and Salmonella on fresh, refrigerated, vacuum-packed chicken breasts and L. monocytogenes on refrigerated, cured roast chicken, and consequently enhanced their safety.

Made available in DSpace on 2014-08-20T13:42:09Z (GMT). No. of bitstreams: 1 Dissertacao_Valmor_Lansini.pdf: 2358701 bytes, checksum: d9bedf4bfd9b299f9a0da63f2f19c198 (MD5) Previous issue date: 2010-11-12 / Broiler production chain is very complex, and when compared with the chain of other domestic animals it has a much shorter cycle for obtainment of the final product (chicken meat and its derivatives), which like any other product from animal origin can be carrier of Foodbourne disease (FBD) causing agents. All stages of a production chain are important to obtain safe food, but some of them, due to their nature and complexity, need special care as they can compromise the product in a definite way, making it impossible to control the risks in the subsequent steps within the processing stages. The aim of this work was to evaluate the efficiency of TIMSEN® sanitizer (N-alkyl dimethyl benzyl ammonium 40% - Stabilized Urea 60%) when added to scalding and pre-chilling water in a poultry abattoir with official inspection in the South of Rio Grande do Sul. Scalding and pre-chilling water, as well as broiler carcasses after scalding and after chilling were sampled for evaluating the presence of Listeria monocytogenes, thermotolerant coliforms and for counting aerobic mesophilic microorganisms. TIMSEN® was efficient in the L. monocytogenes control when added to the scalding as well as pre-chilling water. It was also effective in the reduction of thermotolerant coliforms and aerobic mesophilic microorganisms. / A cadeia de produção de frangos de corte é bastante complexa, e quando comparada com as cadeias das outras espécies de animais domésticos, possui um ciclo muito mais curto para obtenção do alimento final (carne de frango e seus derivados), os quais como qualquer produto de origem animal, podem ser veículos de agentes causadores de Enfermidades Transmitidas por Alimentos (ETA). Todas as etapas de uma cadeia de produção são importantes para obtenção de um alimento seguro, mas algumas delas, pela sua natureza e complexidade, exigem cuidados maiores e especiais, pois podem comprometer o produto de forma definitiva não sendo mais possível diminuir os riscos em etapas seguintes dentro do processamento. Com este trabalho objetivou-se avaliar a eficiência do sanitizante TIMSEN® (N-alquil dimetil benzil amônio 40% - Uréia estabilizada 60%), quando adicionado na água de escaldagem e de pré-resfriamento de frangos em abatedouros com inspeção oficial no sul do Rio Grande do Sul. Foram amostradas a água de escaldagem e de pré-resfriamento, bem como carcaças de frangos, tanto após a escalda quanto após o resfriamento, avaliando-se a presença de Listeria monocytogenes, a enumeração de Coliformes termotolerantes e a contagem de microrganismos mesófilos aeróbios. Verificou-se que o produto TIMSEN® foi eficiente, tanto na água de escaldagem quanto na água de pré-resfriamento, no controle de L. monocytogenes, bem como foi efetivo na redução de coliformes termotolerantes e de microrganismos mesófilos aeróbios.

Listeria monocytogenes continues to challenge the dairy industry in causing post-process contamination of cheeses. To reduce risk of contamination, it is crucial to understand the growth and survival of pathogenic bacteria in cheese products and to develop post-process mitigation strategies. This study evaluated the fate of pathogens in reduced and regular sodium Mozzarella cheese, and the potential of Supercritical Fluid Extraction with CO2 (SFE) to reduce Listeria innocua on Mozzarella and Queso Fresco. The survival of L. monocytogenes, Salmonella, and E.coli O157:H7 (2-3 log CFU/g) in reduced sodium Mozzarella (1.62%), compared to regular sodium Mozzarella cheese (2.15%) at 4ºC and 12ºC for 90 and 30 days, respectively, was evaluated. Salmonella and E. coli O157:H7 populations decreased over incubation time at both temperatures and no difference (pListeria monocytogenes population also decreased during incubation time at 4°C regardless of the sodium concentration in Mozzarella cheese. However, there was a difference in the population of L. monocytogenes for regular and reduced sodium incubated 12°C, and its populations increased 1 log CFU/g in reduced sodium Mozzarella cheese. Additionally, this study determined the bactericidal effect of SFE on the population of L. innocua, a surrogate for L. monocytogenes, in Mozzarella and Queso Fresco cheese (6 log CFU/g) treated with SFE at two pressures and temperatures (120 bar at 40°C and 150 bar at 50°C) for 30 min. SFE treatment at 120 bar, 40°C for 30 min decreased L. innocua by approximately 3.0 and 3.5 log CFU/g in Mozzarella and Queso Fresco cheeses, respectively. SFE at 150 bar and 50°C reduced L. innocua by approximately 3.78 and 5.2 log CFU/g in Mozzarella and Queso Fresco cheeses, respectively. Since SFE had a minimal effect on the physico-chemical characteristics of the cheeses assayed, the results suggest SFE might be used to reduce L. monocytogenes in cheeses without negatively impacting product quality.

[ES] La seguridad alimentaria es una prioridad para la población y en la actualidad cobra mayor importancia por ciertas tendencias alimentarias como el consumo de alimentos crudos y la distribución generalizada de alimentos orgánicos, que pueden ser la causa de enfermedades transmitidas por alimentos. Para garantizar la seguridad alimentaria, la detección de estos microorganismos debe realizarse de manera rápida y eficiente. Par eso, el método de cultivo microbiológico se considera el oficial para la detección de estos patógenos. Sin embargo, adolece de importantes inconvenientes, ya que no solo requiere mucho tiempo, sino que también es laborioso y consume muchos recursos. Además, puede ser limitado con respecto a la detección de bacterias fisiológicamente alteradas y/o estresadas durante el almacenamiento y la conservación. En este trabajo se ha desarrollado un protocolo sencillo y rápido para la detección simultánea de E. coli, L. monocytogenes, S. aureus y S. enterica en alimentos, mediante la combinación de una etapa de co-cultivo en medio líquido y la detección por PCR múltiple. Se ha evaluado la eficiencia de varios medios de enriquecimiento y se seleccionó el agua de peptona tamponada como el medio óptimo para el co-cultivo de las cuatro bacterias diana. También se optimizaron las condiciones de PCR múltiple y se aplicaron tanto a co-cultivos como a muestras de alimentos inoculados artificialmente, lechuga orgánica y carne picada. Después de la optimización, la PCR múltiple desarrollada fue capaz de detectar las cuatro bacterias simultáneamente, hasta con una inoculación inicial de 10^0 UFC/mL. En presencia de ambas matrices alimentarias inoculadas, tras la etapa de co-cultivo, la PCR múltiple pudo detectar simultáneamente las 3 bacterias E. coli, S. enterica y L. monocytogenes, mientras que S. aureus se ha detectado por PCR simplex, a partir del mismo extracto de ADN del co-cultivo. Los resultados obtenidos permiten concluir que el uso de un paso de co-cultivo en Agua peptona tamponada, antes de la detección por PCR simple y múltiple, puede facilitar la detección simultánea de las cuatro bacterias potencialmente presentes en las matrices alimentarias. La presencia o ausencia de la bacteria diana en los alimentos se confirma en unas 30 horas, lo que reduce el tiempo requerido para la detección en comparación con el tiempo mínimo de 7 días por método cultural. Asimismo, permite reducir el número de medios de cultivo y reactivos, para el aislamiento e identificación de bacterias que no son detectadas por PCR y que no están presentes en las matrices alimentarias, lo que supone un importante ahorro económico. / [CA] La seguretat alimentària sempre és una prioritat per a la població i en l' actualitat cobra major importància per certes tendències alimentàries, com el consum d' aliments crus i la distribució generalitzada d' aliments orgànics, que poden ser la causa de malalties transmeses per aliments. Per garantir la seguretat alimentària, la detecció d' aquests microorganismes s' ha de realitzar de manera ràpida i eficient. Per a això, el mètode de cultiu microbiològic es considera l' oficial per a la detecció d' aquests patògens. Però, hi ha importants inconvenients, ja que no només requereix més temps, sinó que també és laboriós i consumeix molts recursos. A més, pot ser limitat pel que fa a la detecció de bacteris fisiològicament alterats i/o estressats durant l'emmagatzematge i la conservació. En aquest treball s'ha desenvolupat un protocol senzill i ràpid per a la detecció simultània d' E. coli, L. monocytogenes, S. aureus i S. enterica en aliments, mitjançant la combinació d' una etapa de co-cultiu en medi líquid i la detecció per PCR múltiple. S'ha avaluat l'eficiència de diversos mitjans d'enriquiment i s'ha seleccionat l'aigua de peptona tamponada com el medi òptim per al co-cultiu dels quatre bacteris diana. També es van optimitzar les condicions de PCR múltiple i es van aplicar tant a co-cultius com a mostres d'aliments inoculats artificialment, enciam orgànic i carn picada. Després de l'optimització, la PCR múltiple desenvolupada va ser capaç de detectar els quatre bacteris simultàniament, fins a una inoculació inicial de 10^0 UFC/mL. En presència d' ambdues matrius alimentàries inoculades, després l' etapa de co-cultiu, la PCR múltiple va poder detectar simultàniament els 3 bacteris: E. coli, S. enterica i L. monocytogenes, mentre que S. aureus s' ha detectat per PCR simple, a partir del mateix extracte d' ADN del co-cultiu. Els resultats obtinguts permeten concloure que l' ús d' un pas de co-cultiu en Aigua de peptona tamponada, abans de la detecció per PCR simple i múltiple, pot facilitar la detecció simultània dels quatre bacteris potencialment presents en les matrius alimentàries. La presència o absència del bacteri diana en els aliments es confirma en unes 30 hores, la qual cosa redueix el temps requerit per a la detecció en comparació amb el temps mínim de 7 dies per mètode cultural. Així mateix, permet reduir el nombre de mitjans de cultiu i reactius, per a l' aïllament i identificació de bacteris que no són detectats per PCR i que no estan presents en les matrius alimentàries, la qual cosa suposa un important estalvi econòmic. / [EN] Food safety is a priority for the population and is nowadays more important than ever due to certain dietary trends such as the consumption of raw foods and the widespread distribution of organic foods, which may be the cause of foodborne diseases. To ensure food safety, the detection of these microorganisms must be done quickly and efficiently. Although, the microbiological culture method is considered to be the official method for the detection of these food-borne pathogens, it suffers from significant drawbacks, such as time-consuming, laborious and expensive, in addition it may be limited regarding the detection of physiologically altered and/or stressed bacteria, during storage and preservation. In this work has been developed a simple and rapid protocol for the simultaneous detection of E. coli, L. monocytogenes, S. aureus and S. enterica in food, by combining a liquid co-culture step and detection by multiplex PCR. The efficiency of several enrichment media was evaluated and buffered peptone water was chosen as the optimal medium for the co-culture of the four target bacteria. Then, optimized multiplex PCR conditions were applied to both the co-cultures and the samples of artificially inoculated foods, organic lettuce and ground meat. After optimization, the developed multiplex PCR was able to simultaneously detect the four bacteria, up to an initial inoculation of 10^0 CFU/mL. In the presence of the two inoculated food matrices, after a co-culture step, the multiplex PCR could simultaneously detect the 3 bacteria: E. coli, S. enterica and L. monocytogenes, whereas, S. aureus has been detected by simplex PCR, from the same co-culture DNA template. The results obtained allow conclusion that the use of a co-culture step in Buffered Peptone Water, before detection by simplex and multiplex PCR, can facilitate the simultaneous detection of the four bacteria potentially present in the food matrices. The presence or the absence of the target bacteria in food is confirmed in approximately 30 hours, which reduce the time required for the detection compared to the minimum time of 7 days by cultural method. Also, it allows to reduce the number of culture media and reagents, for the isolation and identification of bacteria that are not detected by PCR and which are not initially present in the food matrices, which represents a significant economic savings. / Boukharouba, A. (2022). Isolation and Identification of Foodborne Pathogens of Special Interest in Food Safety [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182828