Desarrollo de modelos predictivos de interacción microbiana para Listeria monocytogenes y bacterias del ácido láctico y su aplicación para la optimización de cultivos bio-protectores en productos pesqueros

Minimally processed and ready-to-eat (RTE) fish products that have not undergone any lethal treatment prior to consumption represents for a potential risk of pathogenic microorganisms, such as Vibrio spp. and Listeria monocytogenes. These pathogens cause foodborne infections resulting from consumption of contaminated raw fish or partially cooked fish or products contaminated during food handling and preparation. The genus Vibrio are environmentally ubiquitous in estuarine or marine water, able to reach consumers when products are not properly cooked, while the ability of L. monocytogenes to re-contaminate RTE products, during food handling and processing, and multiply at refrigeration temperatures poses a significant risk to human health. The microbial behaviour in food can be described using predictive mathematical models, taking into account the processing, distribution and home conditions along the food chain. In the first section of this work, an extensive review was elaborated, including the main aspects and concepts covered by the present thesis, which comprise of, among others, information on the aquaculture sector in Andalusia, relevant pathogenic and spoilage microorganisms in fishery products, listeriosis incidence, the use of bioprotective cultures or microbial risk assessment (Chapter 1). In addition, predictive microbiology tools were reviewed, providing an overview of the predictive modelling process, in particular, focused on microbial interaction models, and its application to quantitative microbial risk assessment (QMRA) (Chapter 2). In the experimental and investigation part, the microbiota and foodborne pathogens of Gilthead sea bream (Sparus aurata) and Sea bass (Dicentrarchus labrax) in two estuarine ecosystems were characterized and the shelf-life of both products was estimated using predictive models. Noteworthy, Vibrio parahaemolyticus was isolated from estuarine water and the initial microbiological quality of fish species and estuarine water was demonstrated to impact on product shelf-life (Chapter 3). For the study of biopreservation technology, lactic acid bacteria (LAB) strains were used to inhibit the growth of L. monocytogenes. The data obtained from this study were used to develop predictive microbiology models, able to describe the microbial interaction of both microorganisms in culture media (Chapter 4) and filleted Gilthead sea bream under modified atmosphere packaging at isothermal and non-isothermal conditions (Chapter 5). As result of these studies, an innovative stepwise modelling process was designed to generate predictive models describing interaction of BAL and L. monocytogenes based on the combination of mono and co-culture growth data. In the case of the assay with Lactobacillus sakei CTC494, recognized as a bacteriocin-producing strain, it was evidenced that this bioprotective culture induced an early stationary phase of L. monocytogenes (Chapter 5), inhibiting the pathogen growth or reducing its concentration (Chapter 5). These experiments also confirmed that L. sakei CTC494 could be used, at a level of 4 log cfu/g, as bioprotective culture against L. monocytogenes in the abovementioned Gilthead sea bream product, without negatively affecting the sensory quality of the product. Furthermore, the interaction models generated for this bioprotective culture demonstrated an optimal prediction capacity of the inhibitory effect of L. sakei CTC494 on the pathogen in the fish product (Chapter 5). Overall, results of this work demonstrated the potential of using bioprotective cultures to control L. monocytogenes growth in Mediterranean fish products and enhance product shelf-life. In addition, the approach based on predictive microbiology was shown to be a suitable and effective method to simulate the simultaneous growth of bacteriocinproducing LAB strains and L. monocytogenes in culture media and fish products, presenting itself as a tool for supporting the design and optimization of preservation strategies based on the use bioprotective cultures in minimally processed and RTE fish products.Los productos pesqueros mínimamente procesados y listos para el consumo (RTE) sin tratamiento letal previo al consumo representan un riesgo potencial de presencia de microorganismos patógenos de transmisión alimentaria, como podrían ser Vibrio spp. y Listeria monocytogenes. Esos patógenos pueden estar presentes en el pescado crudo contaminado o pescado parcialmente cocido o bien recontaminarlo durante su manipulación y preparación. Los patógenos del género Vibrio tienen su origen en aguas de estuario o marinas, y pueden llegar hasta el consumidor, si el alimento no ha sido cocinado adecuadamente, mientras que L. monocytogenes puede contaminar los productos RTE, por contaminación cruzada, y multiplicarse a temperaturas de refrigeración, representando, por ello, un riesgo significativo para la salud humana. El comportamiento de los microorganismos en los alimentos puede describirse utilizando modelos matemáticos de microbiología predictiva, considerando las condiciones de procesado, de distribución y de la fase de consumo, cubriendo toda la cadena alimentaria. El propósito de la presente tesis doctoral fue estudiar y cuantificar el efecto bio-protector de cultivos bacterianos seleccionados sobre patógenos alimentarios y optimizar su diseño y uso en productos pesqueros RTE, mediante un enfoque de microbiología predictiva. Para ello, en primer lugar, se abordó una extensa revisión sobre los principales aspectos y conceptos objeto del presente trabajo, incluyendo información sobre el sector de la acuicultura en Andalucía, los tipos de microorganismos patógenos y alterantes de relevancia en productos pesqueros, la incidencia de listeriosis, el uso de cultivos bioprotectores y la evaluación del riesgo microbiano (Capítulo 1). A su vez, se realizó un análisis de las herramientas de microbiología predictiva, proporcionando una descripción general de los procesos de generación de modelos matemáticos, en particular, de modelos de interacción microbiana y de su aplicación en la evaluación cuantitativa del riesgo microbiano (ECRM) (Capítulo 2). En la parte experimental y de investigación del trabajo, se realizó un estudio de caracterización de la microbiota y patógenos alimentarios de la Dorada (Sparus aurata) y Lubina (Dicentrarchus labrax) en dos ecosistemas de estuario, estimándose la vida útil de los productos pesqueros mediante modelos predictivos. Es destacable la presencia de Vibrio parahaemolyticus, que se aisló de agua de estuario, además del impacto demostrado de la calidad microbiológica de las especies pesqueras y del agua en el que se encuentran sobre la vida útil del producto (Capítulo 3). En la parte que estudió y aplicó las tecnologías de bioconservación, se utilizaron cepas de bacterias ácido-lácticas (BAL) para inhibir el crecimiento de L. monocytogenes. Los datos obtenidos sirvieron para generar modelos de microbiología predictiva que permitieron describir, adecuadamente, la interacción microbiana entre ambos microrganismos en medios de cultivo (Capítulo 4) y en productos de filetes de Dorada envasados en atmósfera modificada bajo condiciones isotérmicas y no isotérmicas (Capítulo 5). Como resultado de estos estudios, se diseñó un proceso sistematizado, basado en etapas, para la generación de modelos de interacción entre BAL y L. monocytogenes, combinando datos de crecimiento de mono y cocultivo en caldo y en alimento. En el caso de los experimentos con la cepa de Lactobacillus sakei CTC494, reconocida como productora de bacteriocina, se pudo observar que esta indujo una fase estacionaria temprana sobre el crecimiento de L. monocytogenes (Capítulo 5), reduciendo su crecimiento o, incluso, disminuyendo su concentración. Estos experimentos también confirmaron que L. sakei CTC494 podría aplicarse, a un nivel de 4 log ufc/g, como cultivo bioprotector contra L. monocytogenes en el producto de Dorada previamente mencionado, sin afectar negativamente la calidad sensorial del producto. Además, los modelos de interacción generados para este cultivo mostraron una buena capacidad de predicción del efecto bioprotector de L. sakei CTC494 sobre el patógeno en el producto de Dorada fileteada (Capítulo 5). En general, los resultados de la tesis doctoral demostraron el gran potencial de los cultivos bioprotectores para controlar el crecimiento de L. monocytogenes en productos pesqueros del mediterráneo y mejorar su vida útil. Se comprobó, además, que el enfoque, diseñado en el presente trabajo, basado en la utilización de modelos de interacción microbiana, fue un método valido y efectivo para simular el comportamiento simultáneo de diferentes cepas de BAL, productoras de bacteriocina, y L. monocytogenes en medios de cultivo y productos pesqueros, proponiéndose, por tanto, como una herramienta de gran valor para el diseño y optimización de estrategias de conservación basadas en el uso de cultivos bioprotectores en pescados mínimamente procesados y RTE productos

Efeito do tratamento termoquímico (óleo essencial e calor) no crescimento microbiano durante a vida útil de mexilhões (Perna perna) processados em embalagens flexíveis

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2013.O estado de Santa Catarina é o maior produtor de moluscos bivalves do Brasil. Por ser um alimento altamente perecível, estratégias para prolongar a sua vida útil têm motivado vários estudos. A aplicação de óleos essenciais como conservantes naturais é uma alternativa eficaz na conservação de alimentos, devido ao seu poder antimicrobiano. O tratamento térmico rigoroso, muitas vezes aplicado para garantir a estabilidade microbiológica dos alimentos, pode levar à perda de nutrientes e afetar as características sensoriais desses produtos. Assim, o objetivo deste trabalho foi avaliar o efeito da aplicação de tratamento termoquímico (óleo essencial de orégano (O.E.O.) e tratamento térmico (pasteurização)) sobre a vida útil de mexilhões processados em embalagens flexíveis. Primeiramente, comparou-se a eficácia do óleo essencial de orégano e do óleo essencial de manjericão (O.E.M) em diferentes concentrações (0,2 e 0,4 % (v/m)) sobre os micro-organismos patogênicos e deteriorantes em mexilhões pré-cozidos. Quatro tratamentos foram realizados: P1 (controle), P2 (0,2 % de O.E.O.), P3 (0,4 % de O.E.O.) e P4 (0,4 % de O.E.M.). As análises para patogênicos foram realizadas para amostra P1 (controle) no primeiro e sétimo dia de armazenamento e para as demais no sétimo dia. Os resultados mostraram que nas duas concentrações testadas, os óleos apresentaram potencial antimicrobiano. O O.E.O. na concentração de 0,4 % (v/m) foi ligeiramente melhor no atraso do crescimento dos grupos microbianos estudados. Quatro tratamentos foram realizados para as amostras de mexilhão (Perna perna): A1 (controle, sem O.E. e sem tratamento térmico), A2 (tratamento térmico), A3 (O.E.O. sem tratamento térmico), A4 (O.E.O. com tratamento térmico). Os mexilhões foram massageados com O.E.O.(amostras A3 e A4), embalados em embalagens flexíveis e submetidos à pasteurização (80 °C/10 minutos) e armazenados a 4, 10 e 15 °C. A vida útil dos mexilhões foi acompanhada pelo crescimento microbiano até as contagens atingirem 107 UFC/g (bactérias ácido lácticas, contagem total de mesófilos, psicrófilos e psicrotróficos), pelo pH e comparada sensorialmente (visual e olfativamente), além da análise de patogênicos para amostra A1. As contagens de Coliformes termotolerantes (45 °C) e E. coli, entre o primeiro e o sétimo dia de armazenamento, não ocorreram mudanças no crescimento. As Contagens de Estafilococos Coagulase Positiva e Vibrio parahaemolyticus mantiveram-se constantes em Abstract : Santa Catarina state is the largest producer of bivalve molluscs in Brazil. For being a perishable food, strategies to prolong shelf life have motivated several studies. The application of essential oils as natural preservatives is an effective alternative in food preservation due to its antimicrobial power. The strict heat treatment, often applied to ensure food microbiological stability, can lead to nutrients losses and affect the sensory characteristics of the products. The objective of this work was to evaluate the effect of thermochemical treatment (oregano essential oil (O.E.O.) and heat treatment (pasteurization)) on processed mussel shelf life in flexible packaging. First, it was compared the efficacy of the essential oil of oregano and basil essential oil (B.E.O.) in different concentrations (0.2 and 0.4 % (v/w)) on spoilage and pathogenic microorganisms in pre-cooked mussels. Four treatments were used: P1 (control), P2 (0.2 % O.E.O.), P3 (0.4 % O.E.O.) and P4 (0.4 % B.E.O.). Pathogens were analyzed for sample P1 (control) in the first and seventh day of storage and the other on the seventh day. The results showed that, at tested concentrations, oils exhibited antimicrobial activity. The O.E.O. in the concentration of 0.4 % (v/w) was slightly better in the delay the growth of microbial groups studied. Four associated treatments were performed for samples of mussel (Perna perna): A1 (control), A2 (heat treatment), A3 (O.E.O. without heat treatment), A4 (O.E.O. with heat treatment). The mussels were massaged O.E.O (samples A3 and A4), packaged in flexible packagings and submitted to pasteurization (80 °C/10 min) and stored at 4, 10 and 15 ° C. The shelf life of mussels was accompanied by microbial growth until the counts reach 107 CFU/g (lactic acid bacteria, total count of mesophilic, psychrotrophic and psychrophiles), the pH and compared sensory (visual and olfactory), besides the analysis of pathogenic sample A1. Counts Thermotolerant coliforms (45 °C), and E. coli, between the first and seventh days of storage there were no changes in growth. Counts for Coagulase Positive Staphylococci and Vibrio parahaemolyticus remained constant at <3NMP / g. Results were inconclusive regarding to O.E.O. antimicrobial effects on Listeria monocytogenes. Through sensory evaluation, it was possible to identify odor that appeared at times corresponding to elevated microbial counts. A decrease in the pH for all samples was observed. Baranyi and Roberts model was fitted to growth curves to obtain growth parameters and predict the shelf life of processed mussels. The shelf life of the mussels stored at 4 °C was 21 days for sample A1 (control) and A2 (heat treatment), 31 and 51 days for the samples A3 (O.E.O. without heat treatment) and A4 (O.E.O. with thermal treatment), respectively. The shelf life of the mussels stored at 10 °C was 5 days for samples A1 and A2, 8 days for the sample A3 and 12 days for sample A4. Samples stored at 15 °C had a reduced shelf life, being 2 days to sample A1, 5 days for sample A2 and A3 and 10 days for sample A4. It can be concluded that the O.E.O. had antimicrobial effect on micro-organisms studied and that the thermochemical treatment prolong the shelf life of mussels for another 30 days when stored at 4 °C. The results of this study are useful for describing the variation of microbiological parameters with temperature storage products thermochemically treated and it becomes a useful tool for the seafood processing industries

Modeling the Growth of Six Listeria monocytogenes Strains in Smoked Salmon Pâté

In this study, the growth of six L. monocytogenes strains isolated from different fish products was quantified and modeled in smoked salmon pâté at a temperature ranging from 2 to 20 °C. The experimental data obtained for each strain was fitted to the primary growth model of Baranyi and Roberts to estimate the following kinetic parameters: lag phase (λ), maximum specific growth rate (μmax), and maximum cell density (Nmax). Then, the effect of storage temperature on the obtained μmax values was modeled by the Ratkowsky secondary model. In general, the six L. monocytogenes strains showed rapid growth in salmon pâté at all storage temperatures, with a relatively short lag phase λ, even at 2 °C. The growth behavior among the tested strains was similar at the same storage temperature, although significant differences were found for the parameters λ and μmax. Besides, the growth variations among the strains did not follow a regular pattern. The estimated secondary model parameter Tmin ranged from −4.25 to −3.19 °C. This study provides accurate predictive models for the growth of L. monocytogenes in fish pâtés that can be used in shelf life and microbial risk assessment studies. In addition, the models generated in this work can be implemented in predictive modeling tools and repositories that can be reliably and easily used by the fish industry and end-users to establish measures aimed at controlling the growth of L. monocytogenes in fish-based pâtés

Modelling the interaction of the sakacin-producing Lactobacillus sakei CTC494 and Listeria monocytogenes in filleted gilthead sea bream (Sparus aurata) under modified atmosphere packaging at isothermal and non-isothermal conditions

The objective of this work was to quantitatively evaluate the effect of Lactobacillus sakei CTC494 (sakacin-producing bioprotective strain) against Listeria monocytogenes in fish juice and to apply and validate three microbial interaction models (Jameson, modified Jameson and Lotka Volterra models) through challenge tests with gilthead sea bream (Sparus aurata) fillets under modified atmosphere packaging stored at isothermal and non-isothermal conditions. L. sakei CTC494 inhibited L. monocytogenes growth when simultaneously present in the matrix (fish juice and fish fillets) at different inoculation ratios pathogen:bioprotector (i.e. 1:1, 1:2 and 1:3). The higher the inoculation ratio, the stronger the inhibition of L. monocytogenes growth, with the ratio 1:3 yielding no growth of the pathogen. The maximum population density (Nmax) was the most affected parameter for L. monocytogenes at all inoculation ratios. According to the microbiological and sensory analysis outcomes, an initial inoculation level of 4 log cfu/g for L. sakei CTC494 would be a suitable bioprotective strategy without compromising the sensory quality of the fish product. The performance of the tested interaction models was evaluated using the Acceptable Simulation Zone approach. The Lotka Volterra model showed slightly better fit than the Jameson-based models with 75–92% out of the observed counts falling into the Acceptable Simulation Zone, indicating a satisfactory model performance. The evaluated interaction models could be used as predictive modelling tool to simulate the simultaneous behaviour of bacteriocin-producing Lactobacillus strains and L. monocytogenes; thus, supporting the design and optimization of bioprotective culture-based strategies against L. monocytogenes in minimally processed fish products.info:eu-repo/semantics/acceptedVersio

Quantifying the bioprotective effect of Lactobacillus sakei CTC494 against Listeria monocytogenes on vacuum packaged hot-smoked sea bream

In this study, the bioprotective potential of Lactobacillus sakei CTC494 against Listeria monocytogenes CTC1034 was evaluated on vacuum packaged hot-smoked sea bream at 5 °C and dynamic temperatures ranging from 3 to 12 °C. The capacity of three microbial competition interaction models to describe the inhibitory effect of L. sakei CTC494 on L. monocytogenes was assessed based on the Jameson effect and Lotka-Volterra approaches. A sensory analysis was performed to evaluate the spoiling capacity of L. sakei CTC494 on the smoked fish product at 5 °C. Based on the sensory results, the bioprotection strategy against the pathogen was established by inoculating the product at a 1:2 ratio (pathogen:bioprotector, log CFU/g). The kinetic growth parameters of both microorganisms were estimated in mono-culture at constant storage (5 °C). In addition, the inhibition function parameters of the tested interaction models were estimated in co-culture at constant and dynamic temperature storage using as input the mono-culture kinetic parameters. The growth potential (δ log) of L. monocytogenes, in mono-culture, was 3.5 log on smoked sea bream during the experimental period (20 days). In co-culture, L. sakei CTC494 significantly reduced the capability of L. monocytogenes to grow, although its effectiveness was temperature dependent. The LAB strain limited the growth of the pathogen under storage at 5 °C (<1 log increase) and at dynamic profile 2 (<2 log increase). Besides, under storage at dynamic profile 1, the growth of L. monocytogenes was inhibited (<0.5 log increase). These results confirmed the efficacy of L. sakei CTC494 for controlling the pathogen growth on the studied fish product. The Lotka-Volterra competition model showed slightly better fit to the observed L. monocytogenes growth response than the Jameson-based models according to the statistical performance. The proposed modelling approach could support the assessment and establishment of bioprotective culture-based strategies aimed at reducing the risk of listeriosis linked to the consumption of RTE hot-smoked sea bream.info:eu-repo/semantics/acceptedVersio

A new expanded modelling approach for investigating the bioprotective capacity of Latilactobacillus sakei CTC494 against Listeria monocytogenes in ready-to-eat fish products

Understanding the role of food-related factors on the efficacy of protective cultures is essential to attain optimal results for developing biopreservation-based strategies. The aim of this work was to assess and model growth of Latilactobacillus sakei CTC494 and Listeria monocytogenes CTC1034, and their interaction, in two different ready-to-eat fish products (i.e., surimi-based product and tuna pâté) at 2 and 12 °C. The existing expanded Jameson-effect and a new expanded Jameson-effect model proposed in this study were evaluated to quantitatively describe the effect of microbial interaction. The inhibiting effect of the selected lactic acid bacteria strain on the pathogen growth was product dependent. In surimi product, a reduction of lag time of both strains was observed when growing in coculture at 2 °C, followed by the inhibition of the pathogen when the bioprotective L. sakei CTC494 reached the maximum population density, suggesting a mutualism-antagonism continuum phenomenon between populations. In tuna pâté, L. sakei CTC494 exerted a strong inhibition of L. monocytogenes at 2 °C (<0.5 log increase) and limited the growth at 12 °C (<2 log increase). The goodness-of-fit indexes indicated that the new expanded Jameson-effect model performed better and appropriately described the different competition patterns observed in the tested fish products. The proposed expanded competition model allowed for description of not only antagonistic but also mutualism-based interactions based on their influence on lag time

Modelagem do crescimento de salmonella spp. Em carne de frango armazenada em diferentes condições de temperatura / Modeling the growth of salmonella spp. In broiler meat stored at different temperature conditions

Objetivou-se modelar o crescimento de Salmonella spp. em carne de frango moído (CFM) sob diferentes condições isotérmicas e não isotérmicas. Os dados de crescimento de Salmonella spp. em CFM nas temperaturas entre 15 e 42 ºC de armazenamento foram adquiridos do trabalho de Juneja et al. (2007), disponíveis na base de dados Combase e o modelo de Baranyi e Roberts foi ajustado às curvas de crescimento. Pode-se afirmar que o modelo apresentou ótimo desempenho para descrever o crescimento de Salmonella spp. em CFM nas seis temperaturas avaliadas, com valores de R2 ? 0,99 e Erros Padrão dos ajustes próximos de zero. O modelo da Raiz Quadrada foi selecionado para gerar as predições do crescimento em condições de oscilação de temperatura, destacando a importância na manutenção da cadeia do frio, pois uma mínima alteração nessas condições pode provocar um aumento expressivo no crescimento de micro-organismos patogênicos. Pode-se concluir que a aplicação de modelos preditivos e softwares de fácil utilização pode auxiliar às indústrias de cárneos na determinação da contaminação de alimentos, sem a necessidade de realização de vários experimentos para determinar a qualidade e a segurança dos produtos

The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species’ threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project – and avert – future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups – including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems – www.predicts.org.uk). We make site-level summary data available alongside this article. The full database will be publicly available in 2015