Variability in the response of salmonella and listeria strains to different strategies for inactivation

[ENG] The last EFSA report, notified a total of 5146 food outbreaks in the European Union in 2018. The food industry plays an important role in the food chain and has to adapt and reinvent itself when consumers place certain demands on products. Food safety is the first issue to take into account. There is already a large variety of microbiological species that are capable of developing in food and the safety of food makes it questionable whether or not the product is suitable for consumption. Listeria monocytogenes and Salmonella spp. are characterised by being two ubiquitous microbial species capable of living in borderline conditions of pH, water activity and sodium chloride concentrations. Another circumstance of these two microorganisms is the variability between their different strains and the capacity they have to recover from the different stresses to which they are subjected. For these reasons, it is of utmost importance to know their response to stress in order to control their inactivation and/or development in any situation. This knowledge will enable to efficiently apply technologies and treatments that are capable of inactivating or inhibiting growth of microorganisms, so to prevent or, at least, reduce food contamination. So, in this thesis, the main objective was to study the application of different strategies for the inactivation of L monocytogenes and Salmonella that could reduce the intensity of the treatments, while ensuring food safety. In this thesis, the effect that different inactivation strategies have on L. monocytogenes and Salmonella spp. has been studied. Firstly, the heat resistance of four L. monocytogenes strains -Scott A, CECT 4031, CECT 4032 and 12MOB052- in three different matrices -buffered peptone water (BPW), pH 7 Mcllvaine phosphate citrate buffer and a last one of food origin; semi- skimmed milk- was studied. Under isothermal conditions, there was no between-strains and between-media variability in the resistance. However, when this experiment was carried out under dynamic conditions, some strains were able to develop acclimation to stress, leading to important differences in resistance. Between-strains, the CECT 4031 increased its D-value by 10, with CECT 4032 being the least acclimated and between-media, peptone water buffer and semi- skimmed milk were the mediums where most acclimation was found. These results are of great importance, as they highlight that some mechanisms of heat resistance may not be detected when conditions, such as acclimation.Another part of the research was carried out to evaluate the response to heat when Salmonella Senftenberg and S. Enteritidis - the treatment applied is under isothermal conditions, but they show up under dynamic heating were exposed to a previous acid shock. Both serovars of Salmonella were grown at pH 4.5 and subsequently exposed to heat treatment under isothermal conditions at four different temperatures. The heating medium used was peptone water at pH 4.5 and 7.0. The results showed that both serovars had a different response to a heat treatment after the exposure to acid. On the one hand, S. Senftenberg reduced its heat resistance when subjected to a previous acid shock, while in S. Enteritidis its heat resistance increased. Again, these results bring out the inherent variability in microorganisms, in this case related to a stress response mechanisms. Finally, the effectiveness of the combination of applying electrical pulses (PEF) and oregano essential oil to L. monocytogenes was determined. The medium used was a Mcllvaine buffer at pH 7 with a conductivity of 6 μS/cm. On the one hand, the efficiency of the electric field was evaluated between 5 and 20 kV/cm and it was found that after 60 pulses at 20 kV/cm field strength, 1 Hz frequency and 20 μs pulse width, an inactivation of 2.01 log10 cycles was achieved, while to achieve a similar level of inactivation at 15 kV/cm, 300 pulses were required. Then, PEF treatments were then applied (20 kV/cm field strength, 60 pulses, 1 Hz frequency and 20 μs pulse width) after having exposed the cells to the minimum inhibitory concentration (MIC) of the oregano essential oil (2000 ppm) and these results showed no significant differences (p>0.05) with respect to the PEF treatments applied alone. However, when the same PEF treatment as above was applied first and then the surviving microorganisms were resuspended on the MIC of oregano essential oil, significant differences (p0,05) con respecto a los tratamientos PEAV aplicados solos. Sin embargo, cuando se aplicó primero el mismo tratamiento PEAV que el anterior y luego se resuspendió a los microorganismos sobrevivientes en la CIM del aceite esencial de orégano, se encontraron diferencias significativas (p<0,05) con respecto al FEM aplicado solo y al PEAV después del orégano. También se encontró que al reducir a 1/16 la CIM (125 ppm) el efecto fue el mismo que si aplicáramos la CIM. Para concluir, esta tesis ha investigado cómo pueden combinarse diferentes estrategias de inactivación microbiana para conseguir un alimento seguro con un tratamiento más suave, que en un solo tratamiento convencional. También ha ayudado a abordar las predicciones para futuros estudios y como contribución a la industria alimentaria mejorará la cuantificación de la evaluación de riesgos microbiológicos.Escuela Internacional de Doctorado de la Universidad Politécnica de CartagenaUniversidad Politécnica de CartagenaPrograma de Doctorado de Técnicas avanzadas en investigación y desarrollo agrario y alimentari

Effect of acid shock on the thermoresistance of Salmonella enterica subsp. enterica CECT 4300 at different pH’s.

Salmonella entérica es un microorganismo patógeno alimentario causante de brotes de toxiinfecciones por consumo de alimentos contaminados. Por ello, es de gran importancia, en Seguridad Alimentaria Microbiológica, el conocimiento del efecto de diversos factores, como son el pH y el ClNa del medio en el que podría estar presente este microorganismo sobre su termorresistencia. Se llevaran a cabo estudios sobre la exposición del microorganismo a un choque ácido seguido de tratamientos térmicos a diferentes temperaturas en medios de calentamiento a diferentes pHs y con distintas concentraciones de ClNa. Se trataran de ajustar los datos obtenidos para la obtención de parámetros de esterilización y ver la posible aparición de hombros y colas para garantizar la Seguridad Alimentaria.Escuela Técnica superior de Ingeniería AgronómicaUniversidad Politécnica de Cartagen

Microbiological identification from foods of vegetable origin and the evolution during the processing

[SPA] Debido a la evolución de la industria agroalimentaria y a los grandes avances de ésta, la seguridad alimentaria se considera como una de las principales prioridades. La necesidad de garantizar seguridad y preservar los atributos de calidad (nutricionales y sensoriales) han derivado en la necesidad de contar con herramientas cada vez más sofisticadas para producir alimentos de manera eficiente y en elevadas cantidades. Ello supone un reto para garantizar la estabilidad microbiológica manteniendo estándares muy elevados de calidad. El objetivo de este trabajo ha sido a través del uso de la Reacción en Cadena de la Polimerasa descartar la presencia de microorganismos patógenos como es el caso de Bacillus Cereus. [ENG] Due to the evolution of the agri-food industry and its great advances, food safety is considered one of its main priorities. The need to ensure safety and preserve quality attributes (nutritional and sensory) have led to the need for increasingly sophisticated tools to produce food efficiently and in high quantities. This is a challenge to ensure microbiological stability by maintaining very standards of quality. The objective of this work has been through the use of the Polymerase Chain Reaction to rule out the presence of pathogenic microorganisms such as Bacillus Cereus.Agradecimientos al departamento de Ingeniería de los Alimentos y del Equipamiento Agrícola por toda la ayuda prestada

Effect of the synergy of electric pulses and oregano essential oil in the inactivation of L. monocytogenes

[SPA] Listeria monocytogenes es actualmente una de las principales preocupaciones de la industria alimentaria. Aunque el número de casos reportados en la Unión Europea es relativamente, tiene una gran gravedad, con unas tasas de hospitalización y mortandad del 97% y del 16,7%, respectivamente. Debido a la ubicuidad de este microorganismo y su capacidad de crecer en numerosas matrices alimentarias, las empresas agroalimentarias deben aplicar tratamientos que inactiven las células de L. monocytogenes. En este trabajo, se estudió la cinética de inactivación de L. monocytogenes aislada de productos marinos sometida a un tratamiento combinado de PEAV con un aceite esencial de orégano. Los resultados demuestran esta sinergia, ya que se consiguió una mayor inactivación microbiana con un campo de 20 kV/cm, 1 Hz de frecuencia, 150 pulsos y una concentración de orégano de 1/2 MIC, superior a la obtenida en las muestras control. Llegando a la conclusión de que los PEAV impulsan el efecto del orégano sobre la membrana de la bacteria. [ENG] Nowadays Listeria monocytogenes is one of the main concerns of the food industry. The number of cases reported in the European Union is relatively low it is very serious, with rates of hospitalization and mortality of 97% and 16.7%, respectively. Due to the ubiquity of this organism and its capacity to grow in many food matrices, agri-food companies must apply treatments that inactivate the cells of L. monocytogenes. In this work, it was studied the kinetics of inactivation of L. monocytogenes isolated from marine products subjected to a combined treatment of PEF with an essential oil of oregano. The results show this synergy, since it got a greater inactivation microbial with a field of 20 KV/cm, 1 Hz frequency, 150 pulses and an oregano concentration of 1/2 MIC, higher than that obtained in control samples. Concluding that the PEF promote the effect of oregano on the bacterial cell membrane.Investigación apoyada por el MICINN (es) y FEDER a través de los proyectos AGL2017-86840-C2-1-R y AGL2016-82014-REDT

Variability in the heat resistance of Listeria monocytogenes under dynamic conditions can be more relevant than that evidenced by isothermal treatments

Heterogeneity in the response of microbial cells to environmental conditions is inherent to every biological system and can be very relevant for food safety, potentially being as important as intrinsic and extrinsic factors. However, previous studies analyzing variability in the microbial response to thermal treatments were limited to data obtained under isothermal conditions, whereas in the reality, environmental conditions are dynamic. In this article we analyse both empirically and through mathematical modelling the variability in the microbial response to thermal treatments under isothermal and dynamic conditions. Heat resistance was studied for four strains of Listeria monocytogenes (Scott A, CECT 4031, CECT 4032 and 12MOB052), in three different matrices (buffered peptone water, pH 7 Mcllvaine buffer and semi-skimmed milk). Under isothermal conditions, between-strain and between-media variability had no impact in the heat resistance, whereas it was very relevant for dynamic conditions. Therefore, the differences observed under dynamic conditions can be attributed to the variability in the ability for developing stress acclimation. The highest acclimation was observed in strain CECT 4031 (10-fold increase of the D-value), while the lowest acclimation was observed in strain CECT 4032 (50% increase of the D-value). Concerning the different media, acclimation was higher in buffered peptone water and semi-skimmed milk than in Mcllvaine buffer of pH 7.0. To the knowledge of the authors, this is the first research work that specifically analyses the variability of microbial adaptation processes that take place under dynamic conditions. It highlights that microbial heat resistance under dynamic conditions are sometimes determined by mechanisms that cannot be observed when cells are treated in isothermal conditions (e.g. acclimation) and can also be affected by variability. Consequently, empirical evidence on variability gathered under isothermal conditions should be extrapolated with care for dynamic conditions.</p

The Different Response to an Acid Shock of Two Salmonella Strains Marks Their Resistance to Thermal Treatments

Microbial cells respond to sub-lethal stresses with several physiological changes to increase their chance of survival. These changes are of high relevance when combined treatments (hurdle technology) are applied during food production, as the cells surviving the first hurdle may have greater resistance to subsequent treatments than untreated cells. In this study, we analyzed if Salmonella develops increased resistance to thermal treatments after the application of an acid shock. We compared the heat resistance of acid-shocked (pH 4.5 achieved with citric acid) Salmonella cells with that of cells maintained at pH 7 (control cells). Thermal treatments were performed between 57.5 and 65°C. We observed a differential response between the two strains studied. Acid-shocked cells of Salmonella Senftenberg exhibited reduced heat resistance, e.g., for a treatment at 60.0°C and pH 7.0 the time required to reduce the population by 3 log cycles was lowered from 10.75 to 1.98min with respect to control cells. Salmonella Enteritidis showed a different response, with acid-shocked cells having similar resistance than untreated cells (the time required to reduce 3 log cycles at 60.0°C and pH 7.0 was 0.30min for control and 0.31min for acid-shock cells). Based on results by differential plating (with or without adding the maximum non-inhibitory concentration of NaCl to the recovery medium), we hypothesize that the differential response between strains can be associated to sub-lethal damage to the cell membrane of S. Senftenberg caused by the acid shock. These results provide evidence that different strains of the same species can respond differently to an acid shock and highlight the relevance of cross-resistances for microbial risk assessment