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

Resumen

Resumen de la tesis: Food safety if still a great concern for modern societies. The European Union One Health 2018 Zoonoses Report notified a total of 5146 outbreaks of foodborne diseases in the European Union in 2018. The food industry plays an important role in ensuring the safety along the food chain, guaranteeing that its products are safe and of high quality for the consumer. Food production must account for a wide variety of pathogenic microbiological species that are capable of growing and surviving in food products, being a hazard for human health. As a result, an accurate risk assessment is essential in order to make design safe products. This needs the development of predictive models able to describe the microbial response to the processing treatments. Among the foodborne pathogens, Listeria monocytogenes and Salmonella spp. are of great concern for the food industry. In the 2018 EFSA zoonoses report, it was reported that Listeria monocytogenes has the highest mortality and hospitalization rate among the foodborne pathogens, and Salmonella spp. is the foodborne pathogen with the highest number of outbreaks. Both microorganisms are characterized by being ubiquitous microbial species capable of living in relatively harsh conditions of pH, water activity and sodium chloride concentrations. As a result, the only mean to ensure that food products are free of these pathogens is by introducing a processing step able to inactivate them, reducing their concentration to innocuous levels. In order for these processes to be effective, mitigating over or under-processing, a description of the microbial response to stress is of utmost importance. This knowledge shall be reflected in predictive models, that could be used for process design and microbial risk assessment. This PhD thesis is framed within this need, being its main objective the study of different strategies for the inactivation of L. monocytogenes and Salmonella that could potentially be applied in the food industry. In results chapter I, the heat resistance of four L. monocytogenes strains (Scott A, CECT 4031, CECT 4032 and 12MOB052) in two laboratory media (buffered peptone water [BPW] and pH 7 Mcllvaine phosphate citrate buffer) and a food product (semi-skimmed milk) was studied. Experiments were carried out under isothermal and dynamic conditions. In isothermal treatments, the resistance of the microorganisms was homogeneous between strains and between media. However, when the experiment was carried out under dynamic conditions that enabled the development of stress acclimation, important differences in resistance between strains and between media were observed. Strain CECT 4031 was able to increase its D-value by a factor of 10, whereas strain CECT 4032 was not able to develop stress acclimation. Among the media, the results in BPW were similar to the ones in semi-skimmed milk, observing higher acclimation than in the McIlvaine buffer. These results highlight, that the mechanisms describing variability in the microbial response to stress may be different under dynamic conditions than those observed under isothermal treatments. Result chapters II and III apply combined treatments as a strategy to achieve microbial inactivation using milder processes than with the application of a single treatment. Chapter II evaluates the combination of an acid shock followed by a thermal treatment for the inactivation of Salmonella Senftenberg and S. Enteritidis. An acid shock at pH 4.5 was applied to both Salmonella serovars, followed by an isothermal treatment at four different temperatures at both pH 4.5 and 7.0. The results show that the effect of the acid shock on the resistance to the posterior treatments varies between both serovars. The application of an acid shock reduced the heat resistance of S. Senftenberg, whereas it did not affect or increase the heat resistance of S. Enteritidis. These results point out the added complexity of designing combined inactivation treatments. Bacterial cells are dynamic systems that can respond to stress increasing their resistance. In many cases, these mechanisms are shared between stresses, so the survivors of a mild treatment may have additional resistance to a posterior treatment (cross-resistance). Cross-resistances are especially relevant for the design of combined treatments, as they may make a combined treatment less effective than expected based only on the microbial response to each stress. Therefore, as highlighted in the results, the design of effective combined treatments require not just the description of the bacterial response to each stress, but also an evaluation of possible cross-resistances. Moreover, as shown in this research, cross-resistances may be affected by variability, adding additional complexity to this task. Result chapter III of this PhD thesis proposed a second strategy for microbial inactivation based on combined treatments. In this case, the combination of pulse electric field (PEF) treatments with oregano essential oil (EO) as a natural antimicrobial on the inactivation of Listeria spp. was evaluated. The combined treatment was applied following two strategies: simultaneous (the EO is added during the PEF treatment) and subsequent (the survivors of the PEF treatment are exposed to the EO). Whereas the simultaneous strategy did not improve microbial inactivation with respect to the PEF treatment, the subsequent one was an effective strategy for the inactivation of five of the six Listeria strains tested. However, the efficacy was strongly affected by the exposure time to the essential oil. An exposure of 20 minutes resulted in practically no improvement with respect to the PEF treatment, whereas an exposure of 60 minutes increased the inactivation in approximately 1 log cycle with respect to the PEF method. Therefore, this chapter identifies an effective combined strategy for the inactivation of Listeria spp. that could potentially be used by the food industry. Nevertheless, it also highlights the complexities of designing combined treatments, where many aspects of the process can affect inactivation. To conclude, this thesis has explored different strategies for microbial inactivation that could reduce the intensity of treatments without hampering food safety. The results also highlight the additional complexities associated to these strategies. Namely, the strong impact of variability for thermal treatments and how it can be different from the one observed in isothermal experiments; as well as the added complexities of combined treatments associated to the development of cross-resistances and the impact of the process parameters. Therefore, the development of novel strategies for food processing should be based on a detailed scientific knowledge of the factors identified as relevant in this thesis http://repositorio.bib.upct.es/dspace/