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Abstract:

Low- and non-calorie sweeteners like saccharine, xylitol, acesulfame-K, etc., have been used in the food industry to replace high-calorie sweeteners, e.g., table sugar, since over a century; however, some disadvantages like their thermolability, bitter after-taste or laxative effect, encourage a constant search for better options. Stevia-based sweeteners and tagatose have recently appeared in the market as novel alternatives to traditional non‑/low-calorie sweeteners, with optimized characteristics. Their organoleptic properties, stability and impact on human’s health have been widely reported; however, little is known about their potential effect on microbial growth dynamics of food pathogens. Modifying product formulation might trigger changes in parameters such as chemical composition, aw, etc., which could affect microbiological food safety. Food structure should be also considered when addressing the effect of these sweeteners on microbial growth, since they can be added to a wide variety of liquid and solid-like food products. Although the role of food structure on microbial dynamics has been acknowledged, the combinatory effect of both novel sweeteners and food structure has not been studied. The overall objective of this thesis dissertation is to investigate the effect of stevia-based sweeteners and tagatose on the growth dynamics of Salmonella Typhimurium and Listeria monocytogenes, when added to liquid and solid food (model) systems, and to UHT skimmed milk, (selected as case study). Solid model systems were used over real food product because they are more reproducible, easy to prepare and sterilized. Due to the differences in physicochemical properties between stevia/steviol glycosides and tagatose, these were individually considered. More specifically, commercial stevia (St) and the pure mix steviol glycosides (SG) were studied in comparison to table sugar (TS), while tagatose (Tg) was independently considered. The studies from St/SG vs. TS are presented through two chapters because of the broad amount of information gathered, while results from Tg are compiled in one. The first study focuses on the effect of stevia-based sweeteners in liquid systems. In this case, the effect of different concentration levels of St and SG was compared to sweet-equivalent concentrations of TS and laboratory sucrose (LS). Incubation temperature was another variable taken into account, addressing the effect of the sweeteners at room and refrigeration conditions. The liquid media chosen to address the bacterial growth dynamics were chemically defined minimal media and general culture broth. Results reveal variable responses depending on the microorganism, temperature and sweetener studied. On one hand, the growth of S. Typhimurium is more affected by the presence of the sweeteners, especially at high concentrations and low incubation temperatures. On the other hand, the growth of L. monocytogenes is only altered at 4°C when the sweeteners are present, except with SG. Results from chemically defined minimal media provide useful information regarding the metabolizing ability of the different sweeteners by both bacteria. More specifically, they reveal the key role of the bulk agent used in St, maltodextrin, as both bacteria are able to grow on it. Furthermore, experiments in UHT skimmed milk (UHT SM) enriched with sweetener were conducted at low temperatures. Results reveal a similar behaviour of L. monocytogenes in UHT SM enriched with sweeteners as compared to the results in general liquid media. Nevertheless, the behaviour of S. Typhimurium in this product is very different to that in general liquid culture media, since the presence of TS promotes the growth in UHT SM at 8°C. The second study of this dissertation addresses the effect of stevia-based sweeteners on the growth kinetics of S. Typhimurium and L. monocytogenes in homogeneous and heterogeneous solid systems. As in the previous study, St, SG and TS were added at different concentration values to general media supplemented with gelatine, and a mixture of gelatine and dextran which is known to lead to phase separation. As in the case of liquid media, different results were obtained for each type of bacterium, S. Typhimurium being generally more sensitive to the environmental changes than L. monocytogenes. Results also demonstrate the key role played by the media micro-structure and incubation temperature on the growth of these microorganisms, over the presence of the studied sweeteners. Finally, the effect of Tg at different concentration levels on the growth kinetics of S. Typhimurium and L. monocytogenes was investigated. Incubation temperatures, and the liquid and solid model systems to which Tg was added, were the same as for St/SG/TS. Results demonstrate the inhibitory effect on S. Typhimurium growth when Tg is added to TSBdf and to both solid systems. This effect is stronger at low temperature, as generally observed through the whole thesis. In UHT SM no measurable growth is obtained. The growth of L. monocytogenes remains invariable to the addition of, Tg, except at 4°C, where the presence of Tg facilitates the growth of L. monocytogenes in liquid broth but not in UHT SM. Overall, this work contributes to a better understanding of bacterial kinetics in new low calorie formulations. The different growth behaviour of the two bacteria under study is evident, S. Typhimurium being more sensitive to environmental changes than L. monocytogenes. The use of Tg, that has a hurdle effect on the growth of S. Typhimurium, is here demonstrated to be a better option than replacing TS with St/SG because St/SG can facilitate bacterial growth. Nevertheless, any environmental modification, either the addition of sweeteners or the structure of the media, causes a stronger effect at low temperatures for both microorganisms. These conclusions can be used as a benchmark for the selection of the best low‑/non-calorie sweetener to be used in food industry. The importance of validating predictive models with real food products or more accurate food model systems, including solid systems, has also been demonstrated. Future work should focus on addressing the effect of these sweeteners on the growth dynamics of relevant food spoilage microorganisms, such as Pseudomonas fluorescens and Zyggosaccharomyces bailii, in other food products that are candidates for sugar replacement, e.g., fruit beverages and ketchup sauce. Localized measurements of pH and aw with non-invasive techniques in the solid systems can also contribute to a better understanding of the environmental changes occurring in the media and their effect on microbial behaviour.