Impact of Saccharomyces cerevisiae metabolism on the properties offermenting bread dough

During dough fermentation, yeast converts sugars into carbon dioxide (CO2), ethanol and secondary metabolites such as acetic acid and succinic acid. This leads to the development of the typical aerated character and flavor of leavened bread. However, yeast also affects the structure of bread through its impact on dough rheology. It has been suggested that this impact is mediated by the production and release of hydrogen peroxide (H2O2) by yeast during dough fermentation. Furthermore, addition of yeast metabolites such as ethanol and organic acids to unyeasted dough formula was recently shown to significantly impact the rheology of resulting dough. In spite of available studies, the H2O2-hypothesis deserves scrutiny and the addition approach used to demonstrate the importance of several metabolites does not allow to correctly assess the impact of gradual accumulation of such metabolites in fermenting dough on dough characteristics. The general aim of this dissertation was hence to study the influence of yeast metabolism on characteristics of fermenting dough. More specifically, in first instance the release of H2O2 by yeast in fermenting dough was investigated. In second instance, differences in yeast metabolism caused by differences in the genetic background and the physiological state of yeast strains and by targeted mutations in fermentation related pathways were investigated and then used to study the influence of yeast metabolism on characteristics of fermenting dough. Re-evaluation of the production of H2O2 by yeast in dough and aqueous fermentation broth shows that the previously reported high levels of H2O2 in fermenting dough were most probably due to the lack of specificity of the potassium dichromate/acetic acid-based method used. Using the chemiluminescent HyPerBlu assay, no yeast H2O2 production could be detected in fermented dough or broth. Even though the formation of low levels of H2O2 cannot be ruled out due to the presence of catalase in flour and the fast reaction of H2O2 with gluten proteins, the present results go against the common believe that the changes in dough matrix rheological properties upon fermentation are due to production of H2O2 by yeast. Plausible alternative explanations for the observed rheological effects lie with the production of primary and secondary metabolites such as ethanol and organic acids by yeast during dough fermentation. In a first approach to assess the influence of yeast metabolism on characteristics of fermenting dough, we investigated to what extent different Saccharomyces cerevisiae strains impact dough rheological properties and gas holding capacity to a different extent. We evaluated if such differences are coupled to differences in the metabolites profile of the yeast strains. To this end, the impact of 25 yeast strains from widely differing applications on dough spread, fermentation properties and metabolite profile was analyzed. While the spread test proved an inadequate tool to quantify rheological differences resulting from strains with different fermentation rates, large differences in the fermentation ability and metabolites profile of the 25 strains were observed. Analysis of metabolites in fermented dough confirmed that acetic acid and succinic acid, rather than carbon dioxide (CO2), are responsible for lowering dough pH during fermentation. Results further suggest that the onset of CO2 release from dough is related to dough pH rather than to the volume of CO2 produced within the dough at this time. S. cerevisiae exhibits different growth phases when it is grown on a favorable carbon source. It is well known that the physiological state of yeast cells may affect metabolite production. The aim of this part of the study was to investigate the effect of harvesting yeast at seven different points of its growth profile on its fermentation performance and metabolite production. Coupling of these results to the effect of the yeast on critical dough fermentation parameters, such as gas retention potential, was used to further increase our understanding of the impact of yeast on dough related aspects. Yeast cells harvested during the diauxic shift and the post-diauxic growth phase showed the highest fermentation rate and, consequently, highest maximum dough height compared to yeast cells harvested in the exponential or stationary growth phases. Onset of CO2 loss from fermenting dough was correlated with the fermentation rate of yeast, but not with the amount of CO2 that accumulated up to the onset point. Quantitative analysis of yeast metabolites produced in dough provided a possible explanation for this observation, as they are produced in different levels depending on physiological phase and in concentrations that can influence dough matrix properties. Taken together, results demonstrated a strong effect of yeast physiology at the time of harvest on subsequent dough fermentation performance, and again hinted at an important role of yeast metabolites on dough gas holding capacity. As succinic acid could be one of the yeast metabolites with a large impact on the characteristics of fermenting dough, it was the focus of the last experimental chapter of this study. In first instance, the importance of the central yeast metabolism in succinic acid production was studied. By introducing mutations in the model S288C yeast strain, we show that the oxidative pathway of the TCA cycle and the glyoxylate shunt contribute significantly to succinic acid production during dough fermentation. Mutations in the reductive pathway of the TCA cycle did not affect the fermentation rate and succinic acid production. The changes in the levels of succinic acid produced by selected mutants only resulted in small pH differences in fermented dough, reflecting the buffering capacity of dough at a pH of around 5.1. Moreover, Rheofermentometer analysis using selected mutants revealed no difference in maximum dough height and gas retention capacity compared to the dough prepared with S288C. In conclusion, the results obtained in this dissertation point towards the importance of secondary yeast metabolites on the rheological properties of dough. The impact of differences in physiological state and the genetic diversity of yeast on metabolite production by the yeast during dough fermentation and their subsequent effect on rheological properties of dough were documented for the first time in this work. Due to the complexity of the fermentation process in yeasted dough, further research will be needed to investigate and exploit the potential of yeast in dough fermentation. The analytical approaches developed and used in this study will assist such research.nrpages: 174status: publishe