Investigation of the fermentation capacity of different Saccharomyces cerevisiae strains in bread dough.

The production of tasty leavened bread is almost inseparably linked to the presence of Saccharomyces cerevisiae yeast. The main function of yeast during the fermentation phase is the production of CO2, resulting in dough leavening. Besides CO2, the yeast’s fermentation metabolism also results in the release of ethanol and other minor constituents, such as glycerol, organic acids and flavor components. These metabolites are known to have a major impact on dough rheology and bread texture, aroma and flavor. The amount and type of metabolites produced by yeast during bread making depend on the ingredients (nutrient availability in flour, salt, etc.), fermentation time and temperature as well as on the pregrowth conditions and genetic background of the yeast strain used.

As a consequence of the growing consumer’s interest in a wider variety of tasty and healthy food products, industrial and artisanal bakeries are challenged to improve end products. Strategies to do so include changing dough formulations by, for example, addition of enzymes as bread improvers or wheat bran as a source of dietary fiber. Also yeast technology can be used to change bread properties. The Saccharomyces cerevisiae species embraces a lot of different strains, applied in various industrial fermentation processes, offering a large potential for possible application in bread making.

Preliminary experiments in our lab showed a very limited fermentation ability of several S. cerevisiae strains in wheat flour dough while studying the impact of strains from diverse industrial categories on dough rheology. As this remarkable observation was left unexplored, the first aim of this doctoral research was to gain insight in this inhibition phenomenon by characterizing it and identifying the inhibition-causing factor(s). The second aim was to evaluate the applicability of (non-bakery) S. cerevisiae strains in bread making.

In a first part, the fermentation capacity of a set of 45 S. cerevisiae strains originating from 5 yeast-based industrial categories (bread, beer, bio‑ethanol, sake & spirits and wine) was screened in different fermentation media. In a liquid fermentation medium, with a water activity adjusted to the level in dough, no industrial category differed significantly from the category of bakery strains, suggesting that they all had good fermentation potential. In wheat flour dough, variation in fermentation capacity could be noticed amongst the yeast strains. Most remarkable was the very limited fermentation ability of one fifth of the yeast strains. They produced up to 85% less CO2 than the best bakery strain, clearly demonstrating fermentation inhibition in wheat flour dough. The inhibition-sensitive yeast strains performed significantly better in a gluten‑starch model dough. Replacing the aqueous phase of the model dough by a wheat flour extract induced the same degree of inhibition as did wheat flour dough, allowing to conclude that the water extractable fraction of wheat flour contains a yeast-inhibiting component.

In the next part of this PhD work, the yeast inhibition phenomenon was characterized. The representative of the inhibition-sensitive yeast strains showed an inhibited fermentation capacity in the presence of flour from different wheat varieties. Moreover, rye, maize and barley had a similar effect on the yeast’s fermentation ability. After further analyses, the inhibition-causing factor turned out to be a water extractable wheat flour protein. Starting from a crude wheat flour extract, the inhibitor was isolated by activity-guided fractionation with cation exchange chromatography and reversed phase-HPLC. Identification of the proteins present in the purest inhibition-active fraction with LC/MS-MS and Edman degradation pointed towards the thaumatin-like protein as the inhibition causing factor. This finding was validated by the fact that the recombinantly expressed target protein also showed fermentation inhibition in representative of the sensitive yeast strains. Based on the differences in sensitivity of S. cerevisiae strains towards the inhibitor, a mode of action comparable with that of thaumatin-like tobacco osmotin was suggested, mainly affecting the permeability of the yeast cell wall. Although the inability of brewer’s yeasts to ferment a wheat flour batter was in the early seventies ascribed to purothionins, this was not conform to our results.

In the last part, the applicability of a selection of (non-bakery) S. cerevisiae strains in the bread making process was evaluated. After screening 45 yeast strains for their metabolite and aroma profiles, 10 strains (two from each industrial category), including two inhibition-sensitive strains, were selected for further examination of their impact on dough rheology and their influence on final bread aroma and quality characteristics. As was expected based on the variability in fermentation properties, metabolite analysis of these strains during fermentation also showed different profiles. In spite of these differences and of the fact that individual fermentation metabolites have already been shown to impact rheology in yeastless dough, no clear correlations were found between metabolites produced and rheological changes in yeasted dough. The constant change in density through CO2 production was assumed to have the foremost impact on dough properties. Similarly, significantly differing fermentation capacities did not necessarily result in significantly different bread volumes. Furthermore, no clear differences in crumb texture and structure between a control bakery strain and the other inhibition-insensitive yeast strains were observed. The determined variation in fermentation capacity and metabolite production between yeast strains did not seem sufficiently outspoken to result in significant differences in bread volume and texture. All inhibition-insensitive strains in the selection gave rise to breads with similar characteristics to those obtained with the control bakery strain. Aroma profile analysis hinted towards more significant differences between the different strains.

The research in this PhD study gave insight in a yeast specific inhibition phenomenon, caused by the thaumatin-like protein during wheat flour dough fermentation. This finding is of clear interest for yeast- and cereal-based fermentation industries, although further work is required on inhibitor quantification and its mode of action. The improvement of existing strains by breeding, the creation of new strains through genetic modification, or genetic modification of wheat to eliminate the harmful component might provide some solutions to enable yeast strains with a beneficial impact on dough and bread quality parameters or with a pleasant volatile compound profile, to be used in the bakery industry.