The continuous growth in the craft beer market, and the increased interest and demands of consumers, have directed the efforts of brewers towards the production of differential and innovative beers. In this sense, non-conventional yeasts - other than the traditional domesticated ale (Saccharomyces cerevisiae) and lager (Saccharomyces pastorianus) yeasts - have gained attention as tools for new product development. Of great interest is the cryotolerant species S. eubayanus, isolated for the first time in Patagonia (Argentina) and identified as one of the parents of the hybrid species S. pastorianus (lager yeast). This work sought to contribute to the knowledge about the fermentative behavior of S. eubayanus, develop starter cultures suitable for the brewing industry, and evaluate different strategies for its application on a productive scale.
First, five strains of S. eubayanus (representative of the five geographically structured subpopulations) were tested in laboratory-scale fermentations, evaluating their fermentation performance (fermentation rate, attenuation, sugars consumption) and their organoleptic profile (esters, higher alcohols, phenols, sensory evaluation). The different fermentative characteristics observed and the differential production of volatile compounds between the strains demonstrated intraspecific phenotypic variability of the species. The strain CRUB 1568T was selected due to its good fermentation performance, moderate production of phenols (4-vinylguaiacol and 4-vinylphenol), and its contribution of the fruity ester’s ethyl hexanoate and ethyl octanoate.
From the selected strain, it was sought to establish the propagation conditions for the development of starter cultures for brewing. Different techniques were evaluated to determine the viability and vitality of the biomass produced, selecting the alkaline methylene violet staining method and laboratory-scale fermentations, respectively. A culture medium based on commercial malt extract was selected and optimized by evaluating 9 factors, with two levels for each factor, through an experimental Plackett-Burman design. Two factors showed significant influence on biomass production (yeast extract and acid casein peptone), and were later optimized by a Central Composite Design, through Response Surface Methodology. With the optimized culture medium (malt extract medium with 0.31 % w/v of yeast extract and 0.12 % w/v of acid casein peptone) it was possible to obtain a biomass production equal to that of the non-optimized medium (12 g/L), but with a 40 % a cost reduction. Finally, the propagation was scaled-up to 20 L, being able to obtain pure and quality S. eubayanus cultures, with adequate cell density to inoculate fermenters of up to 700 L.
Prior to transfer the starter cultures to the brewing industry, the interaction of S. eubayanus with hops was evaluated. For this, the fermentation performance of S. eubayanus CRUB 1568T in hopped and unhopped wort was studied, as well as the impact of the yeast on the measured (IBU) and sensory perception of bitterness. S. eubayanus showed tolerance to hop compounds (20 and 60 IBUs), presenting even an increased fermentation rate in hopped worts (34 % and 62 %, respectively). On the other hand, the IBUs obtained for beers fermented with S. eubayanus were 18 % higher than for the industrial strains, with this difference being notorious sensorially and even perceived as a harsh bitterness. These results allow us to adopt criteria for the application of S. eubayanus in brewing, and adequately predict the final profile of the beers produced with this yeast.
Subsequently, S. eubayanus CRUB 1568T was tested on brewing wort at a semi-pilot scale (20-40 L), observing a similar behavior to that obtained in the laboratory. Nevertheless, when this wild yeast was applied on a productive scale (1000-1500 L), it showed a poor fermentation performance. Fermentations were completed in 20 days (almost twice as long as laboratory and semi-pilot scales) with an average attenuation of 30 % (half of what was previously observed).
It was decided to test whether an increase in oxygen supply during fermentation could help improve fermentation performance on a larger scale. An improvement in fermentation performance was found by applying different oxygenation regimens, achieving complete fermentations in 5-7 days, with attenuation levels of 60 %.
This work encourages the use of non-conventional yeasts in brewing. The selection of native yeasts from the environment represents a valuable resource, not only for the development of innovative beers with greater productive differentiation, but also opens the possibility of conferring regional character to the product, with a consequent improvement in competitiveness. It was certainly found that the application of these yeasts in the industry requires different handling than traditional brewing yeasts, and opens a starting point for research and experimentation in relation to the different strategies that can be addressed.