Novel interspecific Saccharomyces hybrids for enhanced brewing starter

Beer is a fermented beverage with a history as old as human civilization and its productive process has been spread all around the world becoming unique in every country and iconic of entire populations. Ales and lagers are by far the most common beers; however, the combination of raw materials, manufacture techniques and aroma profiles are almost infinite, so it is not surprising to notice that there is a large amount of different beer styles, each of them with unique characteristics. One of the major components that can have a deep impact on the final product is yeast, since it is able to pursue the metabolic reaction that can convert carbohydrates in wort, especially maltose and maltotriose, into ethanol, carbon dioxide and other minor compounds. Saccharomyces cerevisiae and Saccharomyces pastorianus are most used in breweries. However, an increasing number of different yeast starter cultures are commercially available, as new starter strains are critical differentiating factors among beers, especially in craft brewing. As consequence, it is becoming popular the research of novel reservoir in which isolate new performant yeasts. An interesting example which is catching the attention of many researchers are sourdoughs, since yeasts obtained from it have resulted strongly performant during wort fermentation. A yeast can be described as suitable for brewing in force of some typical desired traits, such as the ability to ferment maltose and maltotriose, to grow in relative low temperatures, to produce a particular aroma profile avoiding undesired compounds. To obtain all the desired traits in one single yeast cell the main technique used is the hybridization, which is the mating occurring between two different parental strains that can lead to a zygote with enhanced performance regarding both parents. Hybridization can be carried out in different ways, such as spore-to-spore mating, mass mating and rare-mating. In this study, we aimed at de novo creating, by spore-to-spore method, beer hybrids which combine good sugar utilization from S. cerevisiae strains isolated from sourdoughs and cold tolerance from species of Saccharomyces sensu stricto complex, such as S. uvarum and S. eubayanus, which exhibit the ability to stay metabolically active at the relative low temperature of about 12 °C. The aptitude of 7 S. cerevisiae sourdough strains to be exploited in cross-breeding was firstly evaluated by determining spore viability and sporulation efficiency. Spore viability was higher than 45% for except of strains Y17, while sporulation efficiency was higher than 30% for 5 out of 7 tested strains. MAT-PCR of monosporic clones supported that the majority of sourdough strains were HO/ho heterozygous, suggesting that they produce 50% mating-competent progenies. All sourdough strains were able ferment maltose and 6 strains were able to consume maltose as fast as glucose. We constructed two sets of S. cerevisiae x S. uvarum (11 hybrids) and 3 sets of S. cerevisiae x S. eubayanus (32 hybrids). Hybrid validation was carried out by PCR-RFLP of rDNA ITS regions and Pulsed Field Gel Electrophoresis (PFGE). All hybrids displayed ITS and chromosomal additive profiles. Three interspecies hybrids were tested for 15°P wort fermentation at laboratory scale. Hybrids generally performed better than parental strains, while S. cerevisiae x S. eubayanus hybrids were more performant than hybrid S. cerevisiae x S. uvarum under brewing conditions. In conclusion, the present work demonstrated that 1) S. cerevisiae sourdough strains are highly exploitable in cross-breeding for brewing; 2) S. eubayanus is better than S. uvarum when constructing yeast hybrids that express the typical beer yeast phenotype. Finally, we provided a collection of novel interspecies hybrids for future exploitation as beer starter