S. Rainieri

Technological Properties and Temperature Response of Interspecific Saccharomyces Hybrids

Hybrids obtained by crossing cryotolerant and non-cryotolerant strains of Saccharomyces were studied. The hybrids, all sterile, are more vigorous and competitive than their parents over a wide range of temperatures. They develop well both at low temperatures (6°C) and at high temperatures (36°C) and have an optimum temperature range of 27–33°C. Minor fermentation compounds are always produced in medium quantities (while the parent are highly differentiated in this regard): this is true of glycerol, succinic acid, acetic acid and 2-phenylethanol. The hybrids are able to synthesise malic acid but to a lesser extent than their cryotolerant parents. The results regarding cryotolerant and non-cryotolerant parents and their respective hybrids were processed using a one-way analysis of variance and exhibited significant differences. The hybrids are of great enological interest both for their characteristics of competitiveness and stability and for their fermentation products.

Karyotyping of Saccharomyces strains with different temperature profiles

This study examined the karyotype, the fermentation performance and the optimum growth temperature (Topt) of 28 yeast strains all identified as species belonging to Saccharomyces sensu stricto. The strains were isolated from fermented musts, which had not been inoculated, at two temperature ranges: 20–40 °C and approximately 0–6 °C. The results demonstrated a correlation between the Topt and the chromosome organization. In particular, strains with Topt of less than 30 °C showed only two bands in the region between 365 and 225 kb, while those with a Topt greater than 30 °C had three bands in this size range. From a taxonomic viewpoint, the Topt is a better indicator for the Saccharomyces sp. than the ceiling temperature of 37 °C currently used to differentiate cryotolerant Saccharomyces bayanus and S. pastorianus from non‐cryotolerant S. cerevisiae and S. paradoxus strains.

The inheritance of mtDNA in lager brewing strains

In this work, we compared the mtDNA of a number of interspecific Saccharomyces hybrids (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces bayanus) to the mtDNA of 22 lager brewing strains that are thought to be the result of a natural hybridization between S. cerevisiae and another Saccharomyces yeast, possibly belonging to the species S. bayanus. We detected that in hybrids constructed in vitro, the mtDNA could be inherited from either parental strain. Conversely, in the lager strains tested, the mtDNA was never of the S. cerevisiae type. Moreover, the nucleotide sequence of lager brewing strains COXII gene was identical to S. bayanus strain NBRC 1948 COXII gene. MtDNA restriction analysis carried out with three enzymes confirmed this finding. However, restriction analysis with a fourth enzyme (AvaI) provided restriction patterns for lager strains that differed from those of S. bayanus strain NBRC 1948. Our results raise the hypothesis that the human-driven selection carried out on existing lager yeasts has favored only those bearing optimal fermentation characteristics at low temperatures, which harbor the mtDNA of S. bayanus.

Electrophoretic profile of hybrids between cryotolerant and non-cryotolerant Saccharomyces strains.

The chromosomal DNAs of cryotolerant Saccharomyces bayanus, non‐cryotolerant Saccharomyces cerevisiae strains and their intra and interspecific hybrids were separated by pulsed field electrophoresis (PFGE). The cryotolerant and non‐cryotolerant strains gave distinctly different electrophoretic profiles. The hybrids cryotolerant × cryotolerant and non‐cryotolerant × non‐cryotolerant were fertile and they gave the same electrophoretic karyotype as the respective parents. The cryotolerant × non‐cryotolerant hybrids were sterile and gave electrophoretic karyotypes which showed both the bands the parents have in common and those they do not share.

Method for the validation of intraspecific crosses of Saccharomyces cerevisiae strains by minisatellite analysis.

The crossing of Saccharomyces strains by spore conjugation is one of the ways to obtain new starter cultures for the fermentation industry. One of the major difficulties of this practice is the identification of the newly formed hybrids. In this work we describe an effective molecular method for the validation of Saccharomyces intraspecific crosses. The method described is based in the hypothesis that hybrids constructed by spore conjugation contain the sum of the genomes of both parental strains. As a consequence, the conjugation of spores of two yeasts showing different genomic fingerprinting profiles will result in a hybrid culture that will show the sum of both profiles. We demonstrated that the detection of polymorphism in two genes containing minisatellite-like sequences, either SED1 or AGA1, is suitable for this purpose. Using this strategy we were able to validate 15 crosses out of 162 hybridization attempts.

8 – The Brewer's Yeast Genome: From Its Origins to Our Current Knowledge

Brewing yeasts play the fundamental role of converting malt sugars into ethanol, carrying out the alcoholic fermentation that is at the basis of beer manufacturing. Their metabolism is of major importance for establishing the technological and nutritional characteristics of beer, therefore the understanding of their genome is an essential step for optimizing the brewing process, as well as improving the overall beer quality. Many microorganisms and different yeast species can grow and ferment spontaneously in a rich media such as malted barley juice, the fermentation substrate of beer. However, only two types of yeasts have been selected over the years and are currently employed for industrial beer production: ale and lager yeasts. Ale yeasts are polyploid Saccharomyces cerevisiae that differ from S. cerevisiae laboratory strains and show a high degree of interspecific polymorphism. Lager yeasts are now considered as part of the Saccharomyces pastorianus species, even though their classification has always been controversial. Their genome is very complex and seems to be composed of at least two different genomes: one nearly identical to S. cerevisiae and one originating from a non-S. cerevisiae yeast, closely related to Saccharomyces bayanus. This chapter illustrates the development of the complex taxonomical grouping of ale and lager brewing yeasts; it describes the steps that led to the current knowledge on the characteristics of their genome; and finally it attempts at elucidating the way they possibly originated and developed.