Abstract
The beverage industry needs to ensure a high-quality product until the end of shelf life, while consumers demand mild and sustainable preservation methods. These mild preservation methods carry a possible risk to quality, safety and integrity of the products. Strategies to counteract or solve these issues must be effective against
... read more
all spoilage yeasts and all physiological phases. The organism used in this study is Saccharomyces cerevisiae subsp. diastaticus (S. diastaticus), the main fungal cause of spoilage of carbonated fermented beverages in the brewing industry. In this Thesis I studied its ecological niche, its abundance in nature and the built environment, its phylogeny, and its genomic and phenotypic heterogeneity, in particular heat resistance of its vegetative cells and ascospores.
The prevalence of S. diastaticus in nature and industry was explored. Bark, soil and biofilm samples were collected. Data showed that S. diastaticus is a lowly abundant variant of S. cerevisiae in nature, while it accumulates in mixed yeast biofilms in breweries.
The spoilage ability of cells and ascospores of S. diastaticus strain MB523 was assessed. The ascospores were shown to be able to spoil all tested beers and beer-related products. In contrast, cells did only grow in Radler when they were first grown in alcohol free beer or in alcohol free beer mixed with Radler. Conversely, cells from Radler lost their ability to grow in this beer product when they had been growing in a standard medium (YPD) for more than 24 hours. This can be explained by cellular memory of acquired stress resistance.
S. cerevisiae and S. diastaticus strains (identification based on PALL PCR) were collected from breweries, isolated from nature, or obtained from stock centers. Genome sequences positioned 38 of the 43 S. diastaticus strains in the Beer / Mosaic clade and the other 5 strains in 3 other clades. The strains in the Beer / Mosaic clade were generally more heat resistant than the other S. diastaticus and S. cerevisiae strains. Together, this study showed that the S. diastaticus is polyphyletic and also highly heterogeneous with respect to heat resistance. Moreover, neither the STA1 gene nor the commercial PALL PCR are predictive for heat resistance or the position within the S. cerevisiae tree.
Heat resistance of vegetative cells and ascospores of 2 S. diastaticus strains and 1 S. cerevisiae strain was studied. Generally, both S. diastaticus strains were more heat resistant than S. cerevisiae. Transcriptomics analysis revealed genes that are higher expressed in the heat resistant strains. For instance, over 60 genes were higher expressed in both S. diastaticus strains during spore development when compared to S. cerevisiae, including OSW1, CWP1, CWP2 and SRD1. Deletion of these genes impacted various stages of spore formation as well as heat resistance of spores. This shows that these genes indeed play an important role in sporulation and / or the increased heat resistance of these 2 S. diastaticus strains.
To assess whether S. diastaticus can acquire heat resistance by inbreeding, ascospores of S. diastaticus strain MB523 were treated at 60 °C for 10 min followed by vegetative growth of the surviving spores and induction of sporulation. After 8 cycles, not only the resulting ascospores but also the vegetative cells were more heat resistant. In contrast, heat resistance decreased during 8 cycles without heat treatment of the ascospores. Genomic sequencing after each cycle revealed that alleles of 147 genes gradually increased during the cycles in the three heat exposed lineages. Together, these data show that S. diastaticus MB523 can easily acquire increased heat resistance by inbreeding.
show less
