Heat resistance acquirement of the spoilage yeast Saccharomyces diastaticus during heat exposure.

The main fungal cause of spoilage of carbonated fermented beverages in the brewing industry is the amylolytic budding yeast Saccharomyces cerevisiae subsp. diastaticus (Saccharomyces diastaticus). Heat treatment is used to avoid microbial spoilage of the fermented beverages. Therefore, the spoilage capacity of S. diastaticus may be linked to its relative high heat resistance. Here, we assessed whether S. diastaticus can acquire heat resistance when exposed to heat stress. To this end, ascospores of S. diastaticus strain MB523 were treated at 60°C for 10 min followed by growing the surviving spores on a glucose-containing medium. The resulting vegetative cells were then allowed to sporulate again in sporulation medium. This cycle of heat treatment, vegetative growth, and sporulation was performed eight times in three independent lineages. After these eight cycles, the sporulation rate was similar to the start (∼75%) but the resulting ascospores were more heat resistant. The time needed to kill 90% of the population at 60°C (i.e. the D60-value) increased from 6.5 to 9.0 min (p = 0.005). The vegetative cells also showed a trend to increased heat resistance with an increase in the D52-value from 9.2 to 16.2 min (p = 0.1). In contrast, heat resistance of the vegetative cells that had not been exposed to heat during the eight cycles had been reduced with a D52-value of 4.2 min (p = 0.003). Together, these data show that S. diastaticus MB523 can easily acquire heat resistance by inbreeding while subjected to heat stress. Conversely, heat resistance can be easily lost in the absence of this stress condition, indicative of a trade-off for heat resistance.

Presence of Saccharomyces cerevisiae subsp. diastaticus in industry and nature and spoilage capacity of its vegetative cells and ascospores.

Saccharomyces cerevisiae sub-species diastaticus (S. diastaticus) is the main fungal cause of spoilage of carbonated fermented beverages in the brewing industry. Here, prevalence of S. diastaticus in nature and breweries was assessed as well as the spoilage capacity of its vegetative cells and spores. S. diastaticus could only be enriched from 1 out of 136 bark and soil samples from the Netherlands, being the first described natural isolate of this yeast outside South America. On the other hand, it was identified by PCR and selective enrichment in 25 and 21 out of 54 biofilm samples from beer filling halls in Asia, Africa, Europe and North America. ITS sequencing revealed that S. cerevisiae (including S. diastaticus) represented <0.05% of fungal DNA in 17 out of 20 samples, while it represented 0.1, 2 and 32% in samples VH6, VH1 and VH3 respectively. Next, vegetative cells and ascospores of the natural S. diastaticus isolate MB523 were inoculated in a variety of beer products containing 0.0-5.0% alcohol (v/v). Ascospores spoiled all beer products, while vegetative cells did not grow in Radler lemon 0.0, Radler lime mint 0.0 and Radler lemon lime 0.0. Notably, vegetative cells could spoil these Radlers when they first had been grown in alcohol free beer either or not mixed with Radler lemon lime 0.0. Conversely, vegetative cells that had been grown in Radler lemon lime lost their spoilage potential of this beer product when they had grown in YPD medium for more than 24 h. In addition, it was shown that cells grown in alcohol free beer were more heat resistant than cells grown in YPD (D52 40 min and ≤ 10.3 min, respectively). Together, these data show that S. diastaticus is a less prevalent variant of S. cerevisiae in nature, while it accumulates in breweries in mixed biofilms. Data also show that both vegetative cells and spores can spoil all tested beer products, the latter cell type irrespective of its environmental history.