Copper Binding in Sweet Worts Made from Specialty Malts.

Trace levels of copper can impact the flavor stability of beer. The main source of copper is malt, and the wort copper levels are established during mashing and lautering. This study focuses on sweet worts made from experimental roasted and caramel malts. Potentiometric titrations using ion-selective electrodes combined with electron paramagnetic resonance spectroscopy have been used to investigate Cu(II) binding in worts as well as the impact of Cu(II) ions on the wort oxidative stability. High-temperature treatment during malting decreased Cu(II) binding affinities in the worts, with roasted malt worts having lower affinities than caramel malt worts of similar color and pH. Electron paramagnetic resonance spectra indicated dipeptides as the main Cu(II) chelators. A positive correlation between Cu and free amino nitrogen levels in worts is demonstrated. In dark worts with high rates of radical formation, Cu(II) had pronounced antioxidative effects. In contrast, moderate prooxidative effects were observed when adding Cu(II) to pale worts with inherently low rates of oxidation.

Modelling flavour formation in roasted malt substrates under controlled conditions of time and temperature.

Drum roasted products are used to impart colour, flavour and mouthfeel to beers. Here we designed a laboratory-scale roaster (100 g batch size) capable of precise time-temperature control and investigated the impacts of time, temperature and roasting substrate (barley, pale malt or germinated green malt) on formation of 20 key odour active aroma volatiles. Principal Components Analysis (PCA) of flavour volatile data across 37 laboratory roasted and 6 commercial roasted products generated a product flavour space depicting the relationship between roasting conditions and concentrations of these 20 compounds. Response surface models were produced for aroma compound concentrations across the design space of roasting times and temperatures for each substrate. These clearly illustrate the impacts of substrate moisture content and prior history (e.g. whether germinated or germinated and kilned) on flavour formation. In low moisture substrates a steep increase in associated heterocyclic aroma compound production was noted at process temperatures >180 °C.

S. cerevisiae × S. eubayanus interspecific hybrid, the best of both worlds and beyond.

Saccharomyces pastorianus lager-brewing yeasts have descended from natural hybrids of S. cerevisiae and S. eubayanus. Their alloploidy has undoubtedly contributed to successful domestication and industrial exploitation. To understand the early events that have led to the predominance of S. pastorianus as lager-brewing yeast, an interspecific hybrid between S. cerevisiae and S. eubayanus was experimentally constructed. Alloploidy substantially improved the performance of the S. cerevisiae × S. eubayanus hybrid as compared to either parent regarding two cardinal features of brewing yeasts: tolerance to low temperature and oligosaccharide utilization. The hybrid's S. eubayanus subgenome conferred better growth rates and biomass yields at low temperature, both on glucose and on maltose. Conversely, the ability of the hybrid to consume maltotriose, which was absent in the S. eubayanus CBS12357 type strain, was inherited from its S. cerevisiae parent. The S. cerevisiae × S. eubayanus hybrid even outperformed its parents, a phenomenon known as transgression, suggesting that fast growth at low temperature and oligosaccharide utilization may have been key selective advantages of the natural hybrids in brewing environments. To enable sequence comparisons of the parental and hybrid strains, the genome of S. eubayanus CBS12357 type strain (Patagonian isolate) was resequenced, resulting in an improved publicly available sequence assembly.

Functional analysis and transcriptional regulation of two orthologs of ARO10, encoding broad-substrate-specificity 2-oxo-acid decarboxylases, in the brewing yeast Saccharomyces pastorianus CBS1483.

The hybrid genomes of Saccharomyces pastorianus consist of subgenomes similar to those of S. cerevisiae and S. eubayanus, and impact of the genome structure on flavour production and its regulation is poorly understood. This study focuses on ARO10, a 2-oxo-acid decarboxylase involved in production of higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified, three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10. Substrate specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by individually expressing them in a pdc1Δ-pdc5Δ-pdc6Δ-aro10Δ-thi3Δ S. cerevisiae strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived from branched-chain, sulphur-containing amino acids and preferably phenylpyruvate. Expression of both alleles was induced by phenylalanine, however in contrast to the S. cerevisiae strain, the two genes were not induced by leucine. Additionally, LgSeubARO10 showed higher basal expression levels during growth with ammonia. ARO80, which encodes ARO10 transcriptional activator, is located on CHRIV and counts three Sc-like and one Seub-like copies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine induction, revealing 'trans' regulation across the subgenomes. ARO10 transcript levels showed a poor correlation with decarboxylase activities. These results provide insights into flavour formation in S. pastorianus and illustrate the complexity of functional characterization in aneuploid strains.

amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae.

Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, are therefore valuable assets in ambitious metabolic engineering programs. In the present work, the new recyclable dominant marker cassette amdSYM, formed by the Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene (Aspergillus nidulans amdS), is presented. The amdSYM cassette confers S. cerevisiae the ability to use acetamide as sole nitrogen source. Direct repeats flanking the amdS gene allow for its efficient recombinative excision. As previously demonstrated in filamentous fungi, loss of the amdS marker cassette from S. cerevisiae can be rapidly selected for by growth in the presence of fluoroacetamide. The amdSYM cassette can be used in different genetic backgrounds and represents the first counterselectable dominant marker gene cassette for use in S. cerevisiae. Furthermore, using astute cassette design, amdSYM excision can be performed without leaving a scar or heterologous sequences in the targeted genome. The present work therefore demonstrates that amdSYM is a useful addition to the genetic engineering toolbox for Saccharomyces laboratory, wild, and industrial strains.