Repurposing brewery contaminant yeast as production strains for low-alcohol beer fermentation

A number of fungal isolates were recently obtained from a survey of the microbiota of multiple breweries and brewery products. Here, we sought to explore whether any of these brewery contaminants could be repurposed for beneficial use in beer fermentations, with particular focus on low-alcohol beer. 56 yeast strains were first screened for the utilization of different carbon sources, ability to ferment brewer's wort, and formation of desirable aroma compounds. A number of strains appeared maltose-negative and produced desirable aromas without obvious off-flavours. These were selected for further scaled-up wort fermentations. The selected strains efficiently reduced wort aldehydes during fermentation, thus eliminating undesirable wort-like off-flavours, and produced a diverse volatile aroma profile. Sensory analysis of the beer samples using projective mapping identified two strains, Trigonopsis cantarellii and Candida sojae, that produced beers similar to a commercial reference lager beer. 30 L-scale wort fermentations were performed with these two strains together with a commercial Saccharomycodes ludwigii reference strain. Both strains performed comparably to the commercial reference, and the T. cantarellii strain in particular, produced low amounts of off-flavours and a significantly higher amount of the desirable monoterpene alcohol trans-geraniol. The strain was also sensitive to common food preservatives and antifungal compounds, and unable to grow at 37 °C, suggesting it is relatively easily controllable in the brewery, and appears to have low risk of pathogenicity. This study shows how the natural brewery microbiota can be exploited as a source of non-conventional yeasts for low-alcohol beer production.

High Foam Phenotypic Diversity and Variability in Flocculant Gene Observed for Various Yeast Cell Surfaces Present as Industrial Contaminants

Many contaminant yeast strains that survive inside fuel ethanol industrial vats show detrimental cell surface phenotypes. These harmful effects may include filamentation, invasive growth, flocculation, biofilm formation, and excessive foam production. Previous studies have linked some of these phenotypes to the expression of FLO genes, and the presence of gene length polymorphisms causing the expansion of FLO gene size appears to result in stronger flocculation and biofilm formation phenotypes. We performed here a molecular analysis of FLO1 and FLO11 gene polymorphisms present in contaminant strains of Saccharomyces cerevisiae from Brazilian fuel ethanol distilleries showing vigorous foaming phenotypes during fermentation. The size variability of these genes was correlated with cellular hydrophobicity, flocculation, and highly foaming phenotypes in these yeast strains. Our results also showed that deleting the primary activator of FLO genes (the FLO8 gene) from the genome of a contaminant and highly foaming industrial strain avoids complex foam formation, flocculation, invasive growth, and biofilm production by the engineered (flo8∆::BleR/flo8Δ::kanMX) yeast strain. Thus, the characterization of highly foaming yeasts and the influence of FLO8 in this phenotype open new perspectives for yeast strain engineering and optimization in the sugarcane fuel-ethanol industry.

A Control Alternative for the Hidden Enemy in the Wine Cellar

Brettanomyces bruxellensis has been described as the principal spoilage yeast in the winemaking industry. To avoid its growth, wine is supplemented with SO2, which has been questioned due to its potential harm to health. For this reason, studies are being focused on searching for, ideally, natural new antifungals. On the other hand, it is known that in wine production there are a variety of microorganisms, such as yeasts and bacteria, that are possible biological controls. Thus, it has been described that some microorganisms produce antimicrobial peptides, which might control yeast and bacteria populations. Our laboratory has described the Candida intermedia LAMAP1790 strain as a natural producer of antimicrobial compounds against food spoilage microorganisms, as is B. bruxellensis, without affecting the growth of S. cerevisiae. We have demonstrated the proteinaceous nature of the antimicrobial compound and its low molecular mass (under 10 kDa). This is the first step to the possible use of C. intermedia as a selective bio-controller of the contaminant yeast in the winemaking industry.

Influence of yeast autolysis after alcoholic fermentation on the development of Brettanomyces/Dekkera in wine

<p style="text-align: justify;"><em>Brettanomyces</em>, a contaminant yeast, is relatively common in wines and mainly in red wines during barrel aging. The results presented here relate to the effects of yeast lees autolysis on the growth of <em>Brettanomyces</em>. Experiments were realised in a culture medium after alcoholic fermentation, in a hydroalcoholic wine-like solution and in a red wine. <em>Brettanomyces</em> was inoculated at low level : 10² cfu/ml and the growth was controlled by counting on agar appropriate medium. Yeast lees from <em>S. cerevisiae</em> were added to these media in the presence or absence of an exogenous enzymatic preparation containing pectinase and β (1—&gt;3) glucanase activities. <em>Brettanomyces</em> is able to grow in the synthetic media containing yeast lees. The addition of the glucanase preparation on yeast lees was correlated with an enhance in extracellular glucose level. This higher glucose level corresponds to the acceleration of hydrolysis of cell-wall glucans but was not correlated with a higher level of <em>Brettanomyces</em> population. This result confirms that this yeast multiply by fermenting very small quantities of glucose. In red wine the implantation of <em>Brettanomyces</em> is easy. With a low level of inoculation (10² cfu/ml), contaminant yeast grows rapidly to 10⁶ cfu/ml (in 5 days). The presence of <em>S. cerevisiae</em> lees in red wine enhances significantly <em>Brettanomyces</em> population to 10⁷ cfu/ml. During their development, these yeast produced less than 200 µg/1 of 4-ethyl phenol and 4-ethyl gaïacol. Volatile phenol content in red wine containing yeast lees were 3-5 times lower. Yeast lees have probably the particular property to being able to adsorb these volatile phenols by complexion with the cell-walls.</p>

Bioluminescent Standard Curves for Quantitive Determination of Yeast Contaminants in Carbonated Beverages

The bioluminescent adenosine triphosphate (ATP) assay is a rapid and sensitive tool for quantitating contaminant yeast levels in beverage samples. A simple model system is described for generating standard curves relating yeast ATP to conventional colony forming units (CFUs). Bioluminescent standard curves were generated by spiking commercial cola or diet lemon-lime samples with Saccharomyces rouxii ATCC 36141. Yeast cells were concentrated onto filters under vacuum and ATP was subsequently extracted from the cells for analysis. Correlation coefficients for each S. rouxii standard curve indicated strong linear relationships between ATP and CFU levels (r&gt; 0.90). A composite standard curve (r = 0.97) of data collected from all the S. rouxii-spiked studies predicted yeast levels from spiked cola samples in later experiments. When predicted yeast CFU values were plotted against conventional yeast CFU values for three different yeast types, a correlation coefficient of r = 0.82 was obtained.