Phenolic Off-flavor Research Articles

Inhibition of diastatic yeasts by Saccharomyces killer toxins to prevent hyperattenuation during brewing.

Secondary fermentation in beer can result in undesirable consequences, such as off-flavors, increased alcohol content, hyperattenuation, gushing, and the spontaneous explosion of packaging. Strains of Saccharomyces cerevisiae var. diastaticus are a major contributor to such spoilage due to their production of extracellular glucoamylase enzyme encoded by the STA1 gene. Saccharomyces yeasts can naturally produce antifungal proteins named "killer" toxins that inhibit the growth of competing yeasts. Challenging diastatic yeasts with killer toxins revealed that 91% of strains are susceptible to the K1 killer toxin produced by S. cerevisiae. Screening of 192 killer yeasts identified novel K2 toxins that could inhibit all K1-resistant diastatic yeasts. Variant K2 killer toxins were more potent than the K1 and K2 toxins, inhibiting 95% of diastatic yeast strains tested. Brewing trials demonstrated that adding killer yeast during a simulated diastatic contamination event could prevent hyperattenuation. Currently, most craft breweries can only safeguard against diastatic yeast contamination by good hygiene and monitoring for the presence of diastatic yeasts. The detection of diastatic yeasts will often lead to the destruction of contaminated products and the aggressive decontamination of brewing facilities. Using killer yeasts in brewing offers an approach to safeguard against product loss and potentially remediate contaminated beer.IMPORTANCEThe rise of craft brewing means that more domestic beer in the marketplace is being produced in facilities lacking the means for pasteurization, which increases the risk of microbial spoilage. The most damaging spoilage yeasts are "diastatic" strains of Saccharomyces cerevisiae that cause increased fermentation (hyperattenuation), resulting in unpalatable flavors such as phenolic off-flavor, as well as over-carbonation that can cause exploding packaging. In the absence of a pasteurizer, there are no methods available that would avert the loss of beer due to contamination by diastatic yeasts. This manuscript has found that diastatic yeasts are sensitive to antifungal proteins named "killer toxins" produced by Saccharomyces yeasts, and in industrial-scale fermentation trials, killer yeasts can remediate diastatic yeast contamination. Using killer toxins to prevent diastatic contamination is a unique and innovative approach that could prevent lost revenue to yeast spoilage and save many breweries the time and cost of purchasing and installing a pasteurizer.

Differences in Formation Mechanisms of Phenolic Off-Flavor Compounds among Yeast Species

Phenolic off-flavor compounds such as 4-vinylguaiacol (4-VG) affect the quality of fermented foods. In Saccharomyces cerevisiae, 4-VG production requires prenylated FMN (flavin mononucleotide) produced by the mitochondrial protein Pad1. To examine this mechanism further, we expressed Escherichia coli ubiX, whose gene product prenylates FMN in E. coli, in an S. cerevisiae Δpad1 strain, which recovered the ability to produce 4-VG. Because E. coli UbiX is expected to localize to the cytoplasm in the transformed yeast, next, we expressed a truncated Pad1 (tPad1) variant lacking the mitochondrial translocation signal sequence in S. cerevisiae Δpad1. The transformed strain also recovered the ability to produce 4-VG; furthermore, it produced a larger amount of 4-VG than wild-type S. cerevisiae, indicating that 4-VG production in S. cerevisiae may be mediated by the partial mislocalization of Pad1 to the cytoplasm. We also expressed the gene encoding Dekkera (Brettanomyces) bruxellensis phenolic acid decarboxylase (DbPAD) in S. cerevisiae Δpad1. The transformed yeast recovered the ability to produce 4-VG, indicating that the D. bruxellensis phenolic acid decarboxylation reaction does not require prenylated FMN. Sequencing of the cloned DbPAD gene showed that the translation start codon differs from previous reports; therefore, we recloned the DbPAD gene from the position predicted to be the translation initiation point (designated DbPADs). Transformation of S. cerevisiae Δpad1 with DbPADs further increased the amount of 4-VG produced. Collectively, these findings provide insight into the mechanism of off-flavor production by different yeast species.

Assessing Population Diversity of Brettanomyces Yeast Species and Identification of Strains for Brewing Applications.

Brettanomyces yeasts have gained popularity in many sectors of the biotechnological industry, specifically in the field of beer production, but also in wine and ethanol production. Their unique properties enable Brettanomyces to outcompete conventional brewer’s yeast in industrially relevant traits such as production of ethanol and pleasant flavors. Recent advances in next-generation sequencing (NGS) and high-throughput screening techniques have facilitated large population studies allowing the selection of appropriate yeast strains with improved traits. In order to get a better understanding of Brettanomyces species and its potential for beer production, we sequenced the whole genome of 84 strains, which we make available to the scientific community and carried out several in vitro assays for brewing-relevant properties. The collection includes isolates from different substrates and geographical origin. Additionally, we have included two of the oldest Carlsberg Research Laboratory isolates. In this study, we reveal the phylogenetic pattern of Brettanomyces species by comparing the predicted proteomes of each strain. Furthermore, we show that the Brettanomyces collection is well described using similarity in genomic organization, and that there is a direct correlation between genomic background and phenotypic characteristics. Particularly, genomic patterns affecting flavor production, maltose assimilation, beta-glucosidase activity, and phenolic off-flavor (POF) production are reported. This knowledge yields new insights into Brettanomyces population survival strategies, artificial selection pressure, and loss of carbon assimilation traits. On a species-specific level, we have identified for the first time a POF negative Brettanomyces anomalus strain, without the main spoilage character of Brettanomyces species. This strain (CRL-90) has lost DaPAD1, making it incapable of converting ferulic acid to 4-ethylguaiacol (4-EG) and 4-ethylphenol (4-EP). This loss of function makes CRL-90 a good candidate for the production of characteristic Brettanomyces flavors in beverages, without the contaminant increase in POF. Overall, this study displays the potential of exploring Brettanomyces yeast species biodiversity to find strains with relevant properties applicable to the brewing industry.

上面、下面ビール酵母のフェノール臭非産生機構とPAD1，FDC1遺伝子の 変異，欠失の関係

上面、下面ビール酵母のフェノール臭非産生機構とPAD1，FDC1遺伝子の 変異，欠失の関係

Screening for the Brewing Ability of Different Non-Saccharomyces Yeasts

Non-Saccharomyces yeasts have aroused interest in brewing science as an innovative and seminal way of creating new beer flavors. A screening system for potential brewing strains of non-Saccharomyces yeasts was set up to investigate the yeast’s utilization of wort sugars and to examine the effect of hop acids as well as ethanol on the growth of different yeast strains. Additionally, phenolic off-flavor (POF) and sensory odor tests of fermented wort samples were performed. The promising strains were further investigated for their propagation ability and for following fermentation trials. The produced beers were analyzed for secondary metabolites, ethanol content and judged by trained panelists. Subsequently to the screening, it was discovered that among the 110 screened yeast strains, approx. 10 strains of the species Saccharomycopsis fibuligera, Schizosaccharomyces pombe and Zygosaccharomyces rouxii generate promising fruity flavors during fermentation and were able to metabolize maltose and maltotriose as a prerequisite for the production of alcoholic beers. Consequently, the screening method described in this study makes it possible to investigate a tremendous number of different non-Saccharomyces yeasts and to test their brewing ability in a relatively short period of time.

Mutation and Deletion of PAD1 and/or FDC1 and Absence of Phenolic Off-Flavor Production in Top- and Bottom-Fermenting Yeasts

To convert ferulic acid into 4-vinylguaiacol (4-VG), Saccharomyces cerevisiae must have intact PAD1 and FDC1 genes. British-type top-fermenting yeast strains have nonsense mutations in both of these genes; whereas, bottom-fermenting yeast strains have a nonsense mutation in their S. cerevisiae-type FDC1, and have lost their S. eubayanus-type FDC1 and PAD1 genes. Here, top-fermenting yeast transformants in which wild-type PAD1 and FDC1 derived from the laboratory yeast S. cerevisiae S288C were inserted in the genome, exhibited ferulic acid decarboxylation activity. Similarly, bottom-fermenting yeast transformants expressing wild-type S. cerevisiae-type FDC1 or S. eubayanus-type FDC1 also exhibited ferulic acid decarboxylation activity. Thus, the lack of ferulic acid decarboxylation activity in bottom-fermenting yeast is due to mutation of the S. cerevisiae-type FDC1 gene, coupled with absence of the S. eubayanus-type FDC1 gene.

Investigation into the Potential of Lachancea fermentati Strain KBI 12.1 for Low Alcohol Beer Brewing

In brewing research, non-Saccharomyces yeasts have gained attention in recent years, owing to their potential to influence the characteristics and flavor of beer. The Lachancea genus possesses an uncommon trait, the production of significant amounts of lactic acid during alcoholic fermentation. This trait could potentially be harnessed for brewing purposes, particularly for the production of low alcohol beer. In this study, the potential of Lachancea fermentati strain KBI 12.1 was investigated for the production of low alcohol beer in low gravity wort. KBI 12.1 was characterized for sugar utilization, hop sensitivity, phenolic off-flavor (POF) production, and propagation performance. Lab scale fermentation trials in diluted wort (6.6°P) were conducted and compared to a brewers’ yeast, Saccharomyces cerevisiae WLP001. Fermentations were monitored for lactic acid and ethanol production, pH drop, and sugar consumption. In the final beers, amino acid and free amino nitrogen (FAN) content were determined and secondary metabolites were quantified. Lachancea fermentati KBI 12.1 showed to be unable to utilize maltotriose. The strain exhibited no POF production, minor hop sensitivity, and excellent propagation performance. Amino acid and FAN consumption were much lower compared to that of the brewers’ yeast. In the final beer fermented with KBI 12.1, the lactic acid concentration reached 1.3 g/L, giving the beer a sour taste. During sensory analysis, the beer was additionally described to have a fruity character. In conclusion, Lachancea fermentati KBI 12.1 proved to be a suitable strain for brewing purposes, with promising traits with regard to nonalcoholic and low alcohol beer brewing.

Reducing phenolic off-flavors through CRISPR-based gene editing of the FDC1 gene in Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid lager beer yeasts.

Today’s beer market is challenged by a decreasing consumption of traditional beer styles and an increasing consumption of specialty beers. In particular, lager-type beers (pilsner), characterized by their refreshing and unique aroma and taste, yet very uniform, struggle with their sales. The development of novel variants of the common lager yeast, the interspecific hybrid Saccharomyces pastorianus, has been proposed as a possible solution to address the need of product diversification in lager beers. Previous efforts to generate new lager yeasts through hybridization of the ancestral parental species (S. cerevisiae and S. eubayanus) yielded strains with an aromatic profile distinct from the natural biodiversity. Unfortunately, next to the desired properties, these novel yeasts also inherited unwanted characteristics. Most notably is their phenolic off-flavor (POF) production, which hampers their direct application in the industrial production processes. Here, we describe a CRISPR-based gene editing strategy that allows the systematic and meticulous introduction of a natural occurring mutation in the FDC1 gene of genetically complex industrial S. cerevisiae strains, S. eubayanus yeasts and interspecific hybrids. The resulting cisgenic POF- variants show great potential for industrial application and diversifying the current lager beer portfolio.

Selection of Pof-Saccharomyces eubayanus Variants for the Construction of S. cerevisiae × S. eubayanus Hybrids With Reduced 4-Vinyl Guaiacol Formation.

Saccharomyces pastorianus is an interspecies hybrid between S. cerevisiae and S. eubayanus. The identification of the parental species of S. pastorianus enabled the de novo reconstruction of hybrids that could potentially combine a wide array of phenotypic traits. Lager yeasts are characterized by their inability to decarboxylate ferulic acid present in wort, a phenotype also known as Pof- (phenolic off-flavor). However, all known S. eubayanus strains characterized so far produce clove-like aroma specific of 4-vinyl guaiacol, a decarboxylated form of ferulic acid. This study explored a non-GMO approach to construct Pof- S. eubayanus variants derived from the parental strain S. eubayanus CBS 12357. To rapidly screen a population of UV-mutagenized cells two complementary assays were developed. The first assay was based on the difference of light absorption spectra of ferulic acid and 4-vinyl guaiacol, while the second was based on the difference of sensitivity of Pof- and Pof+ strains to cinnamic acid. The S. eubayanus variant HTSE042 was selected and was confirmed not to produce 4-vinyl guaiacol. Whole genome sequencing revealed that this variant lost the subtelomeric region of the CHRXIII right arm that carried the two clustered genes SePAD1- SeFDC1 whose deletion in a naïve S. eubayanus strain (CBS 12357/FM1318) resulted in an identical phenotype. Subsequently, the Pof- variant was crossed with a Pof- S. cerevisiae partner. The resulting hybrid was not able to convert ferulic acid demonstrating the undisputable value of the mutagenized variant HTSE042 to eventually construct S. cerevisiae × S. eubayanus hybrids with phenotypic characteristics of S. pastorianus.

Rapid Screening Method for Phenolic Off-Flavor (POF) Production in Yeast

Because of an increased interest in complex niche products and the ongoing quest for improved brewing efficiency and quality, the interest in novel yeasts is increasing. However, many of these nonconventional yeasts and newly developed hybrids often produce undesirable off-flavors and, in particular, are characterized by strong production of phenolic aromas such as 4-vinyl guaiacol (4VG). Here, we describe a novel high-throughput, low-cost absorbance-based screening method that allows quick determination of the phenolic off-flavor (POF) production capacity of yeasts. We show that this method correlates well with the standard, more laborious GC-based methods and that it can be used to assess POF production of many different strains in parallel with minimal hands-on labor. This new method therefore opens exciting new routes to select rare variants or mutations that combine desirable phenotypes without showing unwanted production of POFs such as 4VG.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

Studies on the Simultaneous Formation of Aroma-Active and Toxicologically Relevant Vinyl Aromatics from Free Phenolic Acids during Wheat Beer Brewing.

During the brewing process of wheat beer, the desired aroma-active vinyl aromatics 2-methoxy-4-vinylphenol and 4-vinylphenol as well as the undesired and toxicologically relevant styrene are formed from their respective precursors, free ferulic acid, p-coumaric acid, and cinnamic acid, deriving from the malts. Analysis of eight commercial wheat beers revealed high concentrations of 2-methoxy-4-vinylphenol and 4-vinylphenol always in parallel with high concentrations of styrene or low concentrations of the odorants in parallel with low styrene concentrations, suggesting a similar pathway. To better understand the formation of these vinyl aromatics, each process step of wheat beer brewing and the use of different strains of Saccharomyces cerevisiae were evaluated. During wort boiling, only a moderate decarboxylation of free phenolic acids and formation of desired and undesired vinyl aromatics were monitored due to the thermal treatment. In contrast, this reaction mainly occurred enzymatically catalyzed during fermentation with S. cerevisiae strain W68 with normal Pof(+) activity (phenolic off-flavor) resulting in a wheat beer eliciting the typical aroma requested by consumers due to high concentrations of 2-methoxy-4-vinylphenol (1790 μg/L) and 4-vinylphenol (937 μg/L). Unfortunately, also a high concentration of undesired styrene (28.3 μg/L) was observed. Using a special S. cerevisiae strain without Pof(+) activity resulted in a significant styrene reduction (<LoQ), but also in low amounts of 2-methoxy-4-vinylphenol (158 μg/L) and 4-vinylphenol (46.7 μg/L), resulting in a less pronounced wheat beer aroma.

Efficient use of DNA molecular markers to construct industrial yeast strains

Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure.

ワインのフェノール性異臭の成因について

ワインのフェノール性異臭の成因について