Use of PCR‐DHPLC (Polymerase Chain Reaction—Denaturing High Performance Liquid Chromatography) for the Rapid Differentiation of Industrial Saccharomyces pastorianus and Saccharomyces cerevisiae Strains

Strain specific detection and control of Saccharomyces pastorianus and Saccharomyces cerevisiae starter cultures is of great importance for the fermentation industry. The preconditions of strain specific fermentation characteristics can be ensured by periodic analysis and confirmation of the strain identity. With regard to industrial S. pastorianus and S. cerevisiae strains and a focus on brewing strains, the differentiation methods most available are time‐consuming and not very discriminative. In this work PCR‐DHPLC analysis was investigated as a novel approach for the differentiation of industrially used S. pastorianus and S. cerevisiae strains. The PCR‐DHPLC‐system was specific for S. cerevisiae strains and S. pastorianus hybrid strains that contain IGS2 rDNA, which originates from the S. cerevisiae ancestor. For the DNA of 177 strains of 41 non‐target species, which are typical for beverage and fermentation surroundings, the absence of PCR‐amplificates could be confirmed by DHPLC analysis. It was shown that single strains of S. cerevisiae and S. pastorianus could be differentiated. A strain specific differentiation within the group of top‐fermenting Saccharomyces cerevisiae strains could also be performed. For the group of bottom fermenting S. pastorianus brewing strains, strain‐to‐strain specific differences in the DHPLC chromatograms could be observed which can be used to differentiate and to compare two single strains with each other, although the comparison of chromatograms of an unknown S. pastorianus strain with a set of known S. pastorianus chromatograms could only reveal tendencies towards grouping into types. The differential DHPLC chromatogram characteristics (fluorescence intensities, number of peaks/side‐peaks/peak‐shoulders) within S. pastorianus are present, but not as distinctive as for S. cerevisiae. Additionally PCR‐DHPLC has advantages compared to other differentiation methods, such as species specificity, speed (2.5 h for one sample) and precision with the described limits.     

The Dynamic Changes of Proton Efflux Rate in Saccharomyces pastorianus Strains During High Gravity or Very High Gravity Brewing

The changes in the proton efflux rate (PER) during fermentation of normal gravity (NG), high gravity (HG) and very high gravity (VHG) wort by a lager yeast (Saccharomyces pastorianus) were monitored using an optimized PER test method. The values of the proton efflux rate in S. pastorianus decreased with increasing initial wort gravity. Moreover, the difference in the proton efflux rate values at the beginning of the fermentation was lower than at the end of fermentation from normal gravity to very high gravity brewing. These results demonstrated that the proton efflux rate in S. pastorianus was inhibited in the later stages of high gravity and very high gravity brewing. Furthermore, the changes of the proton efflux rate in S. pastorianus under the high ethanol concentration conditions appeared to depend on the concentration of ethanol in the fermentation liquid. A better negative correlation (P > 0.001, r = ?0.95) between the ethanol concentration at >4% (w/v) and the proton efflux rate was found. The changes of the proton efflux rate in the cells treated with exogenous ethanol confirmed that higher concentrations of ethanol could significantly inhibit proton efflux in S. pastorianus. This study offers a possible way to monitor and explain the performance of yeast in the complex environment of high gravity and very high gravity brewing.     

Sugar induces death of the bottom fermenting yeast Saccharomyces pastorianus

We confirmed that sugar-induced cell death (SICD) occurs in the bottom fermenting yeast Saccharomyces pastorianus under anaerobic conditions and that mitochondrial DNA is only partly required for SICD. Fermentation tests using different ratios of glucose and non-glucose nutrients demonstrated that SICD is influenced by the balance between these nutrients.     

Identification of bottom-fermenting yeast genes expressed during lager beer fermentation

It has been proposed that bottom-fermenting yeast strains of Saccharomyces pastorianus possess at least two types of genomes. Sequences of genes of one genome [S. cerevisiae (Sc)-type] have been found to be highly homologous (more than 90% identity) to S. cerevisiae S288C sequences, while those of the other [Lager (Lg)-type] are less so. To identify and discriminate Lg-type from Sc-type genes expressed during lager beer fermentation, normalized cDNA libraries were constructed and analysed. From approximately 22 000 ESTs, 3892 Sc-type and 2695 Lg-type ORFs were identified. Expression patterns of Sc- and Lg-type genes did not correlate with particular cell functions in KEGG classification system. Moreover, 405 independent clones were isolated that have no significant homology with sequences in the S288C database, suggesting that they include the bottom-fermenting yeast-specific (BFY) genes. Most of BFY genes have significant homology with the S. bayanus genome.     

Influence of indigenous yeasts on the fermentation and volatile profile of plum brandies

The aim of this study was to determine the influence of different yeasts isolated from fresh blue plum fruits (Aureobasidium sp.) and spontaneously fermenting plum musts (Kloeckera apiculata and Saccharomyces cerevisiae), as well as commercial wine and distillery strains, on the fermentation and chemical composition of plum brandies. Gas chromatography methods were used to detect major volatile components. The most rapid fermentation occurred in musts inoculated with S. cerevisiae. However, the highest concentration of ethanol was detected in samples after spontaneous fermentation (8.40% v/v). Plum brandies obtained after distillation contained from 66.3 (K. apiculata) up to 74.3% v/v ethanol (spontaneous fermentation). The samples after spontaneous fermentation were distinguished by a high content of acetoin, ethyl acetate and total esters, accompanied by a low level of methanol and fusel alcohols. Non-Saccharomyces yeasts were responsible for higher concentrations of esters and methanol, while S. cerevisiae strains resulted in increased levels of higher alcohols. It was also found that isolated indigenous strains of S. cerevisiae synthesized relatively low amounts of higher alcohols compared to commercial cultures. Samples obtained using the distillery strain of S. cerevisiae received the highest score (18.2) during sensory analysis and were characterized by a well-harmonised taste and aroma.     

Co-existence of two types of chromosome in the bottom fermenting yeast,Saccharomyces pastorianus

The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820-kb chromosome and the 1500-kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050-kb and the 1000-kb chromosomes. These chromosomes are thought to be S. cerevisiae-type chromosomes. In addition, these probes also hybridized weakly to the 1100-kb, 1350-kb, 850-kb and 700-kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100-kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350-kb chromosome of S. pastorianus. These results suggested that the 1100-kb and 1350-kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850-kb chromosome, and two gene probes from the left arm hybridized weakly to the 700-kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850-kb and 700-kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co-exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus. © 1998 John Wiley & Sons, Ltd.     

Comparative evaluation of some oenological properties in wine strains of Candida stellata, Candida zemplinina, Saccharomyces uvarum and Saccharomyces cerevisiae

Due to the recent changes in yeast taxonomy, a novel wine-related species Candida zemplinina as well as a “reinstated” species Saccharomyces uvarum have been accepted in addition to Candida stellata, Saccharomyces bayanus and Saccharomyces cerevisiae, and the use of the different taxon names has been inconsistent in the literature of food microbiology. The aim of this work is to make an exact comparison of genetically identified strains of these species, under oenological conditions. Dynamics and some important products of alcoholic fermentation were investigated in laboratory fermentations. The results show that C. zemplinina and C. stellata are similar in their strong fructophilic character. C. stellata produces more glycerol and fare more ethanol, which is comparable with that produced by S. uvarum. Strains of the latter species differed from S. cerevisiae mainly in low acetic acid production and lower ethanol yield. Revision of the oenological traits of these yeasts provides new data for consideration in the control of fermentation, with special regard to botrytized sweet wines, where they are frequently found in mixed population.     

Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation

The biochemical formation of yeast-derived sensory-active metabolites like higher alcohols and esters determines the different characteristics of aroma and taste in fermented beverages. In yeast fermentation process, a large number of environmental factors affecting the production of volatile aroma compounds are abundant. Factors like substrate composition in fermentation media as well as process parameters influencing these flavor-active metabolites have already been described. These factors can act on the expression of yeast genes involved in aroma metabolism resulting in concentration differences in esters and higher alcohols important for flavor and taste. The understanding of the function of genes involved in biosynthetic pathways of aroma-active substances as well as their regulatory mechanisms is needed to control the production of ester and higher alcohol synthesis to create specific aroma profiles in fermented beverages. This review discusses the known regulation and function of several individual genes (ATF1, ATF2, EEB1, EHT1, BAT1, BAT2 and BAP2) described in fusel alcohol and ester synthesis mainly in S. cerevisiae and S. pastorianus var. carlsbergensis. Also, different factors like oxygen and temperature that allow ester and higher alcohol synthesis to be controlled during yeast fermentation are described.     

Saccharomyces pastorianus: genomic insights inspiring innovation for industry

A combination of biological and non‐biological factors has led to the interspecific hybrid yeast species Saccharomyces pastorianus becoming one of the world's most important industrial organisms. This yeast is used in the production of lager‐style beers, the fermentation of which requires very low temperatures compared to other industrial fermentation processes. This group of organisms has benefited from both the whole‐genome duplication in its ancestral lineage and the subsequent hybridization event between S. cerevisiae and S. eubayanus, resulting in strong fermentative ability. The hybrid has key traits, such as cold tolerance and good maltose‐ and maltotriose‐utilizing ability, inherited either from the parental species or originating from genetic interactions between the parent genomes. Instability in the nascent allopolyploid hybrid genome may have contributed to rapid evolution of the yeast to tolerate conditions prevalent in the brewing environment. The recent discovery of S. eubayanus has provided new insights into the evolutionary history of S. pastorianus and may offer new opportunities for generating novel industrially‐beneficial lager yeast strains. Copyright © 2014 John Wiley & Sons, Ltd.     

Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus

This study investigated the formation and utilization of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Time-course papaya juice fermentations were carried out using pure cultures of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 and a mixed culture of the two yeasts at a ratio of 1:1000 (R2:NCYC2251). Changes in S. cerevisiae cell population, Brix, sugar consumption and pH were similar in the mixed culture and in the S. cerevisiae monoculture. There was an early growth arrest of W. saturnus in the mixed culture fermentation. A range of volatile compounds were produced during fermentation including fatty acids, alcohols, aldehydes and esters and some volatile compounds including those initially present in the juice were utilized. The mixed culture fermentation of S. cerevisiae and W. saturnus benefited from the presence of both yeasts, with more esters being produced than the S. cerevisiae monoculture and more alcohols being formed than the W. saturnus monoculture. The study suggests that papaya juice fermentation with a mixed culture of S. cerevisiae and W. saturnus may be able to result in the formation of more complex aroma compounds and higher ethanol level than those using single yeasts.     

Isolation of a lager yeast with an increased copy number of the YCK1 gene and high fermentation performance

The selection of yeast with good fermentation characteristics is critical for producing beer with desirable qualities. A yeast population was selected with an enhanced fermentation rate, referred to as high‐fermentation yeast (HFY), which was derived from the wild‐type Sacchromyces pastorianus yeast population (WTY). To identify genes that contribute to the fermentation performance, we compared the genetic profiles of the WTY and HFY populations by next‐generation sequencing. Several chromosomal regions were found to exhibit markedly different sequence coverage, suggesting chromosomal duplications and deletions, which might have occurred during selection of the HFY population. Among the genes with altered coverage, the copy number of the Saccharomyces eubayanus‐type YCK1 (SeYCK1) gene was almost two times higher in the HFY population than in the WTY population. The gene which is involved in glucose sensing in Saccharomyces cerevisiae was at a higher level in the HFY population throughout fermentation. These findings suggest that the chromosomal duplication of a region including the SeYCK1 gene locus of the HFY population is at least partially responsible for the differences in the fermentation properties between the WTY and HFY populations. © 2018 The Institute of Brewing & Distilling     

Selection of indigenous Saccharomyces cerevisiae strains for Nero d'Avola wine and evaluation of selected starter implantation in pilot fermentation

The present research studied Saccharomyces cerevisiae yeasts isolated from Nero d'Avola grapes, collected in different areas of the Sicily region. RAPD-PCR analysis with M13 primer was used for preliminary discrimination among 341 S. cerevisiae isolates. Inoculated fermentations with S. cerevisiae strains, exhibiting different RAPD-PCR fingerprinting, revealed the impact of selected strains on volatile compound concentration. Two selected strains were used in fermentation at cellar level and the restriction analysis of mtDNA on yeast colonies isolated during fermentation was used to control strain implantation. This study represents an important step to establish a collection of indigenous S. cerevisiae strains isolated from a unique environment, such as Nero d'Avola vineyards. Different starter implantation throughout inoculated fermentation represents an additional character, which might be considered during the selection program for wine starter cultures.     

Production of indole by wine-associated microorganisms under oenological conditions

A high concentration of indole has been linked to ‘plastic-like’ off-flavour in wines, predominantly in wines produced under sluggish fermentation conditions. The purpose of this study was to determine the ability of yeast and bacteria to form indole and whether tryptophan was required for indole accumulation during winemaking. Wine-associated yeast and bacteria species (Saccharomyces cerevisiae, Saccharomyces bayanus, Candida stellata, Hanseniaspora uvarum, Kluyveromyces thermoloterans, Oenococcus oeni, Lactobacillus lindneri, Pediococcus cerevisiae and Pediococcus parvulus) were screened for their potential to generate indole during alcoholic or malolactic fermentation. Tryptophan was required for the accumulation of indole in chemically defined medium, and all yeast and bacteria fermentations were able to accumulate indole. C. stellata showed the greatest potential for indole formation (1033 μg/L) and among the bacteria, the highest concentration was generated by L. lindneri (370 μg/L). Whether primary fermentation is the principle cause of indole formation remains to be determined. We hypothesise that during an efficient fermentation, indole is removed through catabolic metabolism, but, when a sluggish fermentation arises, non-Saccharomyces species might produce excess indole that is still present by end of fermentation.     

Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae

Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).