CLONING OF A YEAST GENE WHICH CAUSES PHENOLIC OFF-FLAVOURS IN BEER

A gene (POF1) has been cloned, which confers upon yeast (Saccharomyces cerevisiae) the ability to decarboxylate phenolic acids such as ferulic and trans-cinnamic acid. This property was previously shown to be a cause of phenolic off-flavour production in wort fermentations. The identity of the cloned gene was confirmed as POF1 by gene disruption techniques. Southern blotting of total genomic DNA revealed that sequences homologous to POF1 are conserved in Pof^? brewing strains of Sacch. cerevisiae. The transformation of a Pof^? lager strain with the cloned POF1 gene led to the production of an aroma characteristic of a phenolic off-flavour, when the transformed strain was used in wort fermentations. This latter observation suggests that the Pof^? phenotype of brewers' yeast is specifically due to the absence of a functional POF1 gene.     

CONTROL OF FERULIC ACID AND 4-VINYL GUAIACOL IN BREWING

Phenolic acids in beer are important because they can be decarboxylated to phenols, which usually impart off-flavours. An improved high performance liquid chromatographic system was used to monitor phenolic acids and phenols during the brewing process. Ferulic acid was the most significant phenolic acid found in beers prepared from malted barley. Extraction of ferulic acid from malt involved an enzymatic release mechanism with an optimum temperature about 45°C. Mashing-in at 65°C significantly decreased the release of free ferulic acid into the wort. Wort boiling produced 4-vinyl guaiacol by thermal decarboxylation, in amounts (0.3 mg/L) close to its taste threshold, from worts that contained high contents of free ferulic acid (> 6 mg/L). The capacity of yeasts to decarboxylate phenolic acids (Pof⁺ phenotype) was strong in wild strains of Saccharomyces and absent in all lager brewing yeast and most ale brewing yeasts. Some top-fermenting strains, especially those used in wheat beer production, possessed a weak decarboxylating activity (i.e. Pof^δ). During storage of beers there were appreciable temperature-dependent losses of 4-vinyl guaiacol. These results indicated that the production of 4-vinyl guaiacol is amenable to close technological control.     

CINNAMIC ACID AS THE BASIS OF A MEDIUM FOR THE DETECTION OF WILD YEASTS

Cinnamic acid (100 μg ml^?1) incorporated in a solid medium was found to inhibit the growth of brewing strains (Pof^?) of yeast while permitting the growth of Pof+ wild yeast contaminants. Typically, colonies of Saccharomyces cerevisiae var. diastaticus (Pof+) mixed with brewing yeast (S. cerevisiae NCYC 240) were visible after 5d incubation at 25°C. The incubation time required to detect a selection of brewery wild yeast isolates was found to vary from 3–12 d.     

A RAPID,SIMPLE AND RELIABLE METHOD OF DIFFERENTIATING BREWING YEAST STRAINS BASED ON DNA RESTRICTION PATTERNS

DNA was isolated from polyploid brewing ale and lager yeast strains using a simple and rapid procedure which was a modification of a previously described method of Seehaus et al.¹⁴ The isolated DNA was cut with a number of restriction enzymes and subjected to agarose gel electrophoresis. Significant differences in banding patterns were observed between a Saccharomyces cerevisiae ale strain DNA and Saccharomyces uvarum (carlsbergensis) lager strain DNA, particularly with the enzyme Hpal. Differences were also observed between the banding patterns of digests from two ale strains, and from two lager strains. Use of this technique with appropriate restriction enzymes should prove useful for the rapid differentiation of brewing yeast strains.     

Formation of 4-vinyl and 4-ethyl derivatives from hydroxycinnamic acids: Occurrence of volatile phenolic flavour compounds in beer and distribution of Pad1-activity among brewing yeasts

This work represents a survey of the occurrence of hydroxycinnamic acids and volatile phenols in a variety of beer styles. The contribution of 4-vinylguaiacol to the overall flavour perception of top-fermented specialty beers was shown. Significant differences in hydroxycinnamic acids (both free and ester-bound) and volatile phenol content between different beers were observed. The variability in volatile phenol content between different beers and beer styles can be explained by the high incidence of Pad1⁺ phenotype and the variability of Pad1 activity observed among top-fermenting brewing yeast strains. The relative importance of thermal versus enzymatic decarboxylation can account for the differences found between bottom and top-fermented beers. Concerning the optimisation of volatile phenol levels in beer, the selection of a suitable brewing yeast strain is the most important means of creating a phenolic taste profile in beer. Given that a considerable amount of hydroxycinnamic acids in beer still occurs in ester-bound form, enhancing the enzymatic release of these phenolic flavour precursors during mashing can greatly enhance the phenolic aroma potential of wort.     

PCR‐Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR‐amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five‐base (ScrFI) and two four‐base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR‐RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP‐PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.     

FERMENTATION PROPERTIES OF BREWING YEAST WITH KILLER CHARACTER

The cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae has been transferred to three different commercially-used brewing yeasts; two ale strains and one lager strain. The ease with which the character can be transferred is very strain dependent. In addition to killer character, mitochondria from the brewing strain have been transferred into the new ‘killer’ brewing strains. Fermentations carried out with the manipulated strains produced beers which were very similar to those produced by the control brewing strains. The beers produced by killer brewing strains containing brewing yeast mitochondria were most like the control beers and could not be distinguished from them in three glass taste tests. In addition to producing good beers the genetically manipulated yeasts killed a range of contaminant yeasts and were themselves immune to the action of Kil-k₁ killer yeasts.     

KAR1-MEDIATED TRANSFORMATION OF BREWING YEAST

Transfer of plasmid between nuclei has been observed in heterokaryons obtained from kar1 × KAR1 crosses³. On this basis, brewing yeast strain CC45 was forced to mate with K5-5A, a kar1 laboratory strain bearing plasmid YEpCR21. This multicopy plasmid carries the gene cyh2 and determines a dominant resistance to cycloheximide . Heteroplasmons having a CC45 nucleus and hosting YEpCR21 were obtained. Plasmid stability and brewing performance have been studied. The utility of kar1-mediated plasmid transference as an alternative method to transform brewing yeast is discussed. Advantages of YEpCR21 as a vector cloning for brewing yeast are considered as well .     

Emergence of [GAR+] cells in yeast from sake brewing affects the fermentation properties

In the traditional (kimoto) method of sake (Japanese rice wine) brewing, Saccharomyces cerevisiae yeast cells are exposed to lactate, which is produced by lactic acid bacteria in the seed mash. Lactate promotes the appearance of glucose-repression-resistant [GAR⁺] cells. Herein, we compared the resistance to glucose repression among kimoto, industrial, and laboratory yeast strains. We observed that the frequencies of the spontaneous emergence of [GAR⁺] cells among the kimoto strains were higher than those among the industrial and laboratory strains. The fermentation ability of a kimoto yeast (strain U44) was lower than that of an industrial strain (K701), as [GAR⁺] cells generally showed slower ethanol production. The addition of lactate decreased the fermentation abilities of the K701 strain by increasing the number of [GAR⁺] cells, but it did not affect those of the U44 strain. These results suggest that lactate controlled fermentation by promoting the appearance of [GAR⁺] cells in the industrial sake strains but not in the kimoto strains.     

Isolation of heterothallic haploid and auxotrophic mutants of Schizosaccharomyces japonicus

The fission yeast Schizosaccharomyces japonicus var. japonicus belong to the genus Schizosaccharomyces, together with Schizosaccharomyces pombe, which has been well studied as a model organism. In contrast, Sz. japonicus is poorly characterized and genetic tools were yet to be developed. We here report the isolation of the heterothallic haploids NIG2017, NIG2025 and NIG2028, which were derivatives of a Sz. japonicus homothallic strain (NIG2008). Based on the genomic sequence of Sz. japonicus, released by the Broad Institute, we found that Sz. japonicus also possesses orthologues of the mating‐type genes of Sz. pombe; two mat‐M (?) and two mat‐P (+) genes. As expected, heterothallic strains were defective in one of the Sz. japonicus mat genes (mat^sj). We confirmed that NIG2017 and NIG2025 strains only expressed mRNA from the mat^sj‐P genes, while homothallic strains expressed both mat^sj‐M and mat^sj‐P. Although the NIG2028 strain expressed both gene products, mat^sj‐P was found mutated, which may have conferred the heterothallic phenotype of the mutant. Thus, we concluded that these were stable heterothallic strains. We designated NIG2017 and NIG2025 as h⁺ and NIG 2028 as h^?, respectively. We also found additional h^? strains (NIG5872 and NIG5873) that arose from the cross between NIG2017 and NIG2028 derivatives. In addition to that, we have constructed a ura4^sj‐deleted strain and an ade6^sj‐mutated strain. We used these heterothallic strains and the auxotroph strains to perform spore dissection analysis to determine the genetic distances between several loci, and found that the mating type loci and ade6^sj locus were linked to centromeres. Copyright © 2009 John Wiley & Sons, Ltd.     

NISIN: A BACTERIOCIN WITH A POTENTIAL USE IN BREWING

Depending on the amount used and the strain of bacteria involved, nisin either kills lactic acid bacteria or inhibits their growth. In medium inoculated with approximately 10⁵ cells ml^?1 of a sensitive strain of Lactobacillus (BSO 375) nisin, added at levels recommended for commercial use (100 International Units ml^?1, killed all the cells in less than 6 h. In the absence of nisin this inoculum grew to a concentration of 10¹⁰ cells ml^?1 in about 50 h. Lower nisin concentrations killed fewer cells but inhibited the growth of those still viable. For the more resistant strain Lactobacillus (BSO 343) growth was only inhibited at the higher nisin concentrations. Nisin maintained its activity against lactic acid bacteria in brewing fermentations. It had no effect on the growth and fermentative performance of the 9 brewing yeast strains tested, and, in a pilot brewery fermentation, had no deleterious affect on the taste of the beer produced. Nisin could be used either as a preventative measure by regular addition to fermentations, or as a remedial measure once contamination by lactic acid bacteria had been detected.     

Isolation of a Schizosaccharomyces pombe Gene Which in High Copy Confers Resistance to the Nucleoside Analogue 5-Azacytidine

Treatment of Schizosaccharomyces pombe with the C5 DNA methyltransferase (C5Mtase) inhibitor 5-azacytidine (5-azaC) has previously been shown to induce G2 checkpoint-dependent cell cycle arrest. S. pombe strains defective in both the checkpoint control pathways and in DNA repair processes are sensitive to 5-azaC. Here we describe the isolation of azr1as a multi-copy suppressor of the 5-azaC sensitivity of G2 checkpoint and DNA repair-deficient strains. azr1⁺ encodes a putative 25 kDa protein with limited homology to a Saccharomyces cerevisiae open reading frame of unknown function. The azr1⁺ gene is not essential and the null mutant shows no alteration in either DNA repair or checkpoint properties. We also report the sequence of the putative fission yeast cytidine deaminase gene, designated pcd1⁺, which lies immediately adjacent to azr1⁺ but which plays only a moderate role in suppression of 5-azaC sensitivity. These data have been deposited with EMBL nucleotide sequence database, Accession Number X98329. © 1997 John Wiley & Sons, Ltd.     

Most,but not all,yeast strains in the deletion library contain the [PIN+] prion

The yeast deletion library is a collection of over 5100 single gene deletions that has been widely used by the yeast community. The presence of a non‐Mendelian element, such as a prion, within this library could affect the outcome of many large‐scale genomic studies. We previously showed that the deletion library parent strain contained the [PIN⁺] prion. [PIN⁺] is the misfolded infectious prion form of the Rnq1 protein that displays distinct fluorescent foci in the presence of RNQ1–GFP and exists in different physical conformations, called variants. Here, we show that over 97% of the library deletion strains are [PIN⁺]. Of the 141 remaining strains that have completely (58) or partially (83) lost [PIN⁺], 139 deletions were able to efficiently maintain three different [PIN⁺] variants despite extensive growth and storage at 4 °C. One strain, cue2Δ, displayed an alteration in the RNQ1–GFP fluorescent shape, but the Rnq1p prion aggregate shows no biochemical differences from the wild‐type. Only strains containing a deletion of either HSP104 or RNQ1 are unable to maintain [PIN⁺], indicating that 5153 non‐essential genes are not required for [PIN⁺] propagation. Copyright © 2009 John Wiley & Sons, Ltd.     

CHARACTERIZATION OF BREWING YEAST STRAINS BY NUMERICAL ANALYSIS OF TOTAL SOLUBLE CELL PROTEIN PATTERNS

Total soluble cell proteins from 33 yeast strains from the brewing industry were extracted and subjected to polyacrylamide gel electrophoresis. Yeast strains were grouped by computerized numerical analysis of protein banding patterns. Three clusters were obtained at r>0.90. Cluster I contained 21 Saccharomyces cerevisiae lager beer strains. Cluster II comprised two strains isolated from beer with a phenolic off flavour and a third strain used for lager beer brewing. Cluster III consisted of two bottom ale yeasts. Protein patterns of yeast strains within each cluster corresponded closely or were identical. However, the intensity of certain bands often varied and the number of peaks recorded was not identical. These minor differences were reproducible and regarded as characteristic for the specific strains. Protein patterns can therefore be used to characterize or fingerprint individual yeast strains.     

The Use of Molecular Methods for Detecting and Discriminating Salmonella Associated with Foods — A Review

The genus Salmonella is composed of two species, Salmonella enterica and Salmonella bongori. Only S. enterica subsp. enterica is considered of human clinical significance and consists of 1478 serotypes. A large number of virulence genes and virulence-enhancing genes have been described for Salmonella. There are more than 30 Salmonella specific genes that have been used for the polymerase chain reaction to detect and characterize Salmonella. The sensitivity of detection of Salmonella from complex matrices such as food and feces by PCR is invariably enhanced using nonselective or selective enrichment, particularly if followed by immuno-magnetic separation in addition to coupling the PCR with ELISA formats. R-plasmids are considered to be the main factors responsible for the horizontal transfer of antibiotic resistance genes in Salmonella. A sizeable number of primer pairs are available for determining by the PCR the presence of many antibiotic resistance genes in Salmonella isolates that are not necessarily specific for Salmonella. The collective PCR detection of members of the genus Salmonella in foods and environmental samples has been achieved by amplification of invA gene sequences that are highly conserved among all Salmonella serotypes in addition to the amplification of his gene sequences also present throughout the genus Salmonella. Amplification of 16S rDNA sequences have also been found useful for genus specific detection of Salmonella. d-Tartrate (dT⁺) fermenting strains have been found to result in less severe gastrointestinal infections than d-tartrate-nonfermenting (dT^-) strains. Primers have therefore been developed for distinguishing between (dT⁺) and (dT^-) strains. Among the molecular techniques available for strain discrimination of Salmonella isolates, pulsed field gel electrophoresis, random amplified polymporphic DNA analysis, ribotyping, multilocus sequence typing, subtracted finger printing, and enterobacterial repetitive intergeneric consensus typing have been found useful. Multiplex PCR has been found effective for simultaneously detecting Salmonella and other pathogens in foods, particularly with real-time PCR.     

Characterization of MEL genes in the genus Zygosaccharomyces

We cloned and sequenced a Zygosaccharomyces cidri MEL gene with a view to investigating the structure and regulation of yeast MEL genes. The amino acid sequence deduced from the nucleotide sequence showed 78·6% and 78·2% similarity to Saccharomyces cerevisiae and Saccharomyces pastorianus α-galactosidases, respectively. The expression of the MEL gene in several Zygosaccharomyces strains was induced by galactose. An electrophoretic karyotype of several Zygosaccharomyces species was obtained using contour-clamped electric field gel electrophoresis. The minimum number of chromosomes was five for Z. cidri, six for Z. fermentati, three for Z. florentinus, and four for Z. microellipsoides. The sizes of the chromosomes were generally larger than those of S. cerevisiae, the smallest containing approximately 0·4 megabase. The MEL gene was located, using the Z. cidri MEL gene as a probe, on the largest chromosome of the Z. cidri strains. In addition, a smaller chromosome (600 kb) in Z. cidri strain CBS4575 showed hybridization to the homologous MEL probe. This chromosome was absent in Z. cidri strain CBS5666. The probe hybridized to the largest chromosome of Mel⁺ Z. fermentati strains but failed to hybridize to any chromosome of Mel⁺ Z. mrakii or Z. florentinus strains. These results suggest the existence of a polymorphic MEL gene family in the yeast Zygosaccharomyces. The sequence has been deposited in the EMBL Data Library under Accession Number L24957.     

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

Nicotinamide adenine dinucleotide (NAD⁺) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD⁺ decomposition products. NAD⁺ biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD⁺ biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD⁺ and NADH (the reduced form of NAD⁺) analyses on BY4742 wild‐type, NAD⁺ salvage pathway knockout (npt1Δ) and NAD⁺ de novo pathway knockout (qpt1Δ) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized ¹⁴C labelled nicotinic acid in the culture media combined with HPLC speciation and both UV and ¹⁴C detection to quantitate the total amounts of NAD⁺ and NADH and the amounts derived from the salvage pathway. We observed that wild‐type and qpt1Δ yeast exclusively utilized extracellular nicotinic acid for NAD⁺ and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions, suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observed that NAD⁺ concentrations decreased in all three strains under CR. However, unlike the wild‐type strain, NADH concentrations did not decrease and NAD⁺: NADH ratios did not increase under CR for either knockout strain. Lifespan analyses revealed that CR resulted in a lifespan increase of approximately 25% for the wild‐type and qpt1Δ strains, while no increase in lifespan was observed for the npt1Δ strain. In combination, these data suggest that having a functional salvage pathway is required for lifespan extension under CR. Copyright © 2009 John Wiley & Sons, Ltd.     

Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus

The bottom‐fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)‐type and two S. bayanus (Sb)‐type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p‐hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G‐418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p‐hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5‐fluoroorotic acid (5‐FOA) plates to generate the desired Ura^– homozygous disruptant (Scura3Δ::TDH3p‐hygro/Scura3Δ::TDH3p‐hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom‐fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1‐overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3? lager strain will be a useful host for constructing strains of relevance to brewing. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.