STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

Differentiation of brewing yeast strains by pyrolysis mass spectrometry and Fourier transform infrared spectroscopy

Two rapid spectroscopic approaches for whole-organism fingerprinting—pyrolysis mass spectrometry (PyMS) and Fourier transform infrared spectroscopy (FT-IR)—were used to analyse 22 production brewery Saccharomyces cerevisiae strains. Multivariate discriminant analysis of the spectral data was then performed to observe relationships between the 22 isolates. Upon visual inspection of the cluster analyses, similar differentiation of the strains was observed for both approaches. Moreover, these phenetic classifications were found to be very similar to those previously obtained using genotypic studies of the same brewing yeasts. Both spectroscopic techniques are rapid (typically 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of the successful discrimination of both ale and lager yeasts. We believe that these whole-organism fingerprinting methods could find application in brewery quality control laboratories. © 1998 John Wiley & Sons, Ltd.     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

Biodiversity of brewery yeast strains and their fermentative activities

We investigated the genetic, biochemical, fermentative and physiological characteristics of brewery yeast strains and performed a hierarchical cluster analysis to evaluate their similarity. We used five different ale and lager yeast strains, originating from different European breweries and deposited at the National Collection of Yeast Cultures (UK). Ale and lager strains exhibited different genomic properties, but their assimilation profiles and pyruvate decarboxylase activities corresponded to their species classifications. The activity of another enzyme, succinate dehydrogenase, varied between different brewing strains. Our results confirmed that ATP and glycogen content, and the activity of the key metabolic enzymes succinate dehydrogenase and pyruvate decarboxylase, may be good general indicators of cell viability. However, the genetic properties, physiology and fermentation capacity of different brewery yeasts are unique to individual strains. Copyright © 2014 John Wiley & Sons, Ltd.     

THE SPORULATION AND MATING OF BREWING YEASTS

A study has been made of the sporulating behaviour of twenty selected brewing strains of yeast, and the mating activity of the products of sporulation. ‘Lager’ yeasts (strains of Saccharomyces carlsbergensis) in general sporulated to a lesser degree and more slowly than ‘ale’ yeasts (strains of Saccharomyces cerevisiae) and produced 1-or 2- spored asci compared with 2-or 3- spored asci for the latter yeasts. Most of the parent strains of S. cerevisiae were shown to be heterozygous for mating type, and they were all probably either triploid or aneuploid. Two of the strains of S. carlsbergensis were apparently homozygous for mating type and also triploid or aneuploid. The compatibility system favours outbreeding of yeasts, ‘ale’ yeasts being more compatible with ‘lager’ yeasts than with other ‘ale’ yeasts.     

INHIBITION OF BEER-SPOILAGE LACTIC ACID BACTERIA BY NISIN

149 strains of bacteria, mostly brewery contaminants able to spoil wort or beer, and 12 brewing strains of yeast (8 ale and 4 lager strains) have been screened using a well-test assay for sensitivity to the food preservative, Nisin (E234), Nisin inhibited growth of 92% of the gram-positive strains, predominantly lactic acid bacteria of the genera Lactobacillus and Pediococcus. In contrast, all 32 gram-negative strains tested, except 3 Flavobacter strains, were Nisin-resistant; in addition none of the brewing yeasts showed Nisin-sensitivity. Therefore. Nisin has potential applications in preventing spoilage of worts or beers by lactic acid bacteria.     

RESTRICTION MAPPING OF THE POF I GENE

The gene (POFI) which imparts to certain yeasts the ability to decarboxylate phenolic acids to corresponding phenolic compounds has been analysed by restriction mapping. New restriction sites have been used to examine differences between Pof⁺ and Pof^? Saccharomyces cerevisiae strains. Southern Blot analysis of selected yeast strains has demonstrated that the POFI gene sequence is highly conserved between the Pof⁺ strain from which the gene was cloned, two Pof^? lager brewing strains and one Pof⁺ Saccharomyces brewery isolate. However, sequence differences have been found between the original Pof⁺ strain, a Pof^?laboratory strain and a Pof^? ale brewing strain.     

BREWING YEAST IDENTIFICATION AND CHROMOSOME ANALYSIS USING HIGH RESOLUTION CHEF GEL ELECTROPHORESIS

Contour-clamped homogeneous electric field (CHEF) gel electrophoresis has been used to study the karyotypes of a range of Saccharomyces cerevisiae yeast strains. The time required from sampling yeast cultures to CHEF analysis was achieved within six hours, making this procedure very useful in reference and quality control work in the brewing industry. Regions of the chromosome profiles were closely studied by adjusting electrophoresis conditions to increase resolution between bands. Both ale and lager strains of brewing yeasts were studied alongside haploid laboratory strains. By comparing different regions of the profiles even very closely related strains of lager yeast could be distinguished. Brewing strains consistently had significantly more chromosome bands than haploid laboratory strains. The electrophoretic karyotypes of brewing yeasts were represented as groups of bands on CHEF gels which apparently comigrated with their haploid chromosomal counterparts.     

FACTORS AFFECTING THE SURVIVAL OF LYOPHILIZED BREWERY YEAST STRAINS

Some of the factors that contribute to the loss of viability of brewery yeast strains during lyophilization (freeze-drying) have been investigated. A lyophilization technique for the maintenance of brewery yeast strains with higher viabilities than those previously reported has been developed. Three lyophilized strains of ale yeast still had a survival rate of 60% after periods of storage of up to three years, while a lager yeast strain maintained a viability of approximately 50% during storage for eighteen months.     

MYGP + COPPER,A MEDIUM THAT DETECTS BOTH SACCHAROMYCES AND NON-SACCHAROMYCES WILD YEAST IN THE PRESENCE OF CULTURE YEAST*

Lin's copper sulphate medium for the detection of non-Saccharomyces wild yeast was modified, mainly by reducing the concentration of copper sulphate, with the aim of producing a medium that could detect a wide range of both non-Saccharomyces and Saccharomyces wild yeast. The performance of the resultant MYGP + Cu medium was compared to that of crystal violet and lysine media using pure cultures of wild yeast, pitching yeast and brewery samples. The growth of the pitching yeasts examined was suppressed more in MYGP + Cu than in crystal violet and the 4 ale yeasts were more susceptible to copper inhibition than the 3 lager yeasts. Neither MYGP + Cu nor crystal violet could detect all the wild Saccharomyces species tested but the proportion detected by each was similar. Twenty-seven out of 28 non-Saccharomyces wild yeasts tested could be detected in MYGP + Cu. When brewery samples were examined, the detection rate by MYGP + Cu compared favourably with that by a combination of crystal violet and lysine. MYGP + Cu is particularly suited to the monitoring of the production of cask-conditioned ales.     

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 INFLUENCE OF WORT GLUCOSE LEVEL ON THE FORMATION OF AROMATIC HIGHER ALCOHOLS

Increasing amounts of glucose either in solid form or in solution were added to all-malt worts, and to worts that contained maize as adjunct. Fermentations were then carried out using either ale or lager yeast. The resultant beers were analysed by gas chromatography for the aromatic higher alcohols tryptophol, tyrosol and phenylethanol; the amyl alcohols were also determined as typical representatives of the aliphatic higher alcohols. Increasing concentration of glucose in the wort resulted in either decreasing levels of tyrosol and phenylethanol (ale yeast) or increasing levels (lager yeast). Tryptophol demonstrated a different pattern both with ale and lager yeasts, by increasing in concentration up to a certain maximum, then decreasing.     

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.     

THE RAPID DETECTION OF BREWERY CONTAMINANTS BELONGING TO THE GENUS SACCHAROMYCES—EXAMINATION OF LAGER YEASTS

For the detection of wild Saccharomyces contaminants in lager yeasts, two different sera combinations, absorbed by two different strains of Sacch. carlsbergensis, are required. The most satisfactory test system applies to lager yeasts belonging to sub-group I, where 60% of Sacch. cerevisiae contaminants are detected, all Sacch. carlsbergensis strains belonging to sub-group II, and all other brewery contaminants belonging to the genus Saccharomyces. The system used for lager yeasts belonging to sub-group II is less satisfactory: 50% of Sacch. cerevisiae strains and all Sacch. carlsbergensis strains belonging to sub-group I were detected, but only 60% of strains belonging to other Saccharomyces spp. The limitations of the antigenic structures ascribed to the various species of this genus are demonstrated, and it is suggested that an immuno-fluorescent test procedure should be used in any further studies relating to antigenic inter-relationships.     

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.     

The Effects of Osmotic Pressure and Ethanol on Yeast Viability and Morphology

The selection of a brewing yeast strain with the required fermentation and recycling characteristics is critical. The yeast strain will influence the rate and extent of fermentation, the flavour characteristics and the overall quality and stability of the finished beer, and consequently, the economic viability of the brewery. Since high gravity worts can have a deleterious effect on yeast fermentation performance, it is imperative that the strain selected be suitable for this environment, which includes a capacity to withstand high osmotic pressures and elevated ethanol levels. Under controlled in vitro osmotic and ethanol induced stresses, there was a decline in mean cell volume in both lager and ale yeast strains. Whilst significant reductions in viability were observed in the lager strains, the ale strains studied were not affected. Cell surface investigations revealed shrinkage of the yeast cells and crenation of the outside envelope under both stresses, although exposure to ethanol had a more marked effect on the yeast cell surface than sorbitol‐induced elevated osmotic pressure.     

APPLICATION OF PULSED FIELD CHROMOSOME ELECTROPHORESIS IN THE STUDY OF CHROMOSOME XIII AND THE ELECTROPHORETIC KARYOTYPE OF INDUSTRIAL STRAINS OF SACCHAROMYCES YEASTS

Pulsed field chromosome electrophoresis is a powerful new technique in yeast genetics which permits the resolution of intact yeast chromosomes in an agarose gel matrix. We utilized contour-clamped homogeneous electric field electrophoresis (CHEF) to survey representative strains of Saccharomyces yeasts from the brewing, baking, distilling, sake and wine industries for their electrophoretic karyotypes. All of the strains tested were found to have a unique chromosomal profile, indicating the potential of this technology for “fingerprinting” prototrophic strains of Saccharomyces yeasts. By employing an ILV2 gene probe specific for chromosome XIII, we determined that all of the industrial strains of Saccharomyces yeasts possessed a chromosome XIII which migrated in an identical fashion to chromosome XIII from a reference haploid strain of Saccharomyces cerevisiae. While one lager yeast strain, Saccharomyces carlsbergensis M244, was found to contain two alleles of ILV2 when digested genomic DNA was probed with ILV2, the presence of a novel independently migrating chromosome XIII could not be detected. A homeologous chromosome XIII in this yeast will therefore have to be determined by genetic analysis. Pulsed field chromosome electrophoresis is concluded to be a technology with immediate application to Quality Control and Research and Development programs in industries using Saccharomyces yeasts.     

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.     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

DETECTION OF WILD YEASTS IN THE BREWERY

The potential of an established culture medium, Wallerstein Laboratories' nutrient agar, for the detection of wild yeasts has been evaluated and recommended for microbiological quality control in brewery laboratories. Wild yeast strains, including Saccharomyces species, can be differentiated from strains of Saccharomyces cerevisiae by the colour, form and rate of growth of their colonies on this medium within 2–3 days. The sensitivity of the method is such that one wild yeast can be detected in a Saccharomyces cerevisiae population of the order of 10° cells.