APPLICATION OF THE POLYMERASE CHAIN REACTION TO THE RAPID ANALYSIS OF BREWERY YEAST STRAINS

The rapid discrimination of closely-related Saccharomyces cerevisiae strains can pose a significant problem to breweries, in particular where closely related strains are being used simultaneously to manufacture different products. In this study, two PCR approaches have been examined to assess their usefulness for the discrimination of brewery ale and lager yeast strains. PCR using arbitrary primers (RAPD PCR) was found unsuitable for such an application since the DNA profiles generated from brewery strains were generally found to be identical, due presumably to the close genetic relatedness of these yeasts. In contrast, PCR using δ sequence primers could rapidly differentiate between many ale and lager strains and characteristic profiles for these were generated. This method could also be applied directly to yeasts isolated from brewery worts or from active dried yeast preparations. Results of such analyses were available within the working day.     

DIFFERENTIATION OF BREWERY YEAST STRAINS BY RESTRICTION ENDONUCLEASE ANALYSIS OF THEIR MITOCHONDRIAL DNA

Mitochondrial DNA (mtDNA) was isolated from different strains of brewery yeast and digested with various restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. Of the twenty restriction endonucleases used, only two—Aval and Haelli—produced different restriction fragment patterns when applied to the mtDNA from two strains of Saccharomyces uvarum. The restriction fragment patterns produced by the other eighteen enzymes were identical. Analysis of mtDNA from a strain of Saccharomyces cerevisiae with the same twenty restriction endonucleases revealed several differences with respect to Saccharomyces uvarum. Taken together, these results indicate that restriction endonuclease fragmentation patterns of mtDNA are useful as diagnostic tools for distinguishing strains of ale and larger yeast.     

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.     

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.     

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.     

AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

Quality assessment of lager brewery yeast samples and strains using barley malt extracts with anti-yeast activity

Membrane active anti-yeast compounds, such as antimicrobial peptides and proteins, cause yeast membrane damage which is likely to affect yeast vitality and fermentation performance, parameters which are notoriously difficult to analyse. In this work the sensitivity of lager brewery yeast strains towards barley malt extracts with anti-yeast activity was assessed with an optimised assay. It was found that yeast, obtained directly from a brewery, was much more sensitive towards the malt extracts than the same yeast strain propagated in the laboratory. Sensitivity to the malt extracts increased during the course of a laboratory scale fermentation when inoculated with brewery yeast. As the assay was able to differentiate yeast samples with different histories, it shows promise as a yeast quality assay measuring the yeast's ability to withstand stress which can be equated to vitality. The assay was also able to differentiate between different lager yeast strains of Saccharomyces cerevisiae propagated in the laboratory when challenged with a number of malt extracts of varying anti-yeast activity. The assessment of yeast strains in the presence of malt extracts will lead to the identification of yeast strains with improved quality/vitality that can withstand malt-associated anti-yeast activity during brewery fermentations.     

THE DETECTION AND IDENTIFICATION OF PEDIOCOCCUS AND MICROCOCCUS IN BREWERIES,USING A SEROLOGICAL METHOD

A commercially-available antiserum for the identification of group D strains of Streptococcus contains antibodies which react with Pediococcus and Micrococcus, and provides a convenient method to detect these genera in yeast, wort and beer. After suitable adsorption it can be used to distinguish between Pediococcus and Micrococcus and, within each genus, to differentiate between groups normally found in a brewery environment and those from other sources.     

ENTEROBACTER AGGLOMERANS—A NEW BACTERIAL CONTAMINANT ISOLATED FROM LAGER BEER BREWERIES

From a large number of bacterial strains isolated from all stages of the brewing process in four different beer breweries in South Africa, fifty-five bacterial strains were identified as Enterobacteriaceae by phenotypic analysis. All enterobacterial species previously reported in brewery samples, as well as eighteen strains of Enterobacter agglomerans were found. E. agglomerans strains were isolated from pitching yeast and fermenting wort samples. The occurrence of E. agglomerans in pitching yeast is significant and indicates that these bacteria survive the brewing process. E. agglomerans is regarded as a potential beer spoilage contaminant in lager beer breweries.     

THE USE OF CONTROLLED LEVELS OF ACTIDIONE FOR BREWING AND NON-BREWING YEAST STRAIN DIFFERENTIATION

The variability of results using actidione media for the identification of beer spoilage bacteria has been shown to be due to the heat sensitivity of this reagent. Critical evaluation shows that with a particular brewery yeast, media containing 0·06 p.p.m. allow the growth of a single brewing strain but inhibit others. This permits the rapid classification of yeast strains in this pitching yeast. In a further brewery, media containing 0·16 p.p.m. differentiate brewing yeast strains from beer spoilage yeasts and this permits the quantitative assessment of these for process control at all stages of brewing. In a particular instance where a culture yeast strain is not inhibited at 0·16 p.p.m. but is p-aminobenzoic acid dependent, a differential medium incorporating this low level of actidione and omitting the vitamin allows the identification of beer spoilage yeasts in process control. These results illustrate the potential of this approach for microbiological control in specific brewery problems.     

THE RAPID DETECTION OF BREWERY CONTAMINANTS BELONGING TO THE GENUS SACCHAROMYCES BY A SEROLOGICAL TECHNIQUE

The cells of contaminant Saccharomyces spp. are rendered fluorescent by means of fluorescein chemically bound to antibody protein. The fluorescent contaminants are observed microscopically, and an estimate of levels of contamination likely to be encountered in the brewery can be obtained within 3 hr. A serum of suitable specificity is obtained by careful cross-absorption with a brewing yeast, and by combining two sera, all brewery contaminants of the genus Saccharomyces so far encountered are detected. Careful selection of the brewing strain used for cross-absorption, and control of the cross-absorption procedure itself, allows the recognition of certain wild-type Sacch. cerevisiae strains.     

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.     

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.     

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.     

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.     

DIFFERENTIATION OF BREWERY YEASTS USING A DISC-DIFFUSION TEST

A simple test, based on the inhibitory effect of a range of compounds (cycloheximide, rhodamine 6G, brilliant green, 2, 3, 5 triphenyl tetrazolium chloride, ethidium bromide, Janus green) towards microorganisms, can be used to discriminate between brewery and brewery-associated yeast strains. Commercially available test discs are used for the tests but the manufacturers' conditions are modified to suit the growth requirements of brewery strains. The test is easy to perform and yields reproducible results.     

RESTRICTION ANALYSIS OF 2 μm DNA FROM DIVERSE STRAINS OF YEAST

2 μm plasmids from an ale yeast, a lager yeast and a strain of Saccharomyces diastaticus have been characterised by restriction analysis. Plasmids were similar to each other and conformed to a pattern which has been reported for well-characterised genetic stocks. The structure of 2 μm DNA, therefore, has been conserved in diverse strains of Saccharomyces. Preparative procedures used here are likely to be applicable to the detection and characterisation of plasmid DNA from a range of yeast genera. The apparent yield of 2 μm was increased when strains were grown at elevated temperature.     

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.     

Investigating flavour characteristics of British ale yeasts: techniques,resources and opportunities for innovation

Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour‐related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production. Copyright © 2014 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.