DETECTION OF WILD YEASTS IN THE BREWERY EFFICIENCY OF DIFFERENTIAL MEDIA

The efficiency of five differential media in the detection of wild yeasts was compared. On the basis of the types of wild yeast they are able to detect, these differential media were classified into two groups. Group I consists of actidione medium and lysine medium, and is suitable for the detection of non-Saccharomyces wild yeasts. In this group, the lysine medium detected more species and a higher percentage of wild yeasts than the actidione medium. Group II consists of crystal violet medium, SDM, and Lin's medium, and is suitable for the detection of Saccharomyces wild yeasts. In this group, Lin's medium is superior to crystal violet medium and SDM for wild yeast detection. It is suggested that lysine medium and Lin's medium be employed together for the detection of wild yeasts in the brewery.     

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.     

THE IMMUNOFLUORESCENT STAINING TECHNIQUE FOR THE DETECTION OF WILD YEASTS - PRACTICAL PROBLEMS

An apparent heavy wild yeast infection in pitching yeast has been detected using the immunofluorescent detection method. This infection could not be detected by conventional liquid forcing or plating techniques. The yeasts responsible were isolated and identified as Saccharomyces cerevisiae. The yeasts were very similar to the pitching yeast but varied in a number of respects associated with the cell wall such as flocculation character and giant colony morphology. The results suggest that the immunofluorescent-positive (IP) yeasts are variants of the culture yeast. As a result of this work it is felt that although immunofluorescence is of value for the rapid detection of infection, it must always be used in association with more conventional microbiological techniques.     

NITRATE REDUCTION AND ATNC FORMATION BY BREWERY WILD YEASTS

Control of NAD(P)H-dependent nitrate reductase (NR) and nitrite reductase (NiR) synthesis and activity in Hansenula anomala, Rhodotorula glutinis, Candida versatilis and Brettanomyces anomalus was investigated. Activities of both enzymes were high in all four yeasts when cultured in a medium containing nitrate as the sole source of cell nitrogen, but ammonia and amino-nitrogen were shown to rapidly repress nitrate assimilation and reduction. Little or no NR or NiR activity was detected in wort or beer-grown cultures. Only B. anomalus was found to excrete nitrite when grown in wort, but not at a concentration which could be chemically reduced to allow formation of a detectable concentration of N-nitrosamines. Cask beer (containing 16 mgl^?1 nitrate) contaminated with nitrate reducing wild yeasts, pre-grown on nitrate, contained < 10 μgl^?1 Apparent Total N-Nitrosocompounds (ATNC) following 10 weeks storage. It was concluded that contamination of wort, fermentation and finished beer by nitrate-reducing wild yeast is unlikely to result in formation of detectable ATNC .     

THE INCIDENCE AND SIGNIFICANCE OF WILD SACCHAROMYCES CONTAMINANTS IN THE BREWERY

The persistence of low levels of contamination by non-brewing Saccharomyces through several batch fermentations establishes the immuno-fluorescent method as a very sensitive procedure for estimating the microbiological purity of pitching yeasts. Trade return figures for draught beers show that in this brewery the principal cause for high rejection rates has, on several occasions, been contamination of pitching yeasts with “wild” Saccharomyces. The recommendation is made that pitching yeasts should be discarded when the level of infection achieves 100 cells of wild Saccharomyces per million cells of brewing yeast.     

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.     

THE VALUE OF LONG-CHAIN FATTY ACID COMPOSITION IN THE IDENTIFICATION OF SOME BREWERY YEASTS

The cellular long-chain fatty acids of 72 strains representing 29 species of brewery yeasts were extracted by saponification and analysed as methyl esters by gas chromatography. With this method, it was possible to distinguish between Saccharomyces cerevisiae and five other groups from the brewery industry, only four hours after they were obtained from 48 h old cultures under standard conditions. This compares favourably with the 7 to 10 days for conventional methods.     

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.     

THE RAPID DETECTION OF BREWERY SPOILAGE MICRO-ORGANISMS

Recent developments in the rapid detection of low concentrations of micro-organisms are discussed and their relevance to improved brewery quality control methods is assessed. A method based on the change in pH of a general purpose growth medium appears to offer the advantages of both time and sensitivity over methods currently in use.     

GROWTH OF AEROBIC WILD YEASTS

Wild yeasts of the genera Debaryomyces, Hansenula and Pichia are commonly considered to be associated with spoilage only under aerobic conditions. However, in pure cultures in either wort or a synthetic medium of yeast nitrogen base + 10% glucose, yeasts of these genera grew as well as a brewing strain of Saccharomyces cerevisiae under anaerobic conditions. Growth of S. cerevisiae was increased by the addition of unsaturated fatty acid (Tween 80) or ergosterol to the medium for anaerobic culture. No equivalent requirement was observed for the wild yeasts examined. Indeed, growth of the wild yeasts was often reduced by the addition of Tween 80, which as a surfactant prevented formation of the surface film of growth. Even under anaerobic conditions, these yeasts grew best with a surface pellicle. Although capable of good anaerobic growth in pure culture, growth of the wild yeasts was suppressed under anaerobic conditions in mixed culture with S. cerevisiae, simulating a contaminated brewery fermentation. However, the contaminants competed successfully with S. cerevisiae under aerobic conditions. There was no evidence of a “killer” effect, but prevention of pellicle formation, or production of inhibitory levels of pH or ethanol under anaerobic conditions could explain the suppression of wild yeasts under anaerobic fermentation conditions.     

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.     

RECENT ADVANCES IN THE RAPID DETECTION OF BREWERY MICRO-ORGANISMS AND DEVELOPMENT OF A MICROCOLONY METHOD

Recent developments in the rapid detection of low concentrations of micro-organisms are reviewed. A modification of the microcolony method, involving uptake of optical brighteners by developing yeast colonies, is described. The method includes the use of incident light microscopy and appears to offer advantages over other detection methods.     

PRODUCTION OF YEAST MASS DURING FERMENTATION OF WORT BY BREWERY YEASTS

The quantity of yeast dry matter present in fermenting wort increases throughout the period of sugar utilization. During the period of exponential production of new cells and yeast mass the yield of yeast dry matter per mol of hexose utilized (Y_hexose) does not vary significantly for the yeasts and worts examined. In the post-exponential phase, however, Y_hexose declines, to an extent which varies with strain and which is particularly marked if exponential growth has previously been restricted by deficiency of non-carbohydrate nutrients. The total yield is primarily determined by the concentration of the nutrient controlling the extent of exponential growth.     

THE DETERMINATION OF GLYCOGEN IN YEASTS

Four methods for the determination of glycogen in yeast have been compared in five strains of Saccharomyces cerevisiae over a range of glycogen contents. A new method has been developed which is specific, precise and more exhaustive than previously published procedures. After extraction with sodium carbonate and perchloric acid, the glycogen was hydrolysed with amyloglucosidase to glucose, which was estimated enzymically. The greater extraction of glycogen using this method cannot be explained by acid hydrolysis of glucans prior to treatment with amyloglucosidase. Further, the older data using the non-specific methods can now be equated with values obtained using a specific method.     

2μm DNA PLASMID IN BREWERY YEASTS

Using an agarose gel screening procedure, 2 μm DNA plasmid was detected in all of 10 brewing strains of Saccharomyces yeast examined and in both of two non-brewing, dextrin-utilising strains. Plasmid DNA was identified in yeast grown with access to air in MYGP medium or in hopped wort, and in yeast harvested from 3-day wort fermentations. The yeast plasmid is a suitable self-cloning vector for the genetic manipulation of brewing yeasts by transformation.     

THE DETECTION OF ENTEROBACTER AGGLOMERANS (ERWINIA HERBICOLA) IN BREWERY WORTS BY GAS CHROMATOGRAPHY

A gas chromatographic procedure is described to detect Enterobacter agglomerans, a bacterial contaminant in beer breweries. Ethanol, acetic acid, acetoin and 2,3-butanediol produced by bacteria in a growth medium was used to detect the contaminant at levels as low as three organisms/ml. A correlation was found between the initial numbers of contaminants present and the amounts of volatiles produced.     

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.     

DETECTION AND IDENTIFICATION OF LACTOBACILLUS LINDNERI FROM BREWERY ENVIRONMENTS

Ten detection media and six cultivation techniques were evaluated for the detection of a harmful brewery contaminant, for which the name Lactobacillus lindneri was recently revived. These bacteria showed slow and weak growth on solid media. However, enrichment of beer with NBB-C or the use of a mixture of MRS and beer (4:1 v/v) reduced the detection time by 2 days or more, depending on the strain, compared to cultivation on solid brewery media. Eight brewery isolates from different origins, the type strain and a test strain were characterized by carbohydrate fermentation tests (API 50 CHL), by a chemotaxonomical (SDS-PAGE) and by a genomic fingerprinting (ribotyping) method. The results obtained by all these methods indicated that the isolates studied form a single group and can be assigned to the species L. lindneri. However, using the current standard reagents, ribotyping cannot discriminate the strains isolated from different sources .     

THE GROWTH OF TOP-FERMENTATION YEASTS IN BINARY MIXTURES OF VARIOUS NITROGEN NUTRIENTS

The growth of English top-fermentation yeasts in various nitrogen sources, used singly—including ammonium phosphate and a number of amino acids—has previously been investigated and reported upon. But malt wort, the natural medium for yeast growth, contains, not a single nitrogen source, but a mixture of many derived from the breakdown of barley proteins. It is therefore important to know whether growth in such mixtures is simply the average of growth in the individual nitrogen sources or whether the different nitrogen sources interact with each other in such a way as to produce enhanced or diminished growth. The object of the work described in this paper was to ascertain whether the admixture of two different nitrogen sources introduces any new effects unpredictable from the known behaviour of the same nitrogen sources used singly. Nutrient media were prepared containing graded proportions of various selected pairs of nitrogen sources. Four different top-fermentation brewery yeasts were cultured in these media, measurements being made of fermentation, nitrogen assimilation and rate and extent of yeast growth. It was thus possible to observe the influence of the percentage composition of any particular binary mixture of nitrogen nutrients upon the growth and fermentation of a variety of yeasts. Among the mixtures investigated there was generally found a slight enhancement of growth and fermentation over what would be expected from the individual behaviour of the nitrogen sources. In a few cases, however, the observed enhancement was very large and was practically independent of the particular variety of yeast used. No important depressive effects were found in any of the mixtures. It may therefore be concluded that the mere admixture of different nitrogen sources can make a special contribution to yeast growth and fermentation; this contribution is generally relatively small but may in some circumstances becomes very significant. A chemical interpretation of some of the phenomena of yeast growth in mixed nutrients has been put forward.     

VARIATION IN THE SEROLOGICAL BEHAVIOUR OF YEASTS OF THE GENUS SACCHAROMYCES TOWARDS FLUORESCENT ANTIBODY

Immuno-fluorescent studies have shown that given populations of many yeasts of the genus Saccharomyces do not react in a uniform manner with anti-yeast sera. The evidence suggests that the antigenic composition of the surface layers of the cell wall is influenced by the physiological state of the cell, the absolute age of the cell being the probable controlling influence. The implications of such variations in response in respect to the practical uses of techniques involving fluorescent antibodies are discussed.