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.     

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 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.     

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 .     

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.     

FORMULATION AND TESTING OF CUPRIC SULPHATE MEDIUM FOR WILD YEAST DETECTION

A new differential medium, cupric sulphate medium, used for the detection of wild yeasts has been formulated and tested. This medium suppressed the growth of culture yeasts and supported that of most non-Saccharomyces wild yeasts. It is not suitable for the detection of Saccharomyces wild yeasts. The contaminating wild yeasts in yeast samples and swab samples were easily detected by this medium. Since Lin's medium or modified Lin's medium is suitable for the detection of Saccharomyces wild yeasts, it is suggested that they be used in conjunction with cupric sulphate medium for detecting a more complete spectrum of wild yeasts.     

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.     

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.     

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.     

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.     

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.     

BIOTIN-ACTIVE COMPOUNDS,THEIR EXISTENCE IN NATURE AND THE BIOTIN REQUIREMENTS OF YEASTS

After a short account of the discovery of biotin and the progress of early biotin research, the natural occurrence of biotin with particular consideration to the raw materials used in the fermentation industry and its products is described. Of the many known biotin derivatives, those appearing in nature and those which can be converted to biotin-active (or inactive) compounds by simple procedures are reported. Ways to by-pass the need for biotin in microbes is discussed. The importance of biotin in yeast production, the biotin requirements of yeast, and the effect of culture conditions on these requirements are reported. The close relationship between the participation of biotinylenzymes and the biotin requirements is noted.     

CLASSIFICATION OF CERTAIN PEDIOCOCCI ISOLATED FROM BREWERY PRODUCTS

New solid and liquid media have been developed for the cultivation of a group of slow-developing pediococci, isolated from brewery products, that have previously proved difficult to cultivate. These media also formed a suitable basis for the production of special media for identification and classification of such pediococci. A physiological and biochemical study indicated that the strains investigated are closely akin to a group of pediococci which by other workers are classified as Pediococcus damnosus.     

HYBRIDIZATION OF NON-SPORULATING AND WEAKLY SPORULATING STRAINS OF BREWER'S AND DISTILLER'S YEASTS

A method for hybridization of strains of industrial yeasts with auxotrophically marked haploid and diploid strains was developed, using the RD-auxotrophic technique of Gunge & Nakatomi. Brewing and distilling strains were hybridized with aα or αα diploids and sporulating, near-tetraploid strains were obtained which could be further subjected to genetic analysis and hybridized by conventional methods. Hybrids could also be obtained between the industrial strains and auxotrophically marked haploid strains, but the viability of the spores obtained from these hybrids was low, and they were probably near-triploids. This method makes possible the genetic analysis of brewing yeast strains and others which do not sporulate, and allows the mapping of characters which are important in industrial practice, so that new yeast strains having extended ranges of desirable characteristics can be obtained by standard genetic techniques.     

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.     

VITAMIN REQUIREMENTS OF CERTAIN PEDIOCOCCI ISOLATED FROM BREWERY PRODUCTS

A medium has been developed for determination of the vitamin requirements of a number of pedicocci, most of which were isolated from brewery products and classified as Pediococcus damnosus. Tween 80 was essential to a number of the P. damnosus strains in the medium used. Folic acid was active only on a test strain, P. cerevisiae NCTC 8066. Biotin had a strong growth-promoting effect on most of the strains. Pantothenic acid was essential or highly stimulating to the growth of all strains. Riboflavin is considered essential to the P. damnosus strains, but was inactive with the two test strains used, P. cerevisiae NCTC 8066 and P. acidilactici NCIB 6990. Pyridoxin had a growth-promoting effect on most of the strains, and was essential to one P. damnosus strain. Ascorbic acid was inactive on the P. cerevisiae and P. acidilactici strains, had a slight growth-promoting effect on one P. damnosus strain, but inhibited, partly or completely, the growth of the other strains. Ascorbic acid was therefore omitted from the vitamin medium. p-Aminobenzoic acid, vitamin B-12 (cyanocobalamin), thiamine, nicotinic acid, nicotinamide, retinol acetate and c. 0·5% v/v alcohol were inactive on all the pediococcus strains studied.     

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.