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.     

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.     

A NOVEL AND PRACTICAL YEAST VITALITY METHOD BASED ON MAGNESIUM ION RELEASE

Using a commercial lager brewing yeast, the immediate release of magnesium, potassium and phosphate ions by cells when inoculated into wort was evaluated to be directly related to its subsequent fermentation performance. Yeast which released appreciable amounts of these ions immediately after inoculation mediated improved fermentations as evidenced by better growth, higher ethanol concentrations and lower diacetyl levels at the end of fermentation. Conversely, yeast that absorbed these ions or released them at very low concentrations performed poorly throughout fermentation producing beers with lower ethanol concentrations and higher diacetyl levels. These observations led to the identification and development of a rapid, practical and highly sensitive method to measure Mg⁺⁺ released or absorbed by yeast as an indicator of its vitality and a predictor of its subsequent fermentative performance. Full method details of the Magnesium Release Test (MRT) are given.     

THE SENSITIVITY OF DIFFERENT BREWING YEAST STRAINS TO CARBON DIOXIDE INHIBITION: FERMENTATION AND PRODUCTION OF FLAVOUR-ACTIVE VOLATILE COMPOUNDS

The sensitivity of brewing yeast strains, with different oxygen demands, to carbon dioxide inhibition was investigated. Laboratory fermentations were performed with, and without, protein-based “yeast foods” to lower dissolved CO₂ during fermentation. Differences were observed in yeast fermentative performance in the presence and absence of “yeast foods” for all yeast strains tested. Fermentation performance was improved with the addition of “yeast foods”. There was improved carbohydrate utilisation and amino acid uptake, while acetaldehyde levels at the end of fermentation were decreased. There was an increase in fusel oil production and acetate ester levels at the end of fermentation. Sulphur dioxide levels at the end of fermentation were unaffected by “yeast food” addition. Different yeast strains displayed differing sensitivity to CO₂ inhibition for all parameters tested. Sensitivity to CO₂ was not found to be related to oxygen demand of the yeast strains.     

A RAPID SPECTROPHOTOMETRIC METHOD TO DETERMINE ESTERASE ACTIVITY OF YEAST CELLS IN AN AQUEOUS MEDIUM

A new rapid method to determine the total esterase enzymatic activity of yeast cells is proposed. In a sodium phosphate buffer a β-naphthol synthetic ester is hydrolized by cells, and the released β-naphthol is coupled with a diazonium salt (Fast Garnet GBC) in the presence of sodium dodecyl sulfate. The whole procedure is carried out in an aqueous buffer medium, and the resulting azo dye is directly evaluated by absorbance measurement at 524 nm. The analytical results from different assays were adjusted to a fixed cell concentration with a statistical procedure. The method shows good repeatability, reproducibility and detectability, and it requires simple equipment and instruments. It is therefore suitable both for routine analysis, as industrial yeast strain screening, and for yeast physiological studies, in order to improve the aromatic quality of fermented drinks.     

STUDIES ON CONJUGATE EFFECTS OF SUBSTRATE (GLUCOSE) AND PRODUCT (ETHANOL) ON CELL GROWTH KINETICS DURING FERMENTATION OF DIFFERENT YEAST STRAINS

The conjugate effect of substrate (glucose) and product (ethanol) has been studied on yeast growth during the course of fermentation. The study was made on five yeast strains Kloeckera apiculata, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces cerevisiae UG5, and Saccharomyces cerevisiae sake. The results attest a different action of added alcohol as compared with produced alcohol for all strains. It is also verified that kinetic behaviour, namely ethanol inhibition, varies with initial substrate concentration, and so a new parameter, the added alcohol concentration at which biomass productivity declines to one-half its value with no added ethanol, is proposed to quantify ethanol inhibition     

A SIMPLE,RAPID METHOD FOR THE DETECTION OF SUBSPECIES OF ZYMOMONAS MOBILIS

Zymomonas strains are simply and rapidly detected after growth in a selective liquid medium by the addition of a Schiff's reagent. This dye reacts with acetaldehyde produced by Zymomonas to give a deep purple colour. The optimal conditions for the test are evaluated and it is shown to be specific for Zymomonas strains. The use of the dye reaction in routine microbiological quality control is described. A comparison is presented of the dye test and the use of an agar medium containing sulphite and lead acetate on which Zymomonas strains which produce H₂S form characteristic colonies.     

A SIMPLE AND RAPID METHOD FOR DETERMINATION OF METAL IONS IN DEVELOPING BARLEY SEEDS

A simple, rapid and accurate procedure involving flame—and flameless—atomic absorption spectroscopy was devised for the extraction and assay of major and trace metal ions in developing embryos and endosperms of barley seeds. Both embryo (maximum dry weight 20 mg) and endosperm (maximum dry weight 300 mg) samples were decomposed by digestion with nitric acid to yield a clear solution from which all the elements were analysed. Potassium, magnesium, calcium, iron, manganese and copper were determined from the embryo digests. Similarly, potassium, magnesium, calcium and manganese were determined from the endosperm digests. The validity of the procedure was confirmed by high recoveries of added metal ions. The sensitivity of analysis was such that sample size was in the range 1–12 mg.     

SCREENING YEAST STRAINS FOR THEIR ABILITY TO PRODUCE PHENOLIC OFF-FLAVOURS: A SIMPLE METHOD FOR DETERMINING PHENOLS IN WORT AND BEER

Amounts of several phenolic compounds in wort and beer were measured by gas-chromatography following extraction with a small volume of chloroform. The procedure described is not specific for phenols and the limit of sensitivity is 0·05–0·1 μg ‘phenol’/ml. However, the precision is reasonable and this relatively rapid method has proved useful for screening yeast strains for their ability to produce phenolic off-flavours. Phenolic-tasting beers contained significant amounts of 4-vinyl guaiacol; whilst this compound may not be the sole cause of the undesirable flavour, its production is symptomatic of the unsuitability of a yeast strain for brewing.     

A RAPID AND SIMPLE METHOD FOR THE DETERMINATION OF STARCH AND β-GLUCAN IN BARLEY AND MALT

A simple and precise method suitable for the routine determination of starch and β-glucan in barley and malt is described. Perchloric acid (50 mM) was used to effect rapid (3 min) and exhaustive extraction of both glucans which were then measured directly from this single extract by specific enzymic hydrolysis of the individual glucans to glucose. The glucose was also measured enzymically. Little or no acid hydrolysis of starch or β-glucan was observed under the extraction conditions used; most or all of the free glucose could be attributed to hydrolysis of sucrose. Complete solubilisation of the gum and hemicellulosic components of β-glucan was achieved. Preincubation of the acid extracts with protease prior to amyloglucosidase digestion resulted in higher measurements (approximately 4% w/w) of starch. The method was used to measure the levels of starch and β-glucan in five varieties of barley with contrasting malting quality, in micro-malts prepared from these samples and in commercial lager and ale malts.     

THE EFFECT OF OSMOTIC PRESSURE ON THE PRODUCTION AND EXCRETION OF ETHANOL AND GLYCEROL BY A BREWING YEAST STRAIN*

When the cells of a lager brewing yeast Saccharomyces uvarum (carlsbergensis) were grown in minimal media containing sucrose and a non-metabolized sugar sorbitol, significant levels of intracellular ethanol were obtained. Intracellular ethanol concentration decreased as the osmotic pressure of the medium was lowered and the proportion of extracellular ethanol increased. A reduction in cell viability occurred when there were high levels of intracellular ethanol. The total amount of glycerol produced increased with increased osmotic pressure, but glycerol diffused out of the cells faster than ethanol.     

KINETIC STUDIES OF THE DECONTAMINATION OF YEAST SLURRIES WITH PHOSPHORIC ACID AND ACIDIFIED AMMONIUM PERSULPHATE AND A METHOD FOR THE DETECTION OF SURVIVING BACTERIA INVOLVING SOLID MEDIUM REPAIR IN THE PRESENCE OF CATALASE

Destruction of Obesumbacterium proteus in pitching yeast slurries is more efficiently accomplished using an acidified ammonium persulphate wash (0·75% w/v; pH 2·8) than by using phosphoric acid at pH 2·1. The effectiveness of both methods against the lactic acid bacteria, acetic acid bacteria and Enterobacter agglomerans is similar. Material cost savings of up to 60% can be achieved through the use of the acidified ammonium persulphatewash. Economy in the use of materials and optimum process efficiency is achieved by determination of the quantity of acid required to adjust the pH of the yeast slurry before washing. Bacteria surviving the yeast wash can be detected reliably using a plating technique involving solid medium repair in the presence of catalase. A buffered diluent must be used to prepare the inoculum.