THE INFLUENCE OF A COLIFORM BACTERIUM ON FERMENTATION BY YEAST

Hopped wort (20°C) was inoculated with brewing yeast alone (10⁷ cells/ml), or with Citrobacter freundii (10⁵ cells/ml) or with both organisms at the concentrations stated. The mixture grew somewhat more quickly than the pure yeast culture, the oxidation-reduction potential and S.G. fell more quickly but the pH less rapidly. The mixed culture produced more ethanol and various organic acids but less volatile carbon. Dimethyl sulphide was produced in the mixed culture but not in the pure yeast culture. It is concluded that C. freundii can have a profound influence on yeast fermentation, consistent with its pure culture characteristics.     

SIMULTANEOUS FLUOROMETRIC MICRODETERMINATION OF HYDROGEN SULPHIDE AND VOLATILE THIOLS IN BEER

A fluorometric method is presented for routine simultaneous determination of hydrogen sulphide and volatile thiols. Reproducibility, sensitivity and simplicity were achieved by the procedure described.     

THE MEASUREMENT OF DIMETHYL SULPHOXIDE IN BARLEY AND MALT AND ITS REDUCTION TO DIMETHYL SULPHIDE BY YEAST

Dimethyl sulphoxide (DMSO) is a normal component of malt and barley. A method is described for its extraction and estimation. DMSO is produced by the oxidation of dimethyl sulphide (DMS), particularly during kilning of malt, and higher levels are found in malts subjected to ale kilning schedules. DMS may also be oxidized during wort preparation. DMSO can be reduced to DMS by yeast in glucose/salts medium, by yeast cell suspensions and by a cell-free extract. Reduction of DMSO is inhibited by methionine sulphoxide. The results suggest that reduction of DMSO may account for the DMS produced during fermentation of ale and lager worts.     

THE USE OF RADIOACTIVE LABELLING TO DEMONSTRATE THE PRODUCTION OF DIMETHYL SULPHIDE FROM DIMETHYL SULPHOXIDE DURING FERMENTATION OF WORT

During fermentation, yeast reduced [¹⁴C] dimethyl sulphoxide (DMSO) to yield radioactive dimethyl sulphide (DMS) even under conditions in which no net formation of DMS could be demonstrated by GLC analysis. It was confirmed that most DMS is produced during fermentation of lager worts at 8°C.     

THE ROLE OF DIMETHYL SULPHOXIDE REDUCTASE IN THE FORMATION OF DIMETHYL SULPHIDE DURING FERMENTATIONS

The ability to produce dimethyl sulphide (DMS) during fermentation of wort is apparently a general characteristic of Saccheromyces cerevisiae and Saccharomyces uvarum. Washed suspensions of these yeasts reduce dimethyl sulphoxide (DMSO) but there is no correlation between DMS formation in fermentation and DMSO reduction in whole cells. Although different strains vary widely in their ability to reduce DMSO they all contain similar levels of NADPH—dependent DMSO reductase. This enzyme is a multi-protein system which closely resembles methionine sulphoxide reductase. Spoilage bacteria including Enterobacter cloacae can reduce DMSO more efficiently than can yeast probably because a different enzyme system is involved.     

RAPID METHODS FOR THE DETECTION OF YEAST AND LACTOBACILLUS BY ATP BIOLUMINESCENCE

Present plating methods for the detection of potential spoilage organisms in packaged beers require 2–3 days for yeast, and 5–7 days for lactic acid bacteria. Simple and inexpensive sterility tests based on bioluminescence have been developed for the detection in broth media of small numbers of yeasts in 1 day, and lactic acid bacteria, in both normal and heat-stressed conditions, in 3 days.     

SYNTHESIS OF DIMETHYL SULPHIDE DURING FERMENTATION BY A ROUTE NOT INVOLVING THE HEAT-LABILE DMS PRECURSOR OF MALT

Pilot scale brewing studies showed that dimethyl sulphide (DMS) can be produced during fermentation substantially in excess of that predicted by measurement of the DMS potential of the wort. This occurred in low temperature fermentations conducted in conical vessels but not if open vessels were used. Neither the type of malt used nor the length of the wort boil substantially influenced the extent of this excess DMS production although they may have affected liberation from the yeast of unidentified material which released DMS on treatment with hot alkali. It is suggested that yeast can synthesise S-methyl-L-methionine (SMM) and that metabolic breakdown of this compound was responsible for some of the DMS produced.     

STORAGE OF PITCHING YEAST FOR PRODUCTION OF WHISKY

The viability of pitching yeast stored at 5°C and at 20°C was compared. The viability of the yeast stored at 5°C was about 80% after 54 d storage and its CO₂ producing activity also remained high. The ethanol production rate of yeast stored for a long period at 5°C was unaffected in a subsequent fermentation test, even though the viability was only about 50%. The ethanol production rate of yeast stored at 20°C decreased in proportion to a rapid decrease in viability during storage. The concentration of intracellular sulphur compounds, important for the production of aromatic components of whisky, of yeast stored at 5°C was higher than that of yeast stored at 20°C. Therefore, the storage of yeast at 5°C seems appropriate for production both of ethanol and aroma.     

METHIONINE AND SULPHATE AS COMPETING AND COMPLEMENTARY SOURCES OF SULPHUR FOR YEAST DURING FERMENTATION

Study of the uptake of sulphate and methionine by an ale yeast from a range of media showed that utilisation of sulphate was fairly strictly controlled but assimilation of methionine was not. Cells never took up more than about 0.3 mMol sulphate per litre whilst methionine, up to an initial concentration of 10 mMol per litre, was completely absorbed. Sulphate-grown cells had low intracellular pools of amino acids and methinonine was never detected. Methionine-grown cells contained methionine in both cytosol and vacuole and the concentration of several other amino acids also increased in such a way to suggest that methionine catabolism was occurring. With mixed sulphur sources methionine prevented uptake of sulphate when the concentration of sulphate was high but not when it was low suggesting the presence of two sulphate transporters with different control properties. Sulphate did not influence uptake of methionine. Addition of other amino acids to the medium did reduce the rate and extent of methionine uptake but not the intracellular pool sizes. Pilot plant studies suggested that SO₂ production in a brewery is more likely to be a reflection of the overall nutritive status of the wort rather than be connected to the initial methionine concentration .     

THE ORIGIN AND OCCURRENCE OF VOLATILE SULPHUR COMPOUNDS IN BRITISH ALES AND LAGERS

A flame-photometric sulphur detector was used to identify, measure and determine the sources of the sulphur volatiles produced during the commercial processing of British ale and lager. Dimethyl sulphide was the main sulphur volatile present in malt but traces of hydrogen sulphide, diethyl sulphide, and dimethyl disulphide were also detected. Hops contained hydrogen sulphide, methyl mercaptan, dimethyl sulphide, diethyl sulphide, methional and dimethyl disulphide. Most of this material extracted into commercial worts was driven off during boiling. Brewing yeasts produced only traces of organo-sulphur volatiles both in laboratory fermentations of wort and during the processing of commercial ales and lagers. In contrast, brewery bacteria, particularly wort spoilage organisms, could generate dimethyl sulphide and sometimes traces of other sulphur volatiles in laboratory cultures. t-Butyl mercaptan was the only organo-sulphur volatile detected in significant concentrations during the primary fermentation and conditioning of commercial beers and this was attributable to the activity of wort-spoilage bacteria. Attempts to identify a volatile compound causing ‘sulphury’ smells in beer were unsuccessful but there was some evidence that it might not contain sulphur.     

THE REDUCTION OF DIMETHYL SULPHOXIDE TO DIMETHYL SULPHIDE DURING FERMENTATION

Dimethyl sulphide (DMS) produced by yeast during fermentation is formed from dimethyl sulphoxide (DMSO) rather than from S-methyl methionine (HADMS). All the yeasts and one of the two spoilage organisms examined formed DMS from DMSO. In fermentations at 8°C only 13–21% of the DMSO is reduced to DMS by yeast and the extent of conversion is greatly decreased by raising the fermentation temperature. The amount of DMS formed increases as the gravity of the wort is raised but is also dependent on the fermentable sugar employed. There is therefore no simple correlation between DMSO content of the wort and DMS formation.     

THE RELATIONSHIP OF DIMETHYL SULPHIDE LEVELS IN MALT,WORT AND BEER

The half-life for the conversion of malt dimethyl sulphide (DMS) precursor to free DMS has been determined at various temperatures and pH values. At pH 5·2 the half-life of the precursor in wort (S.G. 1·060) at its boiling point is 38 min, and is doubled for each 6°C fall in temperature. At pH 5·5 the half-life at the boiling point is 32·5 min. Knowing the stability of the precursor at the various temperatures in the brewing process, the extent of conversion to free DMS in wort at pitching can be predicted for malt of a given precursor content and for a given set of process conditions. The results of DMS analyses of samples taken during brewery trials are in reasonable agreement with the predicted values. This work involved infusion mashing only, but the same principles apply to decoction mashing. The fate of precursor and free DMS during fermentation and conditioning has been followed on a production scale. With some brews, where high levels of free DMS were present at pitching, much free DMS was lost during fermentation. Also, precursor DMS reappeared in the beer after a few days and there was some increase in the level of free DMS. The DMS precursor in green malt has been isolated by ion-exchange and gel-filtration chromatography. A preparation has been obtained which has 0·6 mol potential DMS per mol amino nitrogen. Thin layer chromatography showed that the preparation and its hydrolysis product had the same R_f values as S-methylmethionine and homoserine respectively.     

YEAST BLOTTERS: SURVIVAL OF YEAST AND BACTERIA

The survival of yeast on yeast blotters with and without contaminating bacteria was monitored to determine whether or not microscopic analysis of yeast after shipment on blotters might be influenced by growth of such bacteria. To examine blotter yeast, they were resuspended and roughly balanced to known opacity by colorimeter—then precisely by hemacytometer. Suspensions were then analysed for survival by plating. It was determined that bacteria and yeast both die quickly on blotters but demonstrate varying levels of survival. It was verified that viable counts from blotters are not practical for enumeration purposes although qualitative knowledge can be gained from viable cell assessment.     

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.     

IDENTIFICATION OF THE DIMETHYL SULPHIDE PRECURSOR IN MALT

The heat-labile dimethyl sulphide (DMS) precursor in green malt has been isolated using ion exchange chromatography and gel-filtration under mild conditions. The final preparation had ～0.7 mol potential DMS per mol amino groups. Thin layer chromatography of this preparation, and the gel-filtration behaviour of both crude and purified preparations, showed that the precursor had identical properties to S-methylmethionine (SMM). The DMS precursor in a continental lager malt was also examined. The malt had been kilned under conditions which were expected to lead to a significant content of ‘active’ DMS precursor (reported to be metabolized to free DMS by suitable yeast strains, and contrasting with the ‘inactive’ green malt precursor). The DMS precursor in an extract of this kilned malt had the same elution volume on Sephadex G15 as SMM. There was no evidence for any difference from the DMS precursor in green malt. It is concluded that SMM is the only heat-labile DMS precursor in malt.     

THE PRODUCTION OF AROMA COMPOUNDS BY YEAST

The yeast plasma membrane regulates the movement of compounds into the yeast cell and of yeast metabolites from the cell into the medium. The rate of penetration of organic acids into the yeast cell depends on their lipophilic nature, and on their molecular size and degree of branching. During fermentation yeast synthesizes a vast number of aroma compounds. The numerically and quantitatively largest groups of aroma compounds include fusel alcohols, fatty acids and fatty acid esters. The yeast used and the fermentation conditions can influence the formation of aroma compounds. The yeast also has a profound effect on the formation of other aroma compounds, such as sulphur compounds and phenols. In addition to fermentation, the maturing of a beverage can also influence the aroma. During the maturing lactones, phenols and other compounds are extracted from the oak casks in which the beverage is aged. The presence of the so-called “whisky lactone”, β-methyl-γ-octalactone, is characteristic of a beverage that has been matured in oak.     

THE IDENTITY OF AN INHIBITOR IN WORT OF DIMETHYL SULPHOXIDE REDUCTASE FROM YEAST

Wort contains a substance which inhibits the reduction of dimethyl sulphoxide by yeast. The inhibitor has been purified by dialysis followed by chromatography on Sephadex G-15 and Dowex 50. It has been identified as methionine sulphoxide.     

PRODUCTION OF ESTERS BY DIFFERENT YEAST STRAINS IN SUGAR FERMENTATIONS

The production of the acetates of isoamyl alcohol and phenethyl alcohol and the ethyl esters of the C₆-C₁₀ fatty acids was investigated in semiaerobic sugar fermentations by 56 strains of Saccharomyces cerevisiae and 3 strains of S. uvarum. The S. cerevisiae yeasts generally produced more esters than the S. uvarum yeasts. Isoamyl acetate was the main component in the ester fractions examined and others in decreasing order, were ethyl caprylate, ethyl caproate, ethyl caprate and phenethyl acetate.     

THE EFFECT OF LOW CO2 PRESSURES ON THE ABSORPTION OF AMINO ACIDS AND PRODUCTION OF FLAVOUR-ACTIVE VOLATILES BY YEAST

Laboratory fermentation of 51 batches of wort under CO₂ pressures of 0·5 and 1·0 atm have shown that whilst the speed of fermentation was affected only slightly, absorption of amino acids, production of several flavour-active compounds and some yeast features were affected. The CO₂ pressure led to reduced absorption of Group B amino acids although Group A compounds were unaffected; fusel oil and ester concentration was reduced whereas that of the vicinal diketones and acetaldehyde was increased. Yeast crop and cell viability fell with increase in pressure and at 1·0 atm the mean cell volume was increased. The results suggest that CO₂ could have significant effects on yeast and beer quality within the range of concentration which can be expected to occur in unpressurized commercial fermentations.     

PRODUCTION OF AMYLOLYTIC ENZYMES BY SEVERAL YEAST SPECIES

Several amylolytic yeast species, endomycopsis spp, schwanniomyces spp, pichia spp and saccharomyces spp have been compared for their ability to synthesise α-amylase (E.C. 3.2.1.1), glucoamylase (E.C. 3.2.1.3) and pullulanase (E.C. 3.2.1.9). Endomycopsis fibuligera strain 240 possessed the highest glucoamylase activity (208 nmoles glucose/min) and second highest α-amylase activity (128 nmoles maltose/min) and produced the largest amount of biomass. Schwanniomyces spp were the only yeast species studied which exhibited significant debranching activity. It was found that in endomycopsis spp, schwanniomyces spp and pichia spp, glucose, at a concentration above 3.0 × 10^?3 m , appeared to repress α-amylase activity. However, glucoamylase activity was not repressed at that glucose concentration.