ISOLATION AND PARTIAL CHARACTERIZATION OF THE DIMETHYL SULPHIDE PRECURSOR IN GREEN MALT,AND ITS EFFECT ON BEER DIMETHYL SULPHIDE LEVELS

The component from which dimethyl sulphide (DMS) is produced when green malt worts are heated has been purified and its effects on wort and beer DMS levels studied. The green malt precursor can be split into S-methylmethionine and an as-yet uncharacterized ninhydrin-positive component. When the purified precursor is added to worts derived from kilned malts, it is rapidly taken up by yeast, but there is no resulting increase in DMS production. DMS production during fermentation occurs with worts made from kilned malts but not with worts from green malt; however, this difference is not due to an inhibitor being present in green malt worts. The evidence suggests that malt kilning affects the nature as well as the amount of DMS precursor in malts.     

TRACE VOLATILE CONSTITUENTS OF BEER DIMETHYL SULPHIDE FORMATION IN MALT

Dimethyl sulphide formation in malt occurs primarily during the kilning process, and is significantly dependent upon the maximum kilning temperature. During mashing a considerable fraction of the dimethyl sulphide of malt is transferred to the wort. Since this is not always completely removed by the wort boiling process, the remaining dimethyl sulphide, although decreased during fermentation, may be sufficient to impart a noticeable dimethyl sulphide aroma to the finished beer.     

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.     

CAMBRIDGE PRIZE LECTURE DIMETHYL SULPHIDE—ITS ORIGIN AND CONTROL IN BREWING

The fate of dimethyl sulphide (DMS) and its precursors, S-methylmethionine (SMM) and dimethyl sulphoxide (DMSO), in the malting and brewing processes, has been studied on both a laboratory and a production scale. The information gained has been used to show how the level of DMS in beer may be controlled. The principal determinant of the beer DMS level was the SMM present in malt. DMSO was not a significant precursor of DMS in normal lager production.     

CONTROL OF THE DIMETHYL SULPHIDE CONTENT OF BEER BY REGULATION OF THE COPPER BOIL

In pilot brewing trials where wort boil time was varied from 15 min up to 2 hours the DMS contents of the resultant beers were greatly altered without substantial effect on many other aspects of beer quality. The worts were separately fermented by two yeast strains and a consistent difference between DMS levels of the pairs of beers was found which was almost independent of the boil time. The breakdown of the malt-derived DMS precursor in boiling wort was found to be a first order reaction having a rate constant of about 0.02 min^?1, implying a half life of about 35 min. The breakdown rate of S-methylmethionine in a boiling sucrose solution at pH 5.3 was similar. Free DMS was found to leave this wort substitute rapidly, again as a first order process, but with a rate constant of 0.18 min^?1 (t_1/2 ～ 4 min) when the evaporation rate was 17%/h.     

THE MEASUREMENT OF DIMETHYL SULPHIDE PRECURSOR IN MALTS,ADJUNCTS, WORTS AND BEERS

Dimethyl sulphide in beer originates from a precursor in the malt. A method is described for the measurement of this precursor in brewing raw materials, wort and beer. The precursor levels in various green malts, kilned malts, and adjuncts are given. Excess methionine inhibits precursor uptake by yeast during fermentation. Residual precursor is still present in some commercial bottled beers.     

METHODS FOR THE DETERMINATION OF VOLATILE NITROSAMINES IN MALT,WORT AND BEER

Methods for the analysis of beer, wort and malt for NDMA at the one in 10⁹ level using the thermal energy analyzer have been evaluated. The beer and wort procedure using Preptube extraction has been shown to give excellent results. For malt, the preparation of an aqueous extract, followed by analysis by the beer procedure, also gives excellent results. Vacuum distillation from mineral oil has also been examined for the analysis of malt. Providing sufficient aqueous phase is initially present, this procedure yields results similar to those from aqueous extraction. The methods will also detect NDEA, although no NDEA has been observed in any of the beer, wort or malt samples examined. NDMA has been observed to be present in bound forms in malt and Preptubes and is released by the addition of water     

AN ALTERNATIVE METHOD FOR THE DETERMINATION OF DIMETHYL SULPHIDE IN BEER

A method for the determination of dimethyl sulphide (DMS) in beer and for total DMS precursor in malt is described. The DMS was extracted into chloroform and determined by glass capillary gas liquid chromatography (GLC) using a flame ionisation detector (FID) and diethyl ether as an internal standard. Errors arising from the use of ‘head space’ sampling were thereby avoided. The reproducibility of results and the recovery of DMS at the 100 ppb level were satisfactory but less so at levels below 50 ppb. Beers produced in a pilot brewery from malts containing high levels of total DMS precursor were not found to contain correspondingly high levels of DMS.     

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.     

SEPARATION OF NITROGENOUS CONSTITUENTS OF MALT,WORT AND BEER USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High performance liquid chromatography has been used to separate groups of soluble nitrogenous compounds in malt. Malts are extracted with water at 5°C and the nitrogenous components separated using a column packed with a material designed specifically for the analysis of proteins and peptides. The procedure is suitable for routine use and can be readily adapted to study changes in the development of nitrogenous compounds during the brewing process.     

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.     

THE OCCURRENCE OF TWO GEOMETRICAL ISOMERS OF 2,4,5-TRIMETHYL-1,3-DIOXOLANE IN BEER

Two geometrical isomers of 2,4,5-trimethyl-1,3-dioxolane have been detected in a range of commercial and experimental beers at levels up to ca 0.1 ppm. These compounds, plus a number of other dioxolanes, are present in unboiled wort but are lost by evaporation during wort boiling. The trimethyldioxolanes are then reformed during subsequent fermentation. The flavour threshold of a mixture of the two trimethyldioxolanes was found to be ca 0.9 ppm, at which concentration it produced in beer ‘phenolic’ and ‘astringent/drying’ flavour notes. However, these compounds are not present in sufficiently high concentrations to make a significant contribution to the flavour of beer.     

THE PRESENCE OF TWO DIMETHYL SULPHIDE PRECURSORS IN MALT,THEIR CONTROL BY MALT KILNING CONDITIONS,AND THEIR EFFECT ON BEER DMS LEVELS

The precursor of dimethyl sulphide (DMS) which is formed during barley germination and is present in green malt differs from that present in malt kilned at high (> ～75°C) temperatures in that it cannot be metabolized to DMS by yeast. It is therefore termed ‘inactive’ precursor to distinguish it from the ‘active’ precursor present in kilned malt, which is metabolized to DMS by yeast. During malt kilning, inactive precursor is changed into the active form, but only at temperatures at which destruction of both precursors is also taking place. Therefore, for any kiln there is an optimum temperature range over which maximum conversion of inactive to active precursor can be combined with minimum destruction of both precursors. The DMS in beers brewed from green malt or malts kilned only at low (< ～70°C) temperatures is almost entirely the result of precursor destruction during wort boiling, final levels being governed not only by the conditions of boiling, but also by the extent of losses during fermentation and subsequent processing. In contrast, the DMS in beers brewed from malts kilned at higher temperatures is mostly formed during fermentation, and since beer DMS levels can be related to wort and beer DMS precursor levels, they can be better predicted and controlled. The method used for the measurement of total DMS precursor levels in malts is described in detail.     

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.     

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.     

QUANTITATIVE ANALYSIS OF PURINE NUCLEOSIDES AND FREE BASES IN WORT AND BEER

Low molecular mass compounds containing purine or indole groups exhibit adsorption-based retention characteristics when chromatographed on high performance size exclusion chromatography supports (Superose 12 and Superdex 75). Exploitation of this phenomenon enabled the development of a method for the quantitative analysis of purine nucleosides and free bases in wort and beer. Using this technique, a survey of 18 commercial beers showed the following range of variation of concentrations of these compounds: guanosine + deoxyguanosine 19–110 mg/L, adenosine + deoxyadenosine 3–43 mg/L, xanthine 1–41 mg/L, guanine < 1–11 mg/L and adenine < 1–7 mg/L. Analysis of wort and beer from a commercial brewery demonstrated a decline in both adenine and guanine and an increase in xanthine during the course of a typical fermentation.     

THE INFLUENCE OF ASSIMILABLE NITROGEN COMPOUNDS IN WORT ON THE ABILITY OF YEAST TO REDUCE DIMETHYL SULPHOXIDE

Dimethyl sulphoxide (DMSO) reductase activity increased at the end of the growth cycle of Saccharomyces cerevisiae NCYC 240 on complex medium. On defined medium, DMSO reductase and thioredoxin reductase activities and thioredoxin were all elevated in cultures limiting in nitrogen, but not in nitrogen-sufficient cultures. In nitrogen limiting cultures it was also shown that the reduction of DMSO (measured as disappearance of [¹⁴C] from the medium) was much increased compared with conditions of nitrogen excess. This applied whether ammonium or glutamate was the limiting nitrogen source. When methionine was used as the limiting nitrogen source, DMSO reduction was higher still. Methionine-limited cells accumulated [¹⁷C] methionine much more rapidly than did cells from methionine-sufficient cultures, and [¹⁴C] methionine sulphoxide could be identified in the cells after 1 min incubation with labelled methionine. It is concluded that DMSO reductase activity is controlled by nitrogen catabolite repression and may be involved in methionine uptake by yeast. The amino nitrogen content of wort will in consequence have an important effect on levels of dimethylsulphide (DMS) produced during fermentation.     

POTENTIOMETRIC TITRATION OF CHLORIDE IN BEER AND WORT USING AN ION-SELECTIVE ELECTRODE

A silver electrode — reference electrode pair connected to a pH/mV meter may be used as a potentiometric indicator of the equivalence point in silver nitrate titration of chloride. The method, applied to beer and wort, is sensitive and reproducible and gives good recovery of added chloride.