oc-Keto-ß-methyl-valeric acid a,ß-Dihydroxy-ß-methylvaleric acid a-Aceto-a-hydroxy-butyric acid a-Ketobutyric acid
Serine Cysteine Homocysteine
Homoserine Phosphoric acid
Fig. 3.22 Pathways of assimilation of sulphate, reduction to sulphite and sulphide and incorporation into amino acids.
accumulation occurs. In the fourth phase, sulphite synthesis and accumulation ceases due to depletion of the carbohydrate source.
Several reports (Dufour et al., 1989; Korch et al., 1991; Hansen & Kielland-Brandt, 1995) describe genetic approaches to produce yeast transformants capable of increased sulphite formation. Although details differ, the aim is to produce strains in which the genome has been modified so that the suppressing effects of amino acids on the sulphite producing pathway are relieved. An essential aspect of these methods is to ensure that over-production of sulphite is not accompanied by an equal surfeit of H2S. Fuller details of these experimental approaches are provided in Section 4.3.4.
Another important sulphur-containing beer flavour component is dimethyl sulphide (DMS). At high concentrations this compound has an unpleasant flavour and aroma described as 'cooked sweet-corn' or 'cooked vegetable'. In top-fermented ales its concentration is not usually above its flavour threshold of approximately 30 ppb (Harrison & Collins, 1968) and it is not important in these beers. Higher concentrations are found in lager beers. Here within the range 30-100 ppb, it is considered an essential flavour component contributing to the distinctive flavour and aroma of lagers. At concentrations above 100 ppb it is considered objectionable (Anness & Bamforth, 1982).
DMS arises in beer via two routes. First, from S-methylmethionine (SMM) which decomposes to DMS on heating and, second, via the reduction of dimethyl sulph-oxide (DMSO). The latter reaction is catalysed by yeast during fermentation. The common precursor of all DMS arising in beer is SMM and this derives from green malt. Both DMS and DMSO are formed during the various steps involved in the conversion of green malt to finished malt. The proportions of each are dependent on the conditions used for each step in particular, the temperature of kilning. Thus, significant quantities of DMSO are only formed where a kilning temperature greater than 60°C is used (Anness et al., 1979; Parsons et al., 1977).
DMS is volatile and consequently most of that present in malt is lost during mashing and wort boiling. Conversely, DMSO is heat stable and persists unchanged through the mashing and wort boiling stages. SMM is converted to DMS during the wort boil. Much of this is also lost; however, thermal decomposition of SMM continues during the copper cast and throughout cooling, such that wort in fermenter contains a mixture of SMM, DMS and DMSO. The proportions of each vary depending on the raw materials, conditions of wort production and the nature of the brewery plant employed (Anness & Bamforth, 1982; Dickenson, 1983).
No further conversion of SMM to DMS occurs during fermentation since the temperature is too low for thermal decomposition. SMM is assimilated by yeast where it is converted to methionine. DMSO in wort is reduced to DMS by yeast during fermentation (Anness et al., 1979; Gibson et al., 1985). In a second paper (Gibson et al., 1985), the latter authors demonstrated that the specific activity of dimethyl sulphoxide reductase increased when yeast growth had stopped. The increase in specific activity was associated with nitrogen limitation. In trials using defined media it was demonstrated that DMSO reduction was most rapid in cultures where methionine was the limiting nitrogen source. Furthermore, in experiments using 14[C]methionine, radiolabeled methionine sulphoxide was identified in cells.
From these results it was concluded that DMSO reductase was subject to nitrogen catabolite repression and that it was involved in the uptake of methionine.
Anness and Bamforth (1982) suggested that the reduction of DMSO by yeast is performed by methionine sulphoxide reductase. This complex enzyme utilises thior-edoxin and thioreoxin reductase to transfer electrons from NADPH. These authors conclude that reduction of DMSO may be a fortuitous side reaction.
Anness and Bamforth (1982) summarised the effects of fermentation variables other than wort amino nitrogen on DMSO reduction by yeast. Strains designated as S. cerevisiae were generally more effective than strains of S. uvarum (presumably 'lager' strains). Low temperatures favour formation of DMS by yeast and this may partly explain why levels in lagers are generally higher than ales. Wort concentration and DMS formation by yeast are positively correlated. The relationship is not linear and at very high gravities there is a disproportionate increase in the yield of DMS. Formation of DMS during fermentation is favoured by high pH. High capacity deep fermenting vessels are associated with high DMS levels.
The multiplicity of factors both in wort production and during the fermentation stage allows for great variability in the relative importance of SMM and DMSO as the immediate sources of beer DMS. Thus, in many fermentations the yeast DMSO reductase route appears to be negligible, for example, Dickenson (1983), whereas others have reached a totally opposite conclusion (Leemans et al., 1993).
When yeast is suspended in water it responds by exuding protons. The effect is magnified by the presence of a fermentable sugar (Sigler & Hofer, 1991). The ability to acidify the external medium is utilised in a rapid method for assessing brewing yeast quality, the acidification power test (see Section 18.104.22.168).
It has been observed that under such conditions brewing yeast also excretes a variety of metabolites. This phenomenon has been christened 'shock excretion' (Lewis & Phaff, 1964, 1965). These authors showed that the bulk of the released metabolites were amino acids, derived from the intracellular pool, and a variety of other compounds which absorbed at 260 nm. The process occurred over a period of 1-2 hours and required the presence of exogenous sugar. Glucose was the most effective sugar although maltose could substitute providing the yeast had been grown in the presence of this sugar. Within 4 hours most of the excreted amino acids were re-absorbed; however, other metabolites, including nucleotides, remained in the medium. Differences in the patterns of excretion and re-absorption were observed with individual yeast strains.
It must be assumed that this phenomenon may also occur when yeast is pitched into wort. Presumably, it may reflect a temporary depolarisation of the membrane allowing release of some cell constituents followed by a period of re-equilibration in which some of the released material is again assimilated. It is possible, however, that some of the material that is not re-absorbed, in particular, the nucleotide fraction, might make a subtle contribution to beer flavour.
Brewing Yeast and Fermentation Chris Boulton, David Quain Copyright © 2001 by Blackwell Publishing Ltd
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