Vdk At Peak Fermentation Is

From this, it was concluded that in order to minimise the magnitude of the VDK peak it was necessary to provide a wort with sufficient FAN to ensure a given minimum concentration at the end of fermentation. Since FAN utilisation and yeast growth are positively correlated it was also possible to demonstrate relationships between initial fermentation conditions and the size of the VDK peak. Thus, high wort oxygen concentrations, trub levels, pitching rates and fermentation temperature, all of which favour rapid and extensive yeast growth, also promoted high VDK levels, presumably due to increased FAN utilisation.

In subsequent work (Nakatani et al., 1984b) the same authors confirmed the results of others (Wainwright, 1973), that the valine and isoleucine contents of wort exert specific effects on the formation of a-acetohydroxy acids. Valine and isoleucine inhibit the pathways leading to their biosynthesis by inhibition of the a-acetohydroxy synthetase enzyme and this removes the precursors of diacetyl and 2,3-pentanedione formation. These amino acids are assimilated in the early to middle phase of fermentation and this suppressive effect accounts for the delayed onset of VDK formation. With high wort FAN levels the suppressing effect persists longer because of the increased availability of valine and isoleucine. With low wort FAN levels the requirement for amino acid biosynthesis in middle to late fermentation explains the late large peak of VDK formation.

Amino acids other than valine and isoleucine also modulate the patterns of VDK formation. Barton and Slaughter (1992) concluded that supplementation with valine, leucine, alanine, threonine, glutamate and, surprisingly, ammonium chloride, all decreased VDK concentration in fermentation. These effects were show to be due to inhibition of a-acetohydroxy synthetase. Presumably these effects relate to the complexities of control of amino acid uptake and metabolism in yeast cells. It may be concluded that both total and spectrum of wort FAN affects VDK formation. Since there is little practical control available over FAN spectrum this observation is likely to be of academic but not practical interest.

The non-enzymic oxidative decarboxylation of a- acetohydroxy acids occurs in the wort and (with some caveats, as discussed subsequently) does not require the activity of yeast. Decomposition of a-acetohydroxy acids is rapid under aerobic conditions but the process proceeds under anaerobiosis where copper, ferric and aluminium ions may function as alternative electron acceptors (Inoue et al., 1968a, b). The reactions are also favoured by acidic pH values. Fermentation conditions that produce rapid yeast growth are also associated with a rapid fall in pH and this promotes VDK formation (Inoue et al., 1991). Inoue (1992) reported that non-oxidative decarboxylation of a-acetolactate to acetoin may also occur under appropriate conditions. This may be encouraged by heating at 60 to 70°C under strictly anaerobic conditions but it is suggested that it may also occur at fermentation temperatures providing a sufficiently low redox state pertains. These authors contended that maintenance of low redox during warm conditioning to enhance the proportion of acetolactate non-oxidatively decarboxylated to acetoin could be another essential role of yeast cells in VDK metabolism.

Andries et al. (1997) described a process in which the spontaneous non-oxidative decarboxylation of a-acetolactate to acetoin could be encouraged by passage of green beer through a column containing zeolite resin by a mechanism which was not described. Several different grades of zeolite were tested with very variable results. Conversion rates were temperature dependent; up to 90% at a temperature of 20°C

but only 2% at 5°C. The process was reportedly not affected by the presence of oxygen or the redox state of the beer although no results were provided to support this. No other effects on beer analysis were observed and this appears to be a promising approach for use at production scale.

The reduction of VDK occurs in the late stages of fermentation or during conditioning (see Chapter 6) and requires the presence of viable yeast. The enzymology of the reactions is not well characterised. Hardwick et al. (1976) demonstrated that yeast alcohol dehydrogenase (ADH1) reduced methyl glyoxal, diacetyl and 2,3-pentanedione but at much lower rates than acetaldehyde. No activity with acetoin was observed and this suggested that other specific enzymes might be involved. Several authors have reported the presence of specific diacetyl reductases in yeast (Louis-Eugene et al., 1988; Legeay et al., 1989; Heidlas & Tressl, 1990; Schwarz & Hang, 1994). These enzymes were either NADH or NADPH dependent. In a later study, Murphy et al. (1996) investigated diacetyl reductases in a number of strains of brewing yeasts. A number of different enzymes were detected on the basis of differing thermal lability curves in extracts and mobilities on polyacrylamide gel electrophoresis. Two groups of yeasts could be differentiated on the basis of patterns of dehydrogenases present. Yeasts in one group were primarily bottom fermenting ale types and the other were lager varieties. The lager strains all contained a heat stable acetoin reductase as well as alcohol dehydrogenases which showed no activity with acetoin. The ale types lacked the same acetoin reductase activity but did possess another dehydrogenase which was active with acetoin and diacetyl. These strains also had alcohol dehydrogenases activity different to the lager strains.

Yeast cells are usually capable of rapid diacetyl reduction. The data presented in Fig. 3.19a-e shows the effect of addition of exogenous diacetyl to stirred laboratory high-gravity lager fermentations. In this trial, five identical fermentations were performed. Samples were removed at appropriate intervals and analysed for total VDK using a gas chromatographic procedure. To all but the control fermentation (Fig. 3.19a) diacetyl (1 ppm) was added at the times indicated by the sudden appearance of a peak in the VDK profiles. As may be seen in all cases, the yeast was capable of rapidly reducing the added diacetyl. However, the profiles suggested that there was a progressive decrease in the ability to reduce added diacetyl as a function of time (Fig. 3.19b-d). Interestingly the yeast was capable of reducing exogenous diacetyl during the early part of fermentation where the metabolism was still directed towards production of endogenous VDK (Fig. 3.19e). These data confirm that diacetyl reduction is not the rate-limiting step in the VDK cycle although the apparent decline in activity with time would suggest that the yeast's physiological condition may be of some relevance to diacetyl reduction. In this respect the role of membrane competence could be of importance. It must be assumed that prior to reduction the diacetyl is taken up by the yeast. Since membrane condition will deteriorate during the course of fermentation due to sterol depletion this could in part explain the decline in reduction rates seen in late fermentation. However, it is also entirely possible that there may be a diminution in the activity of the diacetyl reductases during fermentation in a manner analogous to the decline in activity of alcohol dehydrogenase (Fig. 3.15).

An essential aspect of the warm conditioning phase of lager fermentations, if practised, is the necessity to achieve a minimum total VDK concentration (see Section

6.5.3). It is noteworthy that the time taken to achieve this minimum VDK concentration does not necessarily correlate with the magnitude of the peak value. Frequently it may be observed that there is a decline in the rate of decrease in VDK concentration in late fermentation. The inflexion point between rapid and slow decrease in VDK concentration usually occurs near to the threshold value at which cooling is applied to terminate the fermentation. Variability in this slow phase of VDK uptake can be a major cause of inconsistency in vessel turn-round time.

This is illustrated in Fig. 3.20, which shows the terminal phase of decline in total VDK for a number of pilot scale lager fermentations. Fermentation conditions were identical apart from a different batch of wort being used in each case. In the case of these fermentations the specified maximum VDK concentration at which cooling could be applied was 0.1 ppm. As may be seen the most rapid fermentation achieved the VDK specification in approximately 150 hours, whereas the slowest took more than 220 hours. It must be emphasised that the observed variability was not due to differences in suspended yeast count since similar biphasic profiles were also seen with small scale stirred fermentations. Furthermore, the wort gravity attenuation profiles were essentially identical in all cases (data not shown). The reasons for this variability are unclear but appeared to be related to differences in wort composition.

From the previous discussion it is apparent that within the confines of normal fermentation practice little may be done to accelerate VDK removal or to improve the consistency of VDK metabolism other than to apply rigorous control to all aspects of wort production and fermentation management. Other more positive approaches to accelerating VDK removal or reducing its formation have been proposed. Rapid VDK removal using continuous maturation processes is described in Section 5.7.2.

The possibility of spontaneous non-oxidative decarboxylation of a-acetolactate directly to acetoin occurring during fermentation has been discussed previously. In some bacteria this reaction is catalysed by an enzyme, a-acetolactate decarboxylase. Preparations of the bacterial enzyme are available commercially for use as process aids in fermentation (Aschengreen & Jepsen, 1992). Addition of this enzyme to wort at the start of fermentation was shown to reduce diacetyl formation to the extent that it abolished the requirement for a conventional VDK rest. No other effects on fermentation performance or beer quality were reported. A modification to this method was suggested by Dulieu et al. (1997) in which the a-acetolactate decarboxylase preparation was encapsulated in a polymer made from a combination of sodium cellulose sulphate and poly (diallyl dimethyl ammonium) chloride. The claimed advantage was that it would be possible to retain the enzyme within a bioreactor column, which unlike the free enzyme would lend itself to multiple use.

Several reports describe attempts to eliminate or reduce VDK stand-times by genetic modification of brewing yeast strains (see Section 4.3.4). The gene coding for the enzyme a-acetolactate decarboxylase has been cloned from certain bacterial species - Klebsiella terrigena, Enterobacter aerogenes and Acetobacter aceti - and transformed into brewing yeast (Sone et al., 1987, 1988a, b; Suihko et al., 1989; Shimizu et al., 1989; Blomqvist et al., 1991; Tada et al., 1995). In the latest of these studies it is reported that the gene has been integrated into the yeast chromosome to produce a stable transformant in which a-acetolactate decarboxylase expression is regulated by the promoters for the yeast phosphoglycerokinase (PGK1) or alcohol

Fig. 3.19 Contd on next page.

Fig. 3.19 Effect of addition of exogenous diacetyl to VDK profiles of 2-litre stirred laboratory high-gravity wort fermentations. Five identical fermentations were performed and 1 ppm diacetyl was added to fermentations (b) to (e) at the times indicated by the appearance of the transient peak (Box & Boulton, unpublished data).

Fig. 3.19 Effect of addition of exogenous diacetyl to VDK profiles of 2-litre stirred laboratory high-gravity wort fermentations. Five identical fermentations were performed and 1 ppm diacetyl was added to fermentations (b) to (e) at the times indicated by the appearance of the transient peak (Box & Boulton, unpublished data).

Fig. 3.20 End profiles of VDK concentration for a number of pilot scale (8 hi) lager fermentations - see text for explanation (Besford & Boulton, unpublished data).

Fig. 3.20 End profiles of VDK concentration for a number of pilot scale (8 hi) lager fermentations - see text for explanation (Besford & Boulton, unpublished data).

dehydrogenase (ADH1). Pilot scale brewing trials with the transformants have indicated that the necessity for lagering is either eliminated or much reduced. No other aspects of fermentation performance or beer properties were reportedly altered.

A criticism that may be levelled at the genetic modifications of the type applied to the introduction of a-acetolactate decarboxylase, is that foreign DNA has been introduced into brewing yeast strains. Such approaches carry with them an element of commercial sensitivity and, given the public perception of genetic modification, it is unlikely to be implemented in the immediate future. Conceivably, a more acceptable route to controlling VDK production is to manipulate the existing yeast genome to alter carbon flux through the pathways responsible for the formation of a-acetohydroxy acids. In this way no foreign DNA is introduced, although to the authors' knowledge this route has yet to be exploited commercially.

Goossens et al. (1987) reported that in the isoleucine/valine biosynthetic pathway the reductoisomerase and dehydrase enzymes were rate-limiting (Fig. 3.21). It was argued that amplification of the genes responsible, ILV5 and ILV3, respectively, would increase flux through the pathway and reduce the tendency for accumulation of a-acetohydroxy acids. Initial results indicated that increased expression of the ILV5 gene were most effective. Further evidence for the importance of this enzyme was provided by the observation that petite mutants with deficient ILV5 genes over-produced diacetyl (Debourg et al., 1991). In early work gene amplification was achieved by introducing ILV5 on a multi-copy plasmid (Villanueba et al., 1990). However, this

ILV1 (Threonine deaminase)



ILV2 (Acetohydroxy acid synthase)

2,3-Pentanedione M-a-Acetohydroxybutyrate a-Acetolactate

ILV5 (Acetohydroxy acid reductoisomerase)

valerate a,ß-Dihydroxyisovalerate

ILV3 (Acetohydroxy acid dehydratase)

oc-Keto-p-methyl valerate a-Ketoisovalerate



Fig. 3.21 Activities corresponding to the ILV genes and their relationship to amino acid and VDK formation.

proved unstable and in order to overcome this problem transformants were produced in which the gene was integrated into the chromosome and controlled by the constitutive GDP2 (glyceraldehyde 3-phosphate) promoter (Goossens et al., 1991, 1993). Such transformants were stable and in brewing trials reduced diacetyl formation by 50% compared to wild-type strains. No adverse effects on beer quality were reported.

No metabolic functions for the VDK pathway have been proposed. However, the convoluted pathway of excretion of a-acetohydroxy acids and subsequent assimilation of VDK suggests that some metabolic role must be being fulfilled. As with the formation of higher alcohols the only obvious function would be yet another route for maintenance of cellular redox using the NAD (P)H-linked dehydrogenases involved in the terminal steps of VDK reduction.

3.7.5 Sulphur compounds

A number of sulphur containing compounds, both inorganic and organic, contribute both directly and indirectly to beer flavour. Many of these derive directly from wort and persist unchanged in beers but some are influenced by yeast metabolism. Those of particular significance, which are influenced by yeast metabolism during fermentation, are hydrogen sulphide (H2S) and sulphur dioxide (S02). At low concentrations, these may make a positive contribution to beer but at higher levels they may impart undesirable tastes and aromas. For example, a low but detectable concentration of H2S is acceptable and indeed characteristic of some top-fermented ales; however, at high concentrations it would be considered a most undesirable flavour defect.

Indirect flavour effects may arise from the ability of sulphur-containing compounds such as S02 to form reversible adducts with carbonyl compounds. In this way, for example, high sulphite concentrations, which may arise during fermentation, can stabilise beer flavour by binding compounds associated with beer flavour staling such as acetaldehyde and trans-2-nonenal. In addition, sulphite acts as a natural antioxidant. In general there is a positive correlation between the quantity of sulphite formed during fermentation and the concentration of the wort. In low alcohol beers, which may be produced by fermentation of dilute worts or by partial fermentation of normal worts, there may be an insufficiency of sulphite formed and this may affect adversely the staling potential of such beers. Although the shortfall may be corrected for by addition of sulphite to packaged beers, this represents an additional expense and requires specific labelling in some countries. This has provided the impetus for several groups to perform research work into the biochemical and genetic basis of sulphite formation so that methods of maximising the formation of this metabolite can be identified.

Dufour (1991) discussed the significance of carbonyl-sulphite adduct formation during fermentation. This author pointed out that the degree of binding of each type of carbonyl would be a function of the magnitude of the adduct's equilibrium constants. In this regard, the highest affinities would be shown for acetaldehyde, whereas the vicinal diketones would have medium affinities. It was concluded that during fermentation the rate of sulphite formation would regulate the proportion of car-bonyls bound as adducts and that fraction reduced by yeast. Carbonyl adducts persisting in beer are flavour negative; however, under some circumstances they may be released to exert their staling effects. The total concentration of sulphite present will correlate with flavour stability. Thus, if sulphite concentrations are low carbonyls can be released due to competitive irreversible reactions of sulphite with other beer components such as polyphenols and quinones.

As described in Clarke et al. (1991), during the early part of fermentation H2S accumulates and then declines during the later stages. Under some circumstances, the phase of decline may not occur and indeed a further increase is possible, resulting in undesirable levels of this metabolite persisting in the beer after fermentation is completed. The quantity of H2S formed during fermentation is much influenced by the yeast strain (Romano & Suzzi, 1992). However, excessive concentrations may arise through deficiencies in wort composition, inappropriate control of fermentation or use of yeast with stressed physiology.

The influence of wort composition on the formation of H2S and other sulphur volatiles is discussed later in this section. Considerable H2S may be lost during active fermentation because of the effects of gas stripping. Should the vigour of fermentation be lessened for any reason, there will be a concomitant reduction in gas purging effects. Such a situation can arise, for example, due to a failure to provide adequate wort oxygenation. Sticking fermentations may also be a consequence of the use of pitching yeast that is in poor condition. In this event H2S may arise as a result of yeast death and autolysis with subsequent degradation of sulphur-containing amino acids.

Traditionally, excessive H2S was controlled by treatment with copper sulphate solution, thereby forming a precipitate of insoluble copper sulphide. This approach must be treated with caution to avoid poisoning yeast. In addition, use of such potentially dangerous additives is now rightly frowned upon. Interestingly, replacement of copper fermenting vessels with stainless steel types has been associated with an increase in beer H2S (R. Wharton, personal communication). King et al., (1990) reported that supplementation of worts with pantothenate (0.01 ppm) suppressed H2S formation. Conversely, addition of certain amino acids, particularly serine, to cask beers during secondary fermentation was effective at reducing sulphury odours. Supplementation of worts with pantothenate also decreases concentrations of S02 and acetaldehyde during fermentation (Lodolo et al., 1995). These authors suggested that this would improve flavour stability since acetaldehyde binds to S02 in preference to more flavour staling longer-chain aldehydes. It was speculated that for some wort and yeast combinations there is a deficiency of pantothenate which may lead to a shortfall of coenzyme A and this results in overproduction of acetaldehyde.

S02 formation during fermentation is influenced principally by wort composition Dufour (1991). Dufour et al. (1989) demonstrated that a positive correlation existed between sulphite concentration and wort OG. Conversely, increase in wort oxygenation, or elevated wort lipid (in the form of trub) was associated with reduced S02. The yeast's physiological condition was also shown to be influential. In this case it was demonstrated that starvation of yeast prior to pitching was associated with increased S02 accumulation. Thus, a positive correlation between starvation time and S02 concentration formed during fermentation was reported.

These relationships can be explained in terms of yeast growth extent during fermentation and how this influences assimilation of wort nutrients, in particular amino acids and sources of inorganic sulphur such as sulphate. Biosynthesis of S02 from sulphate during fermentation requires metabolic energy, and therefore a source of fermentable sugar is needed. It follows that more concentrated worts offer the possibility of increased accumulation. As a general rule, amino acids inhibit the bio-synthetic route from sulphate, and therefore the ratio of fermentable carbohydrate to amino nitrogen will exert a modulating effect. However, products of carbohydrate catabolism and specific amino acids exert additional specific effects, as discussed subsequently, adding further levels of complexity. Factors which influence the extent of yeast growth and thus utilisation of amino nitrogen will also influence the formation of S02, hence the effects of increasing wort oxygenation and lipids. Conversely, yeast in poor physiological condition grows to a limited extent during fermentation and amino acid assimilation will also be affected.

Beer sulphur-containing components, which are influenced by yeast metabolism, can arise from two routes. First, from the dissimilation of complex organic molecules such as sulphur-containing amino acids and vitamins and, second, from assimilatory reactions involving inorganic sulphur-containing nutrients. The pathway of assimilation of sulphate, its reduction and incorporation into sulphur-containing amino acids is shown in Fig. 3.22. Sulphate enters the cell via a specific permease and is reduced to sulphite and then sulphide in a sequence of energy-requiring reactions. Sulphide is incorporated into the sulphur-containing amino acids cysteine, methionine and S-adenosylmethionine.

Regulation of sulphur metabolism is complex involving feedback inhibition of enzyme activity and repression of gene expression. In particular, S-adenosylmethionine represses transcription of all the enzymes involved in sulphate uptake and reduction to sulphide, as well as those which catalyse S-adenosylmethionine synthesis. Thus, growth in the presence of high concentrations of methionine inhibits sulphite production. The presence of threonine increases sulphite formation, reportedly by feedback inhibition of aspartokinase such that there is a depletion of the pool of O-acetylhomoserine, and hence methionine levels fall and relieve the repression effects exerted by this metabolite. Isoleucine brings about feedback inhibition of threonine utilisation and hence has the same outcome as the presence of threonine alone (Gyllang et ai, 1989).

Korch et al. (1991) demonstrated a correlation between sulphite production and concentration of glucose in wort. Since there was a concomitant increase in ethanol and acetaldehyde these authors proposed that an intermediate of glucose catabolism could be implicated. They suggested that pyruvate and acetaldehyde formed adducts with sulphite and this deprived the methionine synthetic pathway of sulphite. As a result the sulphite synthetic pathway becomes increasingly derepressed.

Dufour (1991) proposed four stages of sulphite production. In the first during very early fermentation the presence of high levels of methionine and threonine repress the sulphite synthetic pathway. In the second phase which equates to the period of active yeast growth the decline of wort methionine and threonine derepresses the sulphite synthetic pathway but the pool so formed is fully utilised for the synthesis of sulphur-containing amino acid required for biomass formation. Hence, no extracellular sulphite accumulates. In the third phase (mid to late fermentation), yeast growth ceases, the sulphite reductase declines to a low level but the continued availability of fermentable sugar allows sulphite biosynthesis to continue unabated and extracellular


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  • milen ermias
    What is vdk and how to reduced in beer?
    3 years ago
  • maurizio palerma
    Does oxygen increase VDK in beer?
    2 years ago
  • Salvador
    What is vdk stand for in beer?
    1 year ago

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