Kinetic study of the chemical reactivity of ?-acetolactate as a function of pH in water, and in fresh and fermented culture media used for Lactococcus lactis spp. lactis bv. diacetylactis cultivation

Ekberg

et al. 2014

Yeast

A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Saccharomyces pastorianus; lager beer; diacetyl; ILV6; acetohydroxyacid synthase IntroductionDiacetyl or 2,3-butanedione is a vicinal diketone (VDK) formed during beer fermentation as well as other yeast fermentations (Inoue, 2008). It is typically perceived as a butter-or butterscotch-like flavour and has a low flavour threshold (20-100 ppb) (Krogerus and Gibson 2013a). In lager beer, which is characterized by a clean flavour profile, diacetyl is generally considered to be an off-flavour and considerable effort is taken to ensure that concentrations are lower than threshold levels before sale. Diacetyl is formed as an indirect by-product of valine biosynthesis in yeast, and the amount formed during fermentation varies greatly, depending on wort valine content and other parameters, such as the yeast strain used, the wort quality (e.g. free amino nitrogen content, initial oxygen content and pH) and the fermentation conditions (e.g. temperature, pressure and yeast pitching rate) (Garcia et al., 1994;Kobayashi et al., 2005;Pugh et al., 1997;Verbelen et al., 2008Verbelen et al., , 2009. The concentration of diacetyl in beer typically peaks during the first half of the fermentation and thereafter is lowered toward the end of the fermentation. Freshly fermented 'green' beer undergoes a time-and resource-consum...

Section: Discussionmentioning

confidence: 99%

Variation inα-acetolactate production within the hybrid lager yeast groupSaccharomyces pastorianusand affirmation of the central role of theILV6gene

Ekberg

et al. 2014

Yeast

“…Hence, it becomes evident that it is not the valine concentration per se that is of central importance regarding the production rate of diacetyl during fermentation, but rather the uptake rate. Since the pH of the semi-synthetic worts was lower during fermentation than that of the all-malt worts (maximum difference 0.4 units), most likely due to loss of buffer capacity through dilution, the lower diacetyl concentrations produced with wort containing a reduced FAN content may also be influenced in this case by an increased reaction rate for the spontaneous decarboxylation of α-acetolactate into diacetyl (Garcia et al 1994; Kobayashi et al 2005a; Rondags et al 1996). …”

Section: Discussionmentioning

confidence: 99%

Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort

Appl Microbiol Biotechnol

2013

Undesirable butter-tasting vicinal diketones are produced as by-products of valine and isoleucine biosynthesis during wort fermentation. One promising method of decreasing diacetyl production is through control of wort valine content since valine is involved in feedback inhibition of enzymes controlling the formation of diacetyl precursors. Here, the influence of valine supplementation, wort amino acid profile and free amino nitrogen content on diacetyl formation during wort fermentation with the lager yeast Saccharomyces pastorianus was investigated. Valine supplementation (100 to 300 mg L−1) resulted in decreased maximum diacetyl concentrations (up to 37 % lower) and diacetyl concentrations at the end of fermentation (up to 33 % lower) in all trials. Composition of the amino acid spectrum of the wort also had an impact on diacetyl and 2,3-pentanedione production during fermentation. No direct correlation between the wort amino acid concentrations and diacetyl production was found, but rather a negative correlation between the uptake rate of valine (and also other branched-chain amino acids) and diacetyl production. Fermentation performance and yeast growth were unaffected by supplementations. Amino acid addition had a minor effect on higher alcohol and ester composition, suggesting that high levels of supplementation could affect the flavour profile of the beer. Modifying amino acid profile of wort, especially with respect to valine and the other branched-chain amino acids, may be an effective way of decreasing the amount of diacetyl formed during fermentation.

“…towards the end of fermentation). Several sources report an increased reaction rate for the oxidative decarboxylation of α ‐acetolactate to diacetyl at lower pH values, which also suggests that the rate‐limiting conversion of α ‐acetolactate to diacetyl is faster at more acidic wort conditions. The maturation time needed for diacetyl reduction could thus be reduced at lower beer pH values, as long as the pH stays within the range that is suitable for a palatable beer.…”

Section: Process Conditions and Diacetyl Reductionmentioning

confidence: 99%

125^thAnniversary Review: Diacetyl and its control during brewery fermentation

2013

J. Inst. Brew.

Diacetyl is a butter-tasting vicinal diketone produced as a by-product of yeast valine metabolism during fermentation. Concentration is dependent on a number of factors including rate of formation of the precursor α-acetolactate by yeast, spontaneous decarboxylation of this acetohydroxy acid to diacetyl and removal of diacetyl by yeast via the action of various reductase enzymes. Lowering concentrations of diacetyl in green beer represents an expensive and time-consuming part of the brewing process and strategies to minimize diacetyl formation or hasten its reduction have potential for improving overall efficiency of the lager brewing system. Here we review the processes that determine diacetyl levels in green beer as well as the various ways in which diacetyl levels can be controlled. The amount of diacetyl produced during fermentation can be affected by modifying process conditions, wort composition or fermentation technique, or by yeast strain development through genetic engineering or adaptive evolution. The process of diacetyl reduction by yeast is not as well understood as the process of formation, but is dependent on factors such as physiological condition, cell membrane composition, temperature and pH. The process of diacetyl removal is typically rate-limited by the reaction rate for the spontaneous decarboxylation of α-acetolactate to diacetyl. Copyright © 2013 The Institute of Brewing & Distilling Keywords: diacetyl; vicinal diketone; amino acid; beer; yeast; fermentation Diacetyl production during fermentationDiacetyl (2,3-butanedione) and 2,3-pentanedione are vicinal diketones (VDK) formed during beer fermentation as byproducts of amino acid synthesis (valine and isoleucine, respectively) in Saccharomyces yeast, and these can have a significant effect on the flavour and aroma of beer. Diacetyl is known for its butter-or butterscotch-like flavour, with a flavour threshold usually reported as around 0.1-0.2 ppm in lager and 0.1-0.4 ppm in ales (1,2), although flavour thresholds as low as 17 ppb (3) and 14-61 ppb (4) have been reported. It has also been suggested that the flavour threshold of diacetyl varies with taster's geographical background, ethnicity and diet (5). The compound 2,3-pentanedione has a similar flavour to diacetyl, although often described as more toffee-like, but with a higher flavour threshold of around 0.9-1.0 ppm (1,2). VDKs are most easily detectable in lighter beers, where the flavour is not masked by malt and hop flavours. The presence of VDKs above their flavour threshold in beer is generally regarded as a defect, since their flavour is undesirable in many beer styles and it can also indicate microbial contamination, for example, by Lactobacillus spp., Pediococcus spp. or Pantoea agglomerans (6-8). Nevertheless, diacetyl at detectable concentrations is acceptable in some beer styles, such as Bohemian Pilsner and some English ales. Martineau et al. (9) found diacetyl concentrations ranging from 0.002 to 0.1 ppm (average concentration of 0.046 ppm) during the analysis of 11 comm...