This is the second part of my mini-essay on diacetyl formation during beer fermentation. You can find the first part here. Most of the text is based on my recently published review on diacetyl in brewery fermentations, so have a look at it as well and please cite the review rather than the text in this blog.

So what kinds of fermentation conditions favour the formation of diacetyl? We ended the previous part by stating that fermentation conditions favouring rapid yeast growth can give rise to increased diacetyl production if wort free amino nitrogen content is insufficient. Why is this then? Since diacetyl is directly linked to the valine biosynthesis pathway, the concentration of valine inside the yeast cell will affect the amount of diacetyl generated during fermentation. It has been shown that valine strongly inhibits the acetohydroxyacid synthase (AHAS) enzyme, responsible for catalysing the formation of α-acetolactate from pyruvate (see the second Figure in the previous part) (25, 26). Hence, the more valine present in the yeast cells, the less α-acetolactate will be synthesized, as the catalysing enzyme is inhibited, and consequently less diacetyl will be formed as well. Studies have shown varying data on the inhibitory effects of other branched-chain amino acids on AHAS. Both Barton and Slaughter (26) and Magee and de Robichon-Szulmajster (25) observed that leucine inhibited the AHAS enzyme’s activity, though not as much as valine. No inhibitory effect was observed with isoleucine. Pang and Duggleby (27) observed the opposite on the other hand, i.e. that isoleucine had a slight inhibitory effect and leucine had no inhibitory effect on AHAS activity.

Nakatani et al. (28) studied the effect of supplementing valine and isoleucine to wort on the production of diacetyl and found that increased wort valine concentrations significantly reduced the amount of diacetyl produced during fermentation. In fermentation trials with lager yeast involving wort of differing original gravities, free amino nitrogen and valine content, Petersen et al. (29) observed that low concentrations of valine in the wort resulted in the formation of double-peak diacetyl profiles (most likely as a result of valine depletion toward the end of fermentation), while high concentrations of valine in the wort resulted in single-peak diacetyl profiles with a lower maximum diacetyl level compared to the worts with low valine concentrations. The results show that the valine concentrations of the wort influence the amount of diacetyl formed, but the trials performed in the study varied in specific gravity and free amino nitrogen, meaning that no definite conclusions regarding the relationship between wort valine concentration and diacetyl concentration can be drawn. Cyr et al. (30) observed in trials with two different lager yeast strains, that diacetyl concentrations in the fermenting wort were constant or decreased when valine uptake increased, while diacetyl concentrations increased when valine uptake decreased or was null. Krogerus and Gibson (31) showed that direct supplementation of wort with valine (100 – 300 ppm) and consequently greater uptake of valine by yeast cells resulted in less diacetyl being formed during fermentation. Other fermentation parameters such as fermentation rate and yeast growth were unaffected (31).

The general free amino nitrogen (FAN) content of the wort may also affect the valine uptake rate and consequently diacetyl production. Krogerus and Gibson (31) reported that when FAN levels were lowered the diacetyl production was also lowered presumably due to faster absorption of preferred amino acids, resulting in an earlier and greater demand for valine and its increased uptake due to less competition for permease interactions. Increasing background levels of initial wort amino acids (while keeping valine concentration constant) resulted in a greater production of diacetyl. This increased production was influenced by which amino acids were increased. Preferred amino acids, i.e. those taken up faster than valine, caused greater diacetyl formation in the first stage of fermentation, while increasing the concentrations of non-preferred amino acids influenced diacetyl levels later in the fermentation and therefore had a greater influence on the diacetyl levels in green beer (31). Pugh et al. (32) also observed that the maximum diacetyl concentration during fermentation decreased as the initial FAN content was increased from 122 to 144 ppm, after which it again increased as the initial FAN content was increased from 144 via 168 to 216 ppm. Verbelen (33) reports a lower diacetyl production rate and simultaneously increased valine uptake rate and BAP2 expression level in lager yeast for fermentations of 18° Plato worts containing adjuncts (FAN contents of around 150-210 ppm) compared to 18° Plato all-malt wort (FAN content around 300 ppm). Nakatani et al. (28) on the other hand report a negative correlation between the initial wort FAN content and the maximum VDK concentration observed during fermentation. These conflicting results are presumably due to differences in valine uptake. At high FAN levels the yeast cell utilizes the preferred amino acids and less valine is taken up as a result (resulting in higher α-acetolactate production), while at very low FAN levels many amino acids will be entirely removed from the system and yeast growth is affected. If valine is depleted in this fashion then the demand for anabolic valine synthesis is increased and the α-acetolactate level increases as a result. It would appear from the values available in the literature that a FAN level of approx. 150 ppm is optimum for low diacetyl production, however this value will vary depending on individual fermentation and process conditions. Lei et al. (34) also observed that the amount of valine absorbed during fermentation decreased when FAN content was increased from 264 ppm to 384, 398 and 433 ppm by adding protease enzymes during mashing, despite the increased in total valine concentration.

Barton & Slaughter (26) investigated the effect of adding individual amino acids and ammonium chloride in excess to wort on the VDK concentration and AHAS activity during fermentation, and found that alanine and ammonium chloride significantly lowered both the amount of diacetyl formed and the AHAS activity, suggesting they have an inhibiting effect on the enzyme. Valine and leucine also showed an inhibiting effect on AHAS (their effect on diacetyl concentration was not studied). The results suggest that alanine, ammonium chloride and possibly leucine could be used in excess together with valine in wort, to minimize the formation of diacetyl during fermentation, and that AHAS activity is vital for the control of diacetyl formation. Dasari and Kölling (35) observed elevated diacetyl production in petite mutants of S. cerevisiae, as a result of cytosolic localization of the AHAS enzyme, suggesting that accumulation of AHAS in the cytosol could result in increased diacetyl production, possibly as a result of increased secretion of α-acetolactate from the cell.

In this part we focused primarily on the theory of how wort amino acids affect diacetyl formation, and in the next part we will continue looking at how other fermentation conditions affect diacetyl formation and some methods brewers can use for reducing the amount of diacetyl formed during fermentation.

References:

• (25) Magee, P., de Robichon-Szulmajster, H., (1968) The regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae – 3. properties and regulation of the activity of acetohydroxyacid synthetase. Eur. J. Biochem. 3, 507-511.
• (26) Barton, S., Slaughter, J., (1992) Amino acids and vicinal diketone concentrations during fermentation. Tech. Q.  Master Brew. Assoc. Am. 29, 60-63.
• (27) Pang, S., Duggleby, R., (2001) Regulation of yeast acetohydroxyacid synthase by valine and ATP. Biochem. J. 357, 749-757.
• (28) Nakatani, K., Takahashi, T., Nagami, K., Kumada, J., (1984) Kinetic study of vicinal diketones in brewing, II: theoretical aspect for the formation of total vicinal diketones. Tech. Q.  Master Brew. Assoc. Am. 21, 175-183.
• (29) Petersen, E., Margaritis, A., Stewart, R., Pilkington, P., Mensour, N., (2004) The effects of wort valine concentration on the total diacetyl profile and levels late in batch fermentations with brewing yeast Saccharomyces carlsbergensis. J. Am. Soc. Brew. Chem. 62, 131-139.
• (30) Cyr, N., Blanchette, M., Price, S., Sheppard, J., (2007) Vicinal diketone production and amino acid uptake by two active dry lager yeasts during beer fermentation. J. Am. Soc. Brew. Chem. 65, 138-144.
• (31) Krogerus, K., Gibson, B.R., (2013) Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort. Appl. Microbiol. Biotechnol. 97, 6919-6930.
• (32) Pugh, T., Maurer, J., Pringle, A., (1997) The impact of wort nitrogen limitation on yeast fermentation performance and diacetyl. Tech. Q.  Master Brew. Assoc. Am. 34, 185-189.
• (33) Verbelen, P., (2009) Feasability of high cell density fermentations – for the accelerated production of beer. Ph.D. thesis. Katholieke Universiteit Leuven.
• (34) Lei, H., Zheng, L., Wang, C., Zhao, H., Zhao, M., (2013) Effects of worts treated with proteases on the assimilation of free amino acids and fermentation performance of lager yeast. Int. J. Food Microbiol. 161, 76-83.
• (35) Dasari, S., Kölling, R., (2011) Cytosolic localization of acetohydroxyacid synthase Ilv2 and its impact on diacetyl formation during beer fermentation. Appl. Environ. Microbiol. 77, 727-731.

In this multi-part mini-essay, I thought I’d write a little about diacetyl (or 2,3-butanedione) and why it is an important flavor compound in beer. Most of the text is based on my recently published review on diacetyl in brewery fermentations, so have a look at it as well and please cite the review rather than the text in this blog.

Diacetyl (2,3-butanedione) and 2,3-pentanedione are vicinal diketones (VDK) formed during beer fermentation as by-products of amino acid synthesis (valine and isoleucine, respectively) in Saccharomyces yeast. VDKs can have a significant effect on the flavour and aroma of beer, and lighter beers especially are more vulnerable. Diacetyl is known for its butter- or butterscotch-like flavour, and its flavour threshold is 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), 14 – 61 ppb (4), and 10 – 40 ppb (5) have been reported. This means that 100 µg (0.0001 g) of diacetyl is detectable in 1 litre of beer. 2,3-pentanedione has a similar flavour to diacetyl, though often described as more toffee-like, but it has 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, and light lager beer can typically be troubled with diacetyl flavours. 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, e.g. 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 (smell a freshly poured glass of Pilsner Urquell and you should be able to detect diacetyl).

Diacetyl concentrations in beer can be determined via a variety of analytical methods, including colorimetric assays (e.g. through complex formation with dimethylglyoxime or o-phenylenediamine), gas chromatography and liquid chromatography (10-12). During analysis, care must be taken in order to avoid interference by 2,3-pentanedione and α-acetolactate (a precursor to diacetyl, which we will come to later). During fermentation, the concentrations of free diacetyl in wort are usually low and α-acetolactate rather constitutes the majority of the ‘total diacetyl’ present (22-24). As a result, diacetyl concentrations are often expressed as ‘total diacetyl’ concentrations, i.e. the sum of the free diacetyl and α-acetolactate (‘potential diacetyl’), during analysis, in order to highlight potential diacetyl concentrations.

So how does yeast produce diacetyl? Well, yeast doesn’t actually produce diacetyl, rather it produces a precursor, which gets converted into diacetyl in the wort. The generally accepted pathways for diacetyl and 2,3-pentanedione formation and reduction in Saccharomyces spp. are presented in the figure (click to enlarge) above  (13-16). Diacetyl and 2,3-pentandione are formed indirectly as a result of valine and isoleucine anabolism, since they arise from the spontaneous non-enzymatic oxidative decarboxylation of α-acetohydroxy acids that are intermediates in the valine and isoleucine biosynthesis pathways. In yeast, valine and isoleucine synthesis is localized in the mitochondria (17). In the valine biosynthesis pathway, the reaction between α-acetolactate and 2,3-dihydro-isovalerate is rate-limiting, which means that during fermentation and yeast growth, some α-acetolactate is secreted out through the cell membrane into the wort (13,16-19). The reasons and mechanisms for α-acetolactate secretion by yeast are not fully understood, but may involve protecting the yeast from carbonyl stress (20). The α-acetolactate then spontaneously decarboxylates, either oxidatively or non-oxidatively, forming either diacetyl or acetoin respectively, and in both cases releasing carbon dioxide. The non-oxidative decarboxylation into acetoin can be encouraged by heating under anaerobic conditions and by maintaining a low redox potential in the wort (21). Diacetyl production thus increases with increasing valine biosynthesis, which in turn depends on the cell’s need for and access to valine and other amino acids. Hence, any fermentation conditions that favour rapid yeast growth can give rise to increased diacetyl production if wort free amino nitrogen content is insufficient, and more specifically if the yeast can’t access and uptake sufficient amounts of valine.

This is the end of the first part of the mini-essay. Upcoming parts will discuss what fermentation conditions favour diacetyl formation, what can be done to reduce diacetyl concentrations in the finished beer, and how yeast cells take up valine. The second part can be read here.

References:

• (1) Meilgaard, M., (1975) Flavor chemistry of beer: part II: flavour and threshold of 239 aroma volatiles. Tech. Q.  Master Brew. Assoc. Am. 12, 151-168.
• (2) Wainwright, T., (1973) Diacetyl – a review. J. Inst. Brew. 79, 451-470.
• (3) Saison, D., de Schutter, D., Uyttenhove, B., Delvaux, F., Delvaux, F.R., (2009) Contribution of staling compounds to the aged flavour of lager beer by studying their flavour thresholds. Food Chem. 114, 1206-1215.
• (4) Kluba, R., de Banchs, N., Fraga, A., Jansen, G., Langstaff, S., Meilgaard, M., Nonaka, R., Thompson, S., Verhagen, L., Word, K., Crumplen, R., (1993) Sensory threshold determination of added substances in beer. J. Am. Soc. Brew. Chem. 51, 181-183.
• (5) Aroxa (2013) Diacetyl beer flavour standard – 2,3-butanedione – butter, butterscotch. [Online] Available at: http://www.aroxa.com/beer/beer-flavour-standard/2-3-butanedione/
• (6) Boulton, C. and Quain, D., (2001) Brewing Yeast and Fermentation. Blackwell Science.
• (7) Priest, F., (2003) Gram-positive brewery bacteria, in Brewing Microbiology, (F. Priest & I. Campbell, eds.), pp. 181-217, New York: Kluwer Academic/Plenum Publishers.
• (8) van Vuuren, H., Cosser, K., Prior, B., (1980) The influence of Enterobacter agglomerans on beer flavour. J. Inst. Brew. 86, 31-33.
• (9) Martineau, B., Acree, T., Henick-Kling, T., (1994) A simple and accurate GC/MS method for quantitative analysis of diacetyl in beer and wine. Biotechnol. Tech. 8, 7-12.
• (10) European Brewery Convention. (2008)  Analytica–EBC. 7th ed. Section 9 Beer Method 9.24 Vicinal Diketones in Beer, Fachverlag Hans Carl: Nürnberg, Germany.
• (11) American Society of Brewing Chemists. (2011)  Methods of Analysis, 14th ed (online). Beer-25 Diacetyl. The Society: St. Paul, MN.
• (12) McCarthy, S., (1995) Analysis of diacetyl and 2,3-pentanedione in beer by HPLC with fluorometric detection. J. Am. Soc. Brew. Chem. 53, 178-181.
• (13) Chuang, L., Collins, E., (1968) Biosynthesis of diacetyl in bacteria and yeast. J. Bacteriol. 95, 2083-2089.
• (14) Radhakrishnan, A., Snell, E., (1960) Biosynthesis of valine and isoleucine. J. Biol. Chem. 235, 2316-2321.
• (15) Strassman, M., Shatton, J., Corsey, M., Weinhouse, S., (1958) Enzyme studies on the biosynthesis of valine in yeast. J. Am. Chem. Soc. 80, 1771-1772.
• (16) Suomalainen, H., Ronkainen, P., (1968) Mechanism of diacetyl formation in yeast fermentation. Nature 220, 792-793.
• (17) Ryan, E., Kohlhaw, G., (1974) Subcellular localization of isoleucine-valine biosynthetic enzymes in yeast. J. Bacteriol. 120, 631-637.
• (18) Dillemans, M., Goossens, E., Goffin, O., Masschelein, C., (1987) The amplification effect of the ILV5 gene on the production of vicinal diketones in Saccharomyces cerevisiae. J. Am. Soc. Brew. Chem. 45, 81-84.
• (19) Haukeli, A., Lie, S., (1971) The influence of 2-acetohydroxy acids on the determination of vicinal diketones in beer and during fermentation. J. Inst. Brew. 77, 538-543.
• (20) van Bergen, B., Strasser, R., Cyr, N., Sheppard, J., Jardim, A., (2006) α,β-dicarbonyl reduction by Saccharomyces d-arabinose dehydrogenase. BBA-Gen. Subjects 1760, 1636-1645.
• (21) Kobayashi, K., Kusaka, K., Takahashi, T., Sato, K., (2005) Method for the simultaneous assay of diacetyl and acetoin in the presence of α-acetolactate: application in determining the kinetic parameters for the decomposition of α-acetolactate. J. Biosci. Bioeng. 99, 502-507.
• (22) Haukeli, A., Lie, S., (1972) Production of diacetyl, 2-acetolactate and acetoin by yeasts during fermentation. J. Inst. Brew. 78, 229-232.
• (23) White, F., Wainwright, T., (1975) Occurrence of diketones and α-acetohydroxyacids in fermentations. J. Inst. Brew. 81, 46-52.
• (24) Landaud, S., Lieben, P., Picque, D., (1998) Quantitative analysis of diacetyl, pentanedione and their precursors during beer fermentation by an accurate GC/MS method. J. Inst. Brew. 104, 93-99.