Tag Archives: diacetyl

Physiology of Finnish Baker’s Yeast (Suomen Hiiva)

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While ale yeast and baker’s yeast belong to the same species, Saccharomyces cerevisiae, they have over time been adapted to different functions. Brewing with baker’s yeast is not a very common practice, nor generally recommend amongst brewers, mostly because these strains might not exhibit favorable fermentation characteristics, such as production of desirable flavour compounds, adequate attenuation, and flocculation. Of the recognized beer styles, it is more or less only in Northern European Traditional ales (such as Sahti, Gotlandsdricka and Kvass) that it acceptable to ferment with baker’s yeast. As there is little information published on the physiological characteristics of Finnish baker’s yeast in brewing, I thought I’d post some recent results from my related research.

hiiva

I performed a series of mini-fermentations (30 ml of wort) on a range of yeast strains earlier this spring for screening purposes, and I included the baker’s yeast from Suomen Hiiva, as well as two common homebrewing strains WLP002 (English Ale) and WLP380 (Hefeweizen IV). These mini-fermentations were performed at two different temperatures, 15 and 20 °C. Fermentation progress was monitored by weight loss, and after fermentation the resulting beer was analyzed for ABV% (Anton Paar Alcolyzer ME), Extract (Anton Paar DMA 5000 M), pH (Anton Paar pH ME), aroma compounds (HS-GC/FID), diacetyl (HS-GC/ECD), and phenolics (HPLC/PAD). A 15 °Plato (OG 1.060) wort was used for all fermentations, and the yeast was pitched at a rate of 2.5 g/L (~ 10 million cells/ml).

What first surprised me was how well the baker’s yeast performed during fermentation. Compared to the other two reference strains presented here, it fermented faster at both temperatures and reached a higher final attenuation. It managed to reach a final attenuation of 85% in just 5 days when fermented at 20 °C. Not bad for an all-malt wort at this strength. So the baker’s yeast can definitely give rise to adequate attenuation. Note, a pure culture of the yeast was used (starting from a similar package as in the picture above), so there was no risk of any lactic acid bacteria contamination, which otherwise is probable when using the yeast directly. The pH values of all beers were quite similar irrespective of yeast strain and temperature, and it seems like the baker’s yeast acidified the wort slightly less than the other two reference strains. In the table below you can find a summary of the Extract, ABV% and pH of the beers. In the figure below you can find a plot of the fermentation progress over time (20 °C squares; 15 °C circles).

sh_beer

sh_fermentation

I have never actually used baker’s yeast for beer fermentation myself, as I’m not that big of a Sahti fan, but all beers (well mainly Sahtis) I’ve tried have had quite a ester-dominated aroma. Especially 3-methylbutylacetate (isoamyl acetate), with its prominent banana aroma, is very pronounced in, and also an integral part of, Sahti. Hence, it comes as no surprise that, compared to the other two reference strains, the baker’s yeast produced more higher alcohols and esters. It was not only 3-methylbutylacetate that was produced in large amounts (even more than the WLP380 Hefeweizen strain), but also the ethyl esters. Ethyl acetate, with its solvent-like aroma, is typically unwanted at higher concentrations in beer, but the other ethyl esters may contribute a fruity aroma to the beer. In the figure below you can see a summary of the flavour impact (i.e. the concentration of the compound in the beer divided by its flavour threshold. An impact above 1 should affect flavour, while an impact between 0.5 and 1 may affect flavour) of the various higher alcohols, esters, and acetaldehyde in the beers fermented with the different yeast strains (20 °C solid; 15 °C striped). As can be seen from the figure, only 3-methylbutanol (the precursor to the banana ester) of the higher alcohols is close to the flavour threshold. As should come to no surprise, less higher alcohols and esters were produced at a lower fermentation temperature. From these aroma compound results, it is clear that the baker’s yeast will produce a fruity, and maybe even slightly solvent-like, beer. Hence, the yeast would probably be suitable for Hefeweizens and Belgian-style beers, and a slightly lower fermentation temperature is probably recommended.sh_aroma

 

Finally we arrive at two other important aroma compounds in beer, diacetyl (butter-like aroma) and 4-vinylguaiacol (clove-like aroma; 4-VG). Diacetyl is always considered an off-flavour (well in some cases in might be acceptable, but in my opinion it is just a sign of poor fermentation practices), while 4-VG is acceptable (and even required) in some styles (such as Hefeweizens and Belgian-style beers). Diacetyl levels decrease towards the end of fermentation, and are highly dependent on fermentation dynamics, so the concentration that was measured here at the end of fermentation doesn’t say that much. The diacetyl concentration was above the flavour threshold (50 ppb) for all strains at both temperatures, which is not that surprising since the measurements were made 120 hours after pitching the yeast, and the lowest flavour impact was observed in the beer fermented with the baker’s yeast at 20 °C. This result again suggests that baker’s yeast is a good candidate for beer fermentations (and especially more rapid ones). The baker’s yeast is also POF+ (i.e. positive for producing ‘phenolic off-flavours’; the yeast produces a phenylacrylic acid decarboxylase enzyme, that decarboxylates ferulic acid (and other phenolic acids) from the wort into 4-vinylguaiacol (and other phenolic compounds)), since 4-VG was observed in the beer. The baker’s yeast produced slightly less 4-VG than WLP380 (the table below displays the percentage of ferulic acid converted into 4-vinylguaiacol; 77% is the theoretical maximum), but it still produced concentrations above the flavour threshold in an all-barley wort (worts made from wheat malts contain more ferulic acid). This slightly limits the beer styles that the baker’s yeast could potentially be used for, but again it could be used for Hefeweizens and Belgian-style beers.

sh_diacetyl_pof

To conclude, it is evident that the Finnish baker’s yeast is perfectly usable for beer fermentations, and it comes to no surprise that it has successfully been used for traditional beer fermentation in the form of Sahti. Of the two reference strains, the baker’s yeast was closer to WLP380 (Hefeweizen IV), and it can be described as a faster-fermenting and fruitier version of it. As a side-note, it can be mentioned that the baker’s yeast flocculated very poorly, which is also similar to the behavior of WLP380. I would not recommend the use of the baker’s yeast for beer styles calling for a clean yeast aroma and no spicy phenolics, but it would be perfectly suitable for a Hefeweizen, Belgian Blond and why not even Belgian Strong Ale (Sahtis are brewed strong, so the baker’s yeast should be quite tolerant to alcohol levels reaching up to 10% ABV). It fermented quite fast at both 20 °C and 15 °C, and I would recommend a lower temperature if you are after a cleaner finish. As mentioned previously, the yeast packages found in the supermarket are all almost certainly contaminated with lactic acid bacteria, so the use of a pure culture is definitely recommended. Good luck with the brewing!

Feel free to contact me if you have any questions!

Reference for flavour thresholds:

  • Meilgaard, M., Prediction of Flavor Differences between Beers from Their Chemical Composition. Journal of Agricultural and Food Chemistry 30 (1982) 1009-1017.

Article on the differences in diacetyl formation between lager yeast strains and the role of the ILV6 gene

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I’ve been researching diacetyl formation and removal by brewing yeasts the past year(s), and we recently did some screening of various lager yeast (Saccharomyces pastorianus) strains for diacetyl production and subsequent transcriptional analysis of key genes in the isoleucine-valine catabolic pathway (diacetyl is formed as an indirect by-product of valine biosynthesis in yeast; more information available here). We did this in order to identify what genes contribute most to the differences in wort total diacetyl concentration we observed between the different strains. We observed that particularly the ILV6 gene, encoding for a regulatory subunit of the acetohydroxy acid synthase enzyme (responsible for converting pyruvate into the diacetyl precursor α-acetolactate), showed greater expression early during fermentation in a strain producing more diacetyl. To confirm the role of this ILV6 gene in controlling α-acetolactate/diacetyl formation, we overexpressed both the S. cerevisiae- and S. eubayanus-form of ILV6 in our standard production lager strain A15, and found that overexpression of either form of ILV6 resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. These results suggest that both forms of the gene influence diacetyl formation, and different levels of transcription (this could be a result of a gene-dosage effect, since different lager yeast hybrids have inherited different amounts of the parental genomes; for a recent review on the subject click here) may contribute to differences in diacetyl production in various lager yeast strains.

Link to the publication: http://onlinelibrary.wiley.com/doi/10.1002/yea.3026/abstract

E-mail me if you are interested in the article, but don’t have access to the full-text.

Abstract:

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.

Diacetyl in beer (Part II): Diacetyl formation and wort amino acids

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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.

Review on diacetyl and its control during brewery fermentations

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I’m happy to announce that my review on the formation and control of diacetyl during brewery fermentations has been published in the Journal of the Institute of Brewing. The review covers how diacetyl is formed during fermentation, what factors affect its formation, how it is removed during fermentation, methods for reducing residual diacetyl content in beer and strain development. Please have a read if you are interested! Diacetyl is still a problem for the big lager breweries (and why not the smaller ones as well), and one of the limiting factors for increasing fermentation speed. Even homebrewers are commonly affected by this off-flavour. I’ll be basing most of my diacetyl mini-essay on the review, and I will be posting the second part soon.

  • Krogerus, K., Gibson, B.R., (2013) 125th Anniversary Review: Diacetyl and its control during brewery fermentation. Journal of the Institute of Brewing 119: 86-97. DOI: 10.1002/jib.84

Diacetyl in beer (Part I): Introduction

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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.

diacetyl1-inverted

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.

Article on relation between wort amino acids and diacetyl production

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I’m really happy to announce that my manuscript entitled ‘Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort’ has been accepted for publication in the Applied Microbiology and Biotechnology journal. As soon as it is published or available online I’ll post a link to it here. It is an interesting read if you are interested in how amino acids in wort affect diacetyl production. Here is the abstract:

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.

  • 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. Applied Microbiology and Biotechnology. In Press. DOI: 10.1007/s00253-013-4955-1