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.