The text below will appear in the upcoming 46th edition of “Piwowar,” a periodical for homebrewers, in a slightly modified form, co-authored with Paweł Masłowski, Head Brewer and co-owner of Moon Lark Brewery.
Introduction
A by-product of alcoholic fermentation, warmly welcomed in the Czech Republic and its lagers, somewhat less favourably viewed in most countries and beer styles. Diacetyl (2,3-butanedione), as it is referred to, is a by-product of alcoholic fermentation. The compound emerges during the synthesis of valine (Val), an essential amino acid found in beer wort. Together with 2,3-pentanedione, they form a group of compounds called Vicinal Diketones (VDK). Though diacetyl has been acknowledged for its profound impact on beer taste, aroma, and overall palatability since the 1960s, its intricate effects continue to baffle brewers and beer enthusiasts alike. In this article, we delve into the cause, effects, and potential remedies for diacetyl-related challenges during beer fermentation.
The Aromas of Diacetyl
Researchers noticed that the flavour threshold of diacetyl in beer for most people ranges from 0.1-0.2 mg/L in bottom-fermented beers and 0.1-0.4 mg/L in top-fermented beers. In contrast, the taste threshold oscillates between 1.4-6.1 mg/L for beers of both types. This suggests that diacetyl is much easier to pick up with the sense of smell than with taste. Therefore, later in this article I will describe the tests worth using to easily assess its presence in a beer sample. Diacetyl aromas are present in concentrations above the detection threshold and may be expressed as butter, buttery popcorn, fudge or Werter’s Original hard candies. All that creates a sensory experience that can both delight and perturb.
Understanding the Culprit: Diacetyl’s Origins and Influence
The formation of VDK compounds is a consequence of Saccharomyces yeast metabolism during its growth and fermentation. Because of the direct relationship between diacetyl and valine formation, the intracellular concentration of this amino acid is inversely correlated to the amount of diacetyl produced. This means that the more valine, the less diacetyl and vice versa. The amount of valine in the beer wort depends primarily on the type of base malt used to make the beer, where oat and barley malt have the most valine (4.7-4.9% and 5.0-5.7% of the total protein content, respectively), while wheat malt contains the least (4.4%).
The final concentration of valine (as well as other amino acids) is also influenced by the supplementation of the wort with yeast extract-based media, e.g. Fermaid-O, or Servomyces, but also by the type and process of yeast production, as discussed later in this article. It was observed that direct supplementation of the wort with valine alone (100-300 mg/L) and consequently better absorption of this amino acid by the yeast, resulted in less diacetyl production during fermentation. Unfortunately, as it happens in nature, excess harms, increasing the risk of fusel (1,2-isobutanol) formation.
If we compare lager yeast and ale yeast in terms of the rate of valine assimilation from the wort, the former is slightly slower. So, it may seem that diacetyl is more intensely perceptible in lagers due to their usually cleaner aroma profile, but also due to the rate of valine metabolism.
Dry Yeast and Diacetyl Divergence
Interestingly, rehydrated, active dry yeast displays a sixfold slower valine uptake than liquid yeast. This discrepancy contributes to approximately 1.5-7 times higher diacetyl concentrations during fermentation. Moreover, beer fermented with certain active dry yeast strains maintains elevated diacetyl levels for up to seven days, attributed to the reduced ability of dry yeast cells to absorb wort constituents – a consequence of damage sustained during the active dry yeast production process.
Other factors influencing diacetyl concentration
The synthesis of α-acetolactate, a precursor for diacetyl, constitutes an intermediate step in valine formation. This compound is released from yeast cells, yielding CO2 upon conversion to diacetyl. Of note, the delayed release of CO2 near to fermentation’s end can also stem from α-acetolactate to diacetyl conversion*. Below in the figure, we can observe the relationship occurring between diacetyl concentration and the stage of a typical lager fermentation (Figure 1.).
Figure 1. Diacetyl metabolism during beer wort fermentation.
The graph highlights the correlation between diacetyl concentration, wort extract, and yeast cell count in suspension.
Numerous factors influence diacetyl concentration in both fermentation and the final product. These include wort’s amino acid composition, as well as Free Amino Nitrogen (FAN) content. When FAN levels are reduced, diacetyl production is also reduced. This is probably due to the fact that valine is absorbed more quickly from the wort by the yeast.
Yeast quantity also plays a great role. A substantial yeast influx augments diacetyl concentrations, evidenced by over a 10-fold diacetyl concentration escalation during lager fermentation with heightened yeast infusion (termed over-pitching). This scenario results from augmented α-acetolactate production and accelerated yeast energy depletion, inhibiting diacetyl reduction (stemming from intensified nutrient competition between yeast cells). If fermentation proceeds correctly (with ample amino acid availability to yeast), raising fermentation temperature initially boosts diacetyl levels. Ultimately, elevated biomass production allows yeast to better counteract diacetyl. Temperature and pH are pivotal in this process, influencing α-acetobutyrate conversion and diacetyl reduction rates. A simplified scheme for the reduction of diacetyl to acetoin and butanediol is shown in Figure 2.
Figure 2. Diacetyl reduction occurs inside the cell with the involvement of enzymes. The products of this transformation are released outside the yeast cell. Adapted from G. Stewart, (2017) The Production of Secondary Metabolites with Flavour Potential during Brewing and Distilling Wort Fermentations
The enzyme responsible for the reduction of diacetyl to acetoin has its optimum of action around pH 3.5, suggesting that it occurs more intensively at lower pH, i.e. towards the end of fermentation when the beer wort reduces its pH level to <4.5.
Diacetyl’s Varied Origins
Diacetyl’s inception extends beyond fermentation. Finished, packaged beer can harbor diacetyl as a result of the Maillard reaction, acetoin and butanediol oxidation, or by other microorganisms, such as lactic acid bacteria resulting from infections, can also contribute to diacetyl in beer. The latter often stems from inaccurate brewery equipment cleaning or glass line maintenance; these infections involve in most cases Lactobacillus and/or Pediococcus. Stressed yeast and improperly conducted fermentation also warrant mention as potential diacetyl sources. Finally, the burgeoning hop creep phenomenon can affect diacetyl levels in beer.
Diacetyl and Hopcreep
Hop creep involves a secondary fermentation triggered by amylolytic enzymes released from hops during dry hopping. An unintended consequence is diacetyl formation and the potential for intensified buttery scents if beer is prematurely cooled. To mitigate diacetyl during hop creep, lengthening fermentation and elevating temperatures to diacetyl rest levels is advised, complemented by a suggested forced diacetyl test
Practical advice for brewers:
For brewers aiming to maintain optimal diacetyl levels, consider the following guidelines:
- Choose the right raw materials: account for valine content in protein dry matter when choosing base malts, and avoid excessive oat malt proportions.
- Control the amount of yeast pitched. Standard pitch-rate for ale beers:
- ≤12P = 0.5 million/ml/P,
- ≤16P = 0.75 million/ml/P,
- ≥18P = 1 million/ml/P,
- for lager beers fermented at low temperatures, use a minimum of 1.5 million/ml/P up to 16P, and >16P 2 million/ml/P.
- Ensure good fermentation conditions: prioritize temperature control, yeast nutrition, oxygenation, and yeast condition for effective fermentation.
- Implement a diacetyl break: it is usually applied for the last 25% of wort attenuation, raising the temperature of the end of fermentation 10% higher than the highest temperature of the fermentation to date. The diacetyl break usually lasts for 1-3 or 4 days.
- Use yeast for Kölsch (YS128 Köln) to avoid the diacetyl problem with hop creep during dry hopping your ale beers with a clean profile. For juicy NEIPAs go for YS107 Manchester Ale, as we have shown it presents good diacetyl reduction properties.
- Use the α-acetolactate enzyme (ALDC) at the beginning of fermentation (commercial name ‘Maturex’) catalyzing the reaction to prevent diacetyl formation:
α-acetobutyrate → acetoin + CO2
- Admittedly, these are not available in the EU, but it is worth noting that there are GMO yeasts that have a gene encoding an ALDC that prevents diacetyl formation. It is not necessary to use the VDK test at that time.
- Introduce a forced diacetyl test before chilling the beer for lagering. Below you will find a protocol for performing such a test:
- Take beer samples from the fermenter (50-200 ml) into two bottles or jars and cap them tightly + recipe (~65 C for 20 min)
- Place one sample in a container of water at ~65 C and hold for 20 min. Leave the other at room temperature
- Equalize the temperatures of both samples by cooling the heated sample
- Perform sensory analysis starting with the heated sample
- If both samples show no buttery flavours, e.g. Werter’s Orginals candies, buttered popcorn – diacetyl has been reduced. When the above aromas are felt in the heated sample, extend the diacetyl break.
Author: Kamil F. Tomaszewski
