Diacetyl: Sources & Solutions
In the family of vicinal diketones (VDKs), there are two that are of the most importance to brewers: diacetyl (2,3-butanedione) and pentanedione (2,3-pentanedione). The flavour threshold for pentanedione is ten times higher than that of diacetyl which makes diacetyl the primary concern. Olfactory tests describe diacetyl as butterscotch or popcorn margarine, and pentanedione has notes of honey, butter, caramel and is generally sweet. They are considered flaws in most styles but are acceptable in some British ales and Czech lagers. It is easier to identify VDKs in less intensely flavoured beers; fuller flavoured lagers may have a diacetyl threshold of 0.1ppm where light lagers may require levels of 0.02ppm or less. (Bamforth, 2014)
Figure 1 - Yeast-Derived Beer Flavour Compounds Formation Map (Briggs, et al., 2004)
The synthesis of Valine and Isoleucine forms diacetyl and pentanedione as by-products. The acetohydroxy acids α-acetolactate and α-acetohydroxybutyrate are formed by yeast during fermentation in the wort. The acetohydroxy acids react in an oxidative decarboxylation to form the VDKs. Later in the fermentation, VDKs are absorbed by the yeast and reduced to create compounds with higher flavour thresholds. Diacetyl forms acetoin and 2,3-butanediol; and 2,3-pentanedione forms 2,3-pentanediol. (Briggs, et al., 2004) Another source of VDKs is from the Maillard reactions that occur in malting and wort boiling. (Cha, et al., 2019)
Additionally, contamination of bacteria of Lactobacillus and Pediococcus should receive an evaluation as they can contribute to diacetyl. (Boulton & Quain, 2001) Controlling the uptake of free amino nitrogen during primary fermentation, reducing the rate of synthesis of Valine and isoleucine, and increasing the rate of reduction during maturation are all potential methods for reducing the total amount of VDKs in the final product.
Figure 2 - Formation Pathways of Common VDKs (Briggs, et al., 2004)
While it is possible to measure VDKs spectrophotometrically, it is not a reliable method as it does not measure the precursor compounds. As yeast can remove diacetyl from the finished product, it is vital to use a gas chromatograph to determine the total VDK as this will be a better indication of the amount of diacetyl in the packaged product after yeast removal from filtering. Heating the sample before testing will be able to identify the total VDKs. (Bamforth, 2014) The heat will accelerate the oxidization of α-acetolactate and α-acetohydroxybutyrate so that the VDKs can be detected as there is not enough time for the yeast to reabsorb the VDKs before testing.
The use of adjuncts in production brewing can change the amount of wort free amino nitrogen (FAN) as adjuncts have lower amounts than barley malt. Brewers should keep FAN levels between 150-350 mg/L, which translates to an adjunct level of under 40%, or when brewing with all barley, malt bills protein levels should be lower than 12.5%. (Carey & Grossman, 2006) Too much Irish Moss (Carrageenan) used in the boil can reduce the amount of free amino nitrogen through coagulation, it is generally not recommended for beers with high adjunct levels. (Palmer, 2017)
High adjunct rates can lead to a product with higher VDK rates. An increase of 20 to 40% adjunct can double the diacetyl, with diacetyl doubling again from 40 to 50%. However, proper maturation techniques can still reduce these to acceptable levels, by ensuring the yeast is healthy, with a high cell count, with a higher temperature, the yeast will complete VDK reduction sooner (Meilgaard, 1999)
Figure 3 - Maillard Reaction Pathways (Briggs, et al., 2004)
One source of the precursor chemicals for VDKs can come from Maillard reactions, a type of non-enzymatic browning that is a reaction between the carbonyl grouping of reactive sugars and amino acids. They occur most readily in environments at high temperatures, low moisture levels, and with increased alkalinity. The malting process is one source of these precursors with crystal and caramel malts having the highest levels: with decoction type mashing and wort boiling being additional sources. (Evans, 2012) During the boil, the fragmentation of dioxyosone leads to α-dicarbonyl, which is a precursor to diacetyl. (Briggs, et al., 2004) Higher levels of VDKs are prevalent in mono-saccharide media than in ones with disaccharide rich worts. (Cha, et al., 2019)
The starting gravity of the wort can affect the overall performance of the fermentation. Higher cell counts and aeration can help before fermentation. Adding a second dose of oxygen between 12-18 hours after the start of fermentation can quicken overall fermentation time and overall attenuation. The yeast can absorb the second dose of oxygen rather quickly for cell membrane regeneration. It can increase primary fermentation speed by up to 33% and aids to decrease diacetyl. (White & Zainasheff, 2010)
Most yeast strains produce VDKs at a similar rate, but the uptake in late fermentation can vary significantly on strain. Highly flocculant and low respiratory yeasts tend to have higher VDK levels post-fermentation. (Carey & Grossman, 2006) Moderate flocculant strains of yeast tend to produce the beers with the least amount of off-flavours as the cells stay in suspension longer as they can perform a higher level of attenuation and reduce VDKs at greater levels. (White & Zainasheff, 2010)
While propagating the yeast, strain health and mutation through re-pitching relies on several factors. Considerations are the potential chromosome mutations that strain has access to, isolate mutation rate and the number of generations used before propagation from the supplier or growth sample. (Large, et al., 2020) Generally, metabolic activity decreases and increases of undesirable flavours such as organic acids, esters, higher alcohols, aldehydes, and diacetyl, after approximately 14 re-pitches. (Kočar, et al., n.d.)
Wort is a media that many organisms can thrive in, so to discourage microbiological contamination and favour a Saccharomyces dominant fermentation, the process is started at a cooler temperature to allow for the yeast to reduce the chance of other organisms from growing. Most yeast growth starts in the first half of fermentation, using cooler fermentation temperatures helps control by-products from yeast growth. Starting too warm can result in a fermentation that accelerates too quickly wholly using up the extract resulting in yeast with low flocculation and diminished yeast health for future batches. (Carey & Grossman, 2006) Fermentations that fail to achieve the proper terminal gravity can leave high amounts of diacetyl in the final product as there was insufficient reabsorption time. A potential cause for this can be a diminished level of oxygen in the wort by less favourable aeration levels at pitching. (White & Zainasheff, 2010)
As the beer finishes the initial fermentation, the yeast starts to reabsorb the VDKs created. As the fermentation slows and the krausen begins to collapse, the yeast will start to flocculate and settle to the bottom of the fermenter. Should this occur without achieving the terminal gravity, the beer should receive a corrective action of rousing the yeast, increasing fermentation temperature, or adding additional yeast. (White & Zainasheff, 2010)
While the beer may have reached the terminal gravity, it is critical to not rush into the secondary aging period too quickly. Cooling the fermenter forces the yeast to enter a dormancy, coagulate, and settle. Applied too early, it prevents the yeast from properly converting any remaining VDKs. Proper cellar management allows for the yeast to metabolize any diacetyl. (White & Zainasheff, 2010)
Figure 4 - Typical Diacetyl Levels During Primary Fermentation (White, n.d.)
The final source of potential VDK production is the introduction of beer spoilage organisms such as Lactobacillus or Pediococcus, both belonging to the group of Lactic Acid Bacteria. Lactic acid bacteria forms lactic acid from sugar, fat, and protein. (Bintus, 2018) Another contaminant in lager is Pediococcus as it contributes diacetyl, turbidity, and acid formation; in conjunction to form extracellular polysaccharides which appear as a “ropey” viscous substance. (Priest, 2012) Both product diacetyl in conjuncture with lactic acid. (Boulton & Quain, 2001)
Ensuring that the grist can provide the proper amount of FAN and specifically Valine is an initial first step to reducing VDKs. However, changing the malt percentage would alter material costs and modify the overall flavour profile of the finished product. Replacing the barley with unmalted adjuncts may have adverse effects on FAN and nitrogen levels of the wort, leading to a higher wort pH. (Schnitzenbaumer, et al., 2012) Beers made with high adjunct levels may have lower FAN and supplementing the wort with 100-300mg/L of L-Valine would aid in fermentation performance with an overall decrease maximum amount of diacetyl and pentanedione. (Krogerus & Gibson, 2013) Other amino acids such as L-Lysine and L-Methionine can aid in the rate of fermentation, but do not have a known pathway for their effect on VDK production. While L-Lysine reduces fermentation time, it increases the rate of production of VDKs. Furthermore, while L-Methionine may reduce VDK formation, it increases the primary fermentation length. (Stewart, et al., 2013)
As adjunct levels of up to 40% typically have acceptable levels of enzymes and amino acids for brewing (Schnitzenbaumer, et al., 2012) the predominant concern is the overall pH of the wort. Decreasing wort pH to ensure that it is near the intended target would allow for the fermenting wort to have an increased reaction rate for the oxidative decarboxylation of α-acetolactate to diacetyl, allowing the yeast to reabsorb VDKs earlier in the primary fermentation. (Krogerus & Gibson, 2013)
Yeast health is critical to proper VDK reduction. Yeast harvesting should occur as soon as it flocculates and be stored at 0-4°C and used within 72 hours. Less healthy yeast increases the chance of higher VDK levels at the end of primary fermentation. Several factors can be low yeast vitality, reduced pitch amounts, and improper aeration of the wort. (Carey & Grossman, 2006) For high gravity wort, ensuring that there is a minimum pitching rate of 1.4 million/mL/°P will aid the overall fermentation, as well as raising the fermentation temperature after 48 hours post-inoculation. (White & Zainasheff, 2010)
Pitching the yeast at a warmer than typical initial fermentation temperature is an unfavourable method of overcoming lower pitch rates. While this method may not create VDKs directly, it does increase the levels of α-acetolactate during the lag phase of fermentation. Using cooler initial fermentation temperatures provides a more controlled cell replication phase, resulting in better yeast health, fewer off flavours crossing cell membranes and an overall cleaner flavour profile. (White & Zainasheff, 2010)
Brewers can estimate the time and temperature required for a VDK rest. While using gas chromatography and forced diacetyl tests are best practice, it is possible to predict the required production schedule for the reduction of VDKs.
Equation 1 - VDK Rest Length Estimation (Carey & Grossman, 2006)
This equation would allow for the cellar department to determine a VDK rest temperature and duration that is favourable for equipment and production scheduling and constraints. (Carey & Grossman, 2006)
Beer with high amounts of diacetyl can be aided in their reduction of by having a longer maturation time or using Krausening. By adding a freshly fermenting beer to the VDK high aging product, it is possible to increase the uptake of VDKs. A standard procedure is to use approximately 10-15% by volume of beer within the first 18-24 hours of fermentation to the ageing product. (Carey & Grossman, 2006)
Additional methods are supplementing the ALDC enzyme that occurs naturally in yeast and using immobilized yeast cells. (Escarpment Labs, 2020) The α -acetolactate decarboxylase catalyzes the non-oxidative decarboxylation of α -acetolactate to acetoin. This reduction of the amount of α -acetolactate prevents the conversion into diacetyl. (Krogerus & Gibson, 2013) Apart from the taboos that exist from the use of production enzymes after the boil (Escarpment Labs, 2020) this method can be very costly on a batch basis and requires heavy initial investment with the application of enzyme encapsulation technology. (Krogerus & Gibson, 2013) Another technological production method is the Sinebrychoff method where the beer passes through immobilized yeast to convert the excess VDKs rapidly. This method can be costly as additional equipment investment is required. (Krogerus & Gibson, 2013) Further challenges are present as this process starves the immobilized yeast, needing constant refreshing. (Escarpment Labs, 2020)
As there are multiple sources for the increases of VDKs in lager production, there are several that can improve product quality with minimal investment. The principal methods of controlling the uptake of FANs during the initial fermentation, reducing the rate of synthesis of valine and isoleucine, and increasing the rate of reduction during maturation all reduce the total amount of VDKs in the final product. The most practical methods for these outcomes are ensuring the wort pH during the boil is consistent, supplying healthy yeast with proper pitching, nutrition, and oxygen levels, and selecting adequate fermentation temperatures will lead to reductions in vicinal diketones.
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