Yeast: Flavours & Aromas
The role of yeast in the formation of aroma and flavour compounds in brewery products.
Introduction: Effects of Fermentation Parameters
The manipulation of amino acids from malt by yeast provides the pathways for the conversion of wort sugars that cause some of the most significant alterations in beer's overall flavour and aroma. By controlling several vital areas, the final olfactory profile can be modified. Including areas such as the fermentation temperature, hydrostatic pressure, initial gravity, free amino nitrogen levels, and wort aeration can either promote healthy fermentations and balance the levels of vicinal diketones, aldehydes, fusel alcohols, and esters, or increase these flavours and aromas to undesirable levels and promote high levels of sulfur.
Due to slow fermentation kinetics, a significantly low pitching wort temperature might impede Fermentation and produce unwanted components like acetaldehyde and vicinal diketones. Increased activity, worsening of foam stability and beer colour, drop in pH, and loss of bitter chemicals can all be caused by a rise in temperature. Higher fermentation temperatures may also increase the levels of fusel alcohols and ester profiles (Kucharczyk & Tuszyński, 2018).
Tall fermenters produce much hydrostatic pressure, which raises the amount of carbon dioxide dissolved in the beer. Excess carbon dioxide limits yeast growth by disrupting decarboxylation processes, crucial in producing higher alcohols or acetyl-CoA. In addition, ts pressure unbalances beer flavour by reducing the substrate available for ester formation, as acetyl-CoA is the primary precursor of acetate esters (Pires, et al., 2014).
High-gravity brewing uses worts with higher sugar concentrations than average, generally 16–18°P. This allows the brewery to increase volumetric production while reducing labour and energy expenses without making additional investments. However, raising the starting wort gravity can change the final flavour and aroma component composition, making it difficult to compare to beers made with lower gravities. In addition, the initial gravity can contribute to higher levels of Vicinal Diketones, aldehydes, fusel alcohols, and ester profiles (He, et al., 2014).
Free Amino Nitrogen (FAN)
The amount of fermentable amino acids in the wort significantly impacts the beer's overall flavour and aroma. The necessary mechanisms for the conversion of wort sugars to alcohol are provided by FAN, which is a complex combination of amino acids. FAN affects aldehydes, esters, Diacetyl, sulphur compounds, and higher alcohols. FAN levels that are too high or too low have been associated with off-flavours such as isoamyl alcohol, propanol, and isobutanol. (Hill & Stewart, 2019).
Oxygen & Unsaturated Fatty Acids
Unsaturated fatty acids and sterols, which are both required for anaerobic development and cell division, are produced by yeasts aerobically. Therefore, lack of oxygen in the wort can delay or impede primary Fermentation and produce higher undesirable components in the new beer aroma, such as acetaldehyde and vicinal diketones (Kucharczyk & Tuszyński, 2017).
Sulfur compounds essential to beer flavour develop in part from synthesizing amino acids and reducing inorganic sulfur. In general, the concentration of these yeast produced compounds increases with increasingly unfavourable fermentation conditions. Among these compounds are sulfur dioxide and offensive hydrogen sulfide resembling the aroma of rotten eggs (Holle, 2019).
Figure 1 - Yeast Derived Aroma & Flavours Pathways (Briggs, 2004)
Two compounds in the family of vicinal diketones (VDKs) are the most important for beer. Olfactory tests describe Diacetyl as popcorn margarine or butterscotch candy, and pentanedione is generally sweet and has notes of honey, butter, caramel. VDKs are typically viewed as off-flavours; low levels are allowed in some British-style ales and Czech-style lagers. The mild profiles of lagers and lighter-flavoured beers ease the identification of VDKs (Bamforth, 2014).
Valine and Isoleucine are synthesized and form Diacetyl and Pentanedione as by-products. As the Fermentation progresses, the yeast converts Diacetyl into acetoin and 2,3-butanediol; and pentanedione into 2,.3-pentanediol, which have lower olfactory thresholds (Briggs et al., 2004).
VDK Precursor Compounds
VDK precursor compounds are made from malting, wort boiling and decoction type mashing; the highest levels are crystal and caramel malts. Another source of VDK precursors is from the Maillard reactions that occur during malting and wort boiling (Cha, et al., 2019). Stabilizing the consumption of free amino nitrogen during primary Fermentation and increasing the reduction rate during secondary Fermentation are methods to reduce the final VDKs level in the beer. In addition, Lactobacillus and Pediococcus contamination should be evaluated as they can create Diacetyl (Boulton & Quain, 2004). Finally, VDK reabsorption time contributes to one of the reasons why brewery fermentations are batch-type reactions instead of continuous cycles (Dennis, 2010).
Managing VDK Levels
The initial wort gravity can affect the rate of Fermentation. Proper dissolved oxygen and cell counts and can help when added before Fermentation. Supplementing oxygen at 12-18 hours after the start of Fermentation can reduce fermentation length. Moderate flocculant yeast strains tend to create beers with lower by-products as the cells are suspended longer (White & Zainasheff, 2010).
As the consumable sugars decrease, VDKs are reabsorbed by the yeast. As the secondary Fermentation starts at colder temperatures, yeast will enter dormancy, coagulate, and drop to the bottom of the fermenter. Cooling temperatures applied too early reduces the ability of the yeast to convert VDKs. Even if terminal gravity has been reached, it is critical to allow the yeast to uptake the off-flavours in due time (White & Zainasheff, 2010). The maximum total amount of Diacetyl in a finished product should never be more than 0.1mg/L (Kunze, 2014).
Figure 2 - Vicinal Diketone Formation Pathways (Briggs, 2004)
Aldehydes & Organic Acids
The most crucial aldehyde in brewing is acetaldehyde, which occurs as an intermediate product in brewery fermentations. It is produced during the first 72 hours of Fermentation and is responsible for the cellar and musty flavours familiar with young beer (Liu, et al., 2018). During this point of the Fermentation, the range for acetaldehyde is approximately 20-40mg/L, and it will decrease to 8-10mg/L throughout ageing (Kunze, 2014).
Levels of acetaldehyde are typically higher in situations with quick fermentations, higher temperatures during the initial Fermentation, improper yeast pitching rates, higher osmotic pressure, low dissolved oxygen levels, and wort contamination. These can be resolved by ensuring a proper maturation period at a warmer temperature, with plenty of dissolved wort oxygen and healthy yeast over the cold stabilization period (Kunze, 2014).
Other Common Aldehydes
The flavour thresholds of the straight-chain saturated aldehydes regularly decrease towards a minimum at eleven-carbon aldehydes. When these compounds increase to above seven carbons, there is a sharp drop in threshold; 2-heptenal, 2-octenal and 2-nonenal have low thresholds and unpleasant, cardboard-like flavours. A branched-chain appears to cause an increase of the threshold above 0.1 ppm for long-chain aldehyde. The presence of hydroxy- and carboxy-groups cause significant increases in the thresholds of all compounds, as already mentioned (Meilgaard, 1975).
Staling AldehydesIn beer, stale flavour formation is accompanied by increased volatile carbonyls, or aldehydes, which can be removed with hydroxylamine. (E)-2-nonenal has been cited as an essential staling compound, but it is just a part of the overall picture. The overall stale flavour is caused by many compounds, including 2,4-dinitrophenylhydrazine and 3,5-methylcyclohexane (Baert, et al., 2012).
However, most staling aldehydes precursor compounds are formed during malting and boiling; they are eventually converted into aldehydes with lower olfactory thresholds through the oxidization of unsaturated fatty acids and Strecker degradation, and not through cellular respiration (Baert, et al., 2012).
Figure 3 - Acetaldehyde In Fermentation, Synthesis of Fusel Alcohols and Esters (Holt, et al., 2019)
The pH of the beer is determined by the rate at which amino acids are broken down into organic acids that are volatile and non-volatile, such as pyruvic, malic, citric, and lactic acids. Fermentations above atmospheric pressure can expect increased levels of these compounds. These compounds cause a yeasty odour and impair head retention. However, the amounts can be controlled by the general fermentation conditions through wort composition, aeration, and selecting a proper yeast strain (Eßlinger, 2009).
During Fermentation, yeast consumes nitrogen through several sources, the essential ammonium-based compounds being a primary contributor. As the conversion of ethanol progresses, these are metabolized into organic acids resulting in a decrease in acidity. As a result, farmhouse strains can reduce the pH more than English Ale or Lager yeasts strains, which can alter the flavour of the product as a lower beer pH is associated with a thinner body and higher hop astringency (Escarpment Labs, 2019).
The non-aromatic aliphatic acids can impart unique flavours reminiscent of rancidity and mustiness. Unlike other compounds, Organic acids show the lowest thresholds when having four- and five-carbon structures. The oxygenated acids such as lactic, pyruvic, fumaric, and tartaric acids show olfactory thresholds near 400 ppm; at higher levels, the pH change is significant enough to be tasted (Meilgaard, 1975).
The main factors to the final beer acidity will be the starting wort pH and yeast and bacteria selection. Some yeast varieties may increase organic acids, so the wort pH should be monitored and modified to reach specific end goals (Escarpment Labs, 2019).
As many as forty-five higher alcohols are found in beer, containing two carbon atoms or more, resulting in a higher boiling point than ethanol. They contribute a significant portion of the volatile and non-volatile compounds in brewery products. They should not be detected in lagers but may increase complexity in ales. In addition, these types of aromatics contribute to the heat found in the aroma and flavour, which creates a sensation on the pallet (Spedding, 2012).
The most commonly identified fusel flavour includes variants of propanol and butanol, which convey alcoholic, sometimes solvent, and vinous notes—additionally, amyl alcohol presents the characteristic "fusely" or boozy pungent note. As the strength of the beer increases with styles such as Double IPAs, Imperial Stouts, and Barleywines, the levels of Fusel alcohol will be higher. However, fusel alcohols are considered a flaw in the majority of styles (Spedding, 2012).
Lager yeast generally produces 60-90 mg of fusel alcohol and ale, often more than 100 mg/L, but ales are known for their spicy character relative to lagers (Holle, 2019).
Fusel alcohols originate from amino acid catabolism from a mechanism called the Ehrlich Pathway. Fusel oil was discovered during spirits' distillation, and its name comes from the German for "bad liquor". These compounds and their respective esters make an essential contribution to the flavours and aromas of fermented foods and beverages (Hazelwood, et al., 2008).
Figure 4 - Main Enzyme Catalyst & Respective Genes in The Ehrlich Pathway (Pires, et al., 2014)
Initial Transamination Reaction
The preliminary step in the Ehrlich pathway involves four enzymes that transfer amines between α-keto acids and their corresponding amino acids, using glutamate and α-ketoglutarate as a donor and acceptor. While Bat1- and Bat2-encrypted enzymes are involved in the transamination of amino acids, Aro8p and Aro9p were first described as amino acid transferases and not genes. However, all phenylalanine, methionine and leucine amino acids are activated by the transcription of Aro9 and Bat2 genes. (Pires, et al., 2014).
Five genes belong to yeast that is similar to thiamine diphosphate decarboxylase genes. The first three genes (PDC1, PDC5, and PDC6) provide pyruvate decarboxylases, while the remaining two (ARO10 and THI3) are alternative Ehrlich pathways genes (Hazelwood, et al., 2008).
Reduction of Higher Alcohols
The final stage of the Ehrlich Pathway is a reduction into fusel oils by alcohol dehydrogenases. There are five alcohol dehydrogenases encoded by Adh1, Adh2, Adh3, Adh4 and Adh5 genes, and a formaldehyde dehydrogenase belonging to Sfa1, which can catalyze the conversion of fusel aldehydes into higher alcohols. Acetaldehyde is reduced into ethanol during Fermentation by the constitutive and cytoplasmic form of an Adh1-encoded enzyme. Contrary to cytosolic counterparts, the Adh3p enzyme reduces acetaldehyde inside the mitochondria and balances reduction-oxidizing reactions during anaerobic growth (Pires, et al., 2014).
Fusel Alcohol Regulation
Fusel alcohol production can be controlled and kept at the desired level under certain conditions. Industrial wort treated with ultrasonic waves before Fermentation produces fewer fusel alcohols but more esters. Judicious selection of yeast strain, low fermentation temperature, proper pitching rate, adequate wort aeration, balanced wort composition, and low initial wort pH can all contribute to the production of a beer with the desired amount of fusel alcohols (Van Gheluwe, et al., 1975).
Esters & Phenols
Esters contribute a large portion of the flavour compounds in alcoholic beverages, providing the majority of "fruity" aromas and flavours. In brewery products, esters are formed through the reaction of organic acids and alcohols created over Fermentation, with ales having higher levels of ester formation than lagers. The most common compounds occurring are isoamyl acetate, ethyl acetate, ethyl caprylate, ethyl caproate and phenylmethyl acetate.
In comparison, phenolic flavours are produced by the decarboxylation of fatty acids. Brewers manage their brewing processes and select specific yeasts to produce these phenols, but phenols are primarily unwelcome in most styles. In some German and Belgian ales, 4-vinyl guaiacol is desired and formed by the decarboxylation of ferulic acid. 4-vinyl guaiacol gives beers aromas and flavours described as being clove-like, spicy, or herbal. However, as these beers age, these phenols start breaking down, giving a vanilla-like character and losing the signature characteristic. Other yeasts, such as Brettanomyces, produce 4-ethyl guaiacol, which gives beer smoked meat or a clove, spicy character (West, et al., 1965).
Figure 5 - Main Genes Involved in Ester Formation (Pires, et al., 2014)
The alcohol acetyltransferases I and II synthesize acetate esters from alcohol and acetyl‐CoA as reactants. ATF1 and ATF2 enzymes form a wide range of less volatile esters, such as ethyl acetate, propyl acetate, pentyl and isobutyl. Different yeast strains produce different levels of these enzymes, leading to different production rates for the individual esters. This indicates that differences in the aroma profiles produced by yeast strains may be partially due to specific mutations in their ATF genes (Saerens, et al., 2010).
Medium-chained fatty acids are the main precursors for ethyl ester biosynthesis. During alcoholic fermentation, short-chain and medium-chain fatty acid intermediates are prematurely released from the cytoplasmic fatty acid synthase complex. The key enzyme in the regulation of fatty acid biosynthesis is acetyl-CoA carboxylase. In yeast, overexpression of the FAS1 and FAS2 fatty acid synthetic genes triggered more medium-chain fatty acid formation (Saerens, et al., 2008).
Esters with Aging Beer
The ester profile of a given beer may change drastically during storage. Hop-derived components are oxidized to form ethyl esters that impart the aged beer a winy aroma. In addition, some esters such as isoamyl\racetate are known to be hydrolyzed during beer storage. For all reasons mentioned above, ageing beers tend to lose fresh fruity aromas, giving place to less fruity and more earthy (Pires, et al., 2014).
Figure 6 - Aroma & Flavour Formation Over Fermentation and Inital Aging Cycles (MBAA, 2006)
The elements that have the most impact on the overall flavour and aroma of the beer have been discussed. These can be influenced by factors such as beer fermentation temperature, hydrostatic pressure, starting gravity, free amino nitrogen levels, and wort aeration. Vicinal Diketones, aldehydes, fusel alcohols, and ester profiles can all be increased from a higher starting gravity. The amount of fermentable amino acids in the wort has a significant impact on the flavour and aroma of the beer. In addition to the fermentation stresses on the yeast from difficult growing conditions, removing these barriers for the optimal conversion of wort sugars would diminish the chance of off-flavours out of favourable levels will increase the quality and consistency of brewery products.
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