Fermentation & Flavor Compound Science
During fermentation, yeast cells degrade sugars into alcohol and carbon dioxide. However, other minor pathways are also active and yeast produce other compounds while a beer ferments. In the previous article in this series we discussed diacetyl. Now we will turn to other compounds and the often inter-related ways in which they are produced.
Higher alcohols are a very important beer flavor. They are essentially more complex forms of alcohol than the simple, neutral-flavored ethanol that accounts for most of the alcohol in beer. Ethanol is a two-carbon molecule whose formula is C2H5OH. Higher alcohols are simply alcohols with more carbon molecules. For example, propanol (C3H7OH) has three carbon molecules and butanol (C4H9OH) has four. (Higher alcohols also have more hydrogen molecules, as the hydrogens are attached to the “carbon skeleton” of the molecule.)
Higher alcohols are volatile and intensely flavored. At elevated levels, they may impart intensely fruity or solvent-like characters to beer. The balance between the levels of higher alcohols and esters is an important factor in determining the beer’s drinkability. Higher alcohols are implicated as the cause of hangover headaches.
Organic acids are part of the background flavor of beer. Yeast require amino acids to build new proteins and enzymes within the cell. If we’ve done our job in wort production, and the maltsters did theirs in the malthouse, then there should be plenty of them available in the wort. However, some are more easily absorbed by yeast than others.
Yeast absorb most of the amino acids they need directly from the wort. They are also able to produce many of the amino acids needed for growth from other, more easily-absorbed amino acids, using transamination reactions. Yeast will remove the amino group from an amino acid and attach it to an organic acid already inside the cell, creating a new amino acid. This leaves the original amino acid without an amino group and now is an oxo-acid or a keto-acid. This molecule can be converted into an aldehyde by the loss of a CO2 molecule, and then reduced to a higher alcohol. A particular amino acid will eventually produce a particular higher alcohol following transamination and reduction. The formation of higher alcohols reduces the potential toxicity of the oxo-acids and at the same time regenerates important cofactors needed for other reactions.
The presence of surplus of a particular amino acid in wort may inhibit the formation of the corresponding higher alcohol; likewise, a shortage of a particular amino acid in the wort will lead to over-production of its corresponding higher alcohol. Too many amino acids in the wort can lead to increases in higher alcohol production, as the yeast grows healthily. The faster yeast grow, the more rapid is the production of amino acids and the more oxo-acids — and hence higher alcohols — are produced.
Since some of the total oxo-acid contents within the cell are also derived from carbohydrate metabolism, some higher alcohol production is coming directly from carbohydrate metabolism. Around 80% of the higher alcohols are formed during primary fermentation, and — unlike vicinal diketones or aldehydes — they will not be removed during maturation.
Factors Increasing Higher Alcohols in Beer:
The production of higher alcohols is influenced by many variables that the brewer can control. Most of these variables are linked to yeast growth.
Higher fermentation temperatures encourage yeast growth, so if the temperature is increased then amino acid synthesis increases. More amino acids mean a larger pool of higher alcohol precursor molecules in the cell.
A vigorous fermentation with lots of bubble formation encourages movement and mixing in the fermenting wort. Good motion in the fermenter encourages yeast growth.
Intensive, early wort aeration encourages yeast growth. This is especially important when a large-scale brewer fills his fermenter with repeated wort additions. Several wort additions has the effect of extending the yeast’s active growth phase since each addition of aerated wort adds new oxygen to the fermenter.
When amino acids are in short supply in the wort, then yeast will use the transamination reactions to produce its own amino acids. Worts with reduced levels of amino acids have a variety of causes, including the two below.
Higher wort gravities, above 13° P, encourage good growth even though the amount of amino acids in that wort may not be proportionally higher. So, more amino acid production may be needed in higher-gravity worts.
A very high proportion of non-malt adjuncts dilutes the amino acid content of the wort. Brewers using malted barley with high levels of protein (6-row) may use up to 50% adjunct in order to dilute the amino acid content of the wort. Belgian beers brewed with a lot of sugar in the kettle have a lot of higher alcohols, although this may be more directly related to the higher fermentation temperatures that are employed by brewers in Belgium.
Yeast will tend to grow until there are a certain density in the wort. If you pitch a lower number of yeast, then they will grow more to reach that level, increasing the higher alcohol level.
Factors Decreasing Higher Alcohols in Beer:
Having top pressure on the early phase of fermentation decreases higher alcohol production. Carbon dioxide (CO2) pressure early in the fermentation will inhibit the reactions that result in CO2 production. The step that results in aldehyde production produces CO2.
So, in summary, higher alcohol levels are important to beer flavor. Large breweries interested in limiting higher alcohols in their beers are skilled at manipulating wort qualities to control their production. Too few amino acids in wort will force yeast to manufacture rather than directly utilize amino acids, which in turn will result in an increase in higher alcohol production. Too many amino acids in the wort will also produce surplus higher alcohols. In general, all-malt beers made with relatively low-nitrogen barley will have sufficient amino acids to produce a well-balanced beer. High nitrogen barley malts require dilution with adjuncts to prevent excessive solvent-like character in the beer.
Aldehydes:
Aldehydes are similar to ketones, but they contain a double-bonded oxygen on the outermost carbon in their structure. Complex aldehydes are found in beer from the pathways that lead to higher alcohols. However, the most common aldehyde in beer is acetaldehyde, an intermediate in the fermentation pathway.
Green beer flavors are simply the flavors associated with young beer. Acetaldehyde and diacetyl are considered green beer flavors because, as a beer ages in the presence of yeast, they disappear. Acetaldehyde is excreted by the cell in the first three days of fermentation and is considered a green beer flavor. Like diacetyl (and other vicinal diketones), acetaldehyde can be removed by healthy yeast during the maturation phase.
Acetaldehyde has a flavor threshold of around 15 ppm in beer. Acetaldehyde formation is generally favored by conditions of high metabolism coupled with low growth. It is “leaked” from the cell because yeast can only convert it to alcohol when sufficient amounts of a particular cofactor are present. This compound is sometimes limiting and acetaldehyde is excreted to keep the glycolytic pathway moving forward. Zinc appears to be a cofactor in the conversion of acetaldehyde to ethanol, so trace amounts of this metal are required for conversion. Carbon dioxide pressure can lead to toxic levels of the gas in yeast cells, which changes their permeability and affects their ability to convert acetaldehyde to ethanol. Early wort aeration will result in healthy, active yeast that are capable of converting acetaldehyde to ethanol.
Lipids And Esters:
Esters are the class of compounds responsible for fruity flavors and aromas in beer. Some beers taste like bananas, apples, strawberries or pineapple and esters are largely responsible for that. They are a major component of the flavor profile of ales rather than lagers, and I once read a technical journal that advocated using ester level as a way to differentiate between ales and lagers now that modern fermentation practices make the traditional definition somewhat obsolete. The traditional definition revolves around yeast strain and yeast reclamation, which in this day and age are similar. So, in ales esters are a desired characteristic, while lager brewers see them as a fault.
Lipids (particularly unsaturated fatty acids and sterols) are formed early in fermentation to provide material for membrane production. As the need for lipid production ends, the intermediates still being produced may be shunted off to form esters.
Esters are a combination of alcohols and fatty acids, the most common of which is ethyl acetate. Under fermentation conditions, the simple combination of alcohols and fatty acids will occur very slowly, if at all.
The reaction is catalyzed by CoEnzymeA, which will attach to the fatty acid and “activate” it. CoEnzymeA (sometimes shown as CoASH to denote an open sulfur group) carries small fatty acids around the cell, where they participate in important reactions. When attached to a fatty acid the complex is known as Acyl CoA.
During fermentation acetic acid forms the specific Acyl CoA known as Acetyl CoA, which is involved in many reactions. Since Acetyl CoA is the most common acid and ethanol is the most common alcohol, the most common ester produced during fermentation is ethyl acetate. This ester has a characteristic nail polish aroma. Beer contains around 60 different esters, but only a few are important to beer flavor and aroma. To understand the factors that affect ester formation, we must look at the compounds that are the key precursors in ester formation.
Acetyl CoA is required by the cell for the production of fatty acids, phospholipids and sterols, key components in cell membranes. As we have already seen, high yeast metabolism will lead to more higher alcohols, as by-products from the transamination of amino acids. High yeast metabolism, coupled with relatively slow growth (i.e. high- gravity worts) will also lead to an excess of Acetyl CoA. Since the yeast cell does not now have high requirements for fatty acids and phospholipids, this leads to a bottleneck in the pathway that results in AcetylCoA or Acyl CoAs combining with higher alcohols to form esters. It is important to note that proper aeration of the wort, especially several hours after the start of fermentation, will result in lower ester production as more of the AcetylCoA or Acyl CoA is diverted for the production of unsaturated fatty acids and sterols for new cell material.
So as long as lipids are being formed for membrane production, ester production is inhibited. So, esters generally develop later in the primary fermentation. It is advantageous for yeast to form esters since it provides a method of removing some toxic long-chain fatty acids from the cell. It also allows the cell to regenerate CoEnzyme A to catalyse other reactions. High-gravity brewing — brewing with worts of high gravities (often over 18° P) that then are diluted post-fermentation — causes an increase in ester production, which causes flavor problems with subsequent dilution. This is probably due to the decreased solubility of oxygen in stronger worts and hence proportionally less yeast growth. This can be helped by introducing more oxygen to the fermenting wort to encourage more yeast growth.
Factors Influencing Ester Formation:
The researchers and the literature are divided on the issue of ester formation and its relationship to temperature. Logically it would seem that increasing the fermentation temperature would increase the rate of yeast growth, and therefore decrease the availability of acetyl CoA for ester formation, but experiments have frequently shown that esters actually are increased at higher fermentation temperatures. One experiment showed that changing the temperature from 59°F (15° C) up to 77° F (25° C) increased the measured esters by 75%. There are several theories that attempt to explain this paradox.
Ester production is increased by increasing the gravity above 18° P, increasing the attenuation limit, restricting wort aeration, restricting yeast growth, increasing fermentation temperature and decreased motion during fermentation. Conversely, ester production is decreased by lower gravity, increased wort aeration, decreased attenuation limit, decreasing fermentation temperature, increased pressure during primary fermentation and underpitching.
Sulfur compounds:
Sulfur compounds are responsible for some of the most dramatic beer flavors. Some of the most aromatic aroma compounds have minute flavor thresholds and so, even though they are present in small quantities, can dramatically effect a beer’s flavor.
Sulfur compounds have their origins in the ingredients we use. Sulfate, sulfite and sulfide ions in the water, and sulfur compounds present in malt and hops, can all lead to sulfur flavors in the beer. Some, such as the frequently encountered DMS, have their origins in the malthouse or the brewery. Others, such as the skunky 3-methyl-2-butene-thiol, are formed from the reaction between light and hop compounds in the package.
Of the compounds actually formed during fermentation there are two main compounds of interest. Hydrogen sulfide (H2S) is the aroma of rotten eggs. Its flavor threshold is only around 4–10 parts per billion but it may be found in beer at levels of 200 parts per billion. At low concentrations, it rounds out the flavor of pale lager beers but at higher concentrations is definitely an off flavor. Some yeast strains are prone to producing more H2S than others, and lager brewers in Europe may base their choice of yeast strain on the amount of sulfur it produces. Growing yeast cells require sulfur for amino acid production, protein structure, and CoEnzymeA formation. Sulfate ions in wort are actively taken up by yeast and biochemically reduced to hydrogen sulfide. Once the need for those certain sulfur containing amino acids is met, the excess H2S is excreted from the cell. If the wort is lacking any nutrient, that may affect yeast growth then sulfur may be produced in excess.
Luckily, H2S is very volatile and is removed with evolving carbon dioxide during fermentation. Capping a fermentation vessel too early can trap H2S in beer, and higher fermentation temperatures can increase the levels. Some commercial brewers experience problems when they increase batch size as they expand production. Taller tanks can cause a pressure differential at the tank base and increase sulfur production. Wort spoiling micro-organisms can also produce copious amounts of H2S.
Sulfur dioxide (SO2) is the drying, struck match flavor and aroma sometimes found in beer. It is rare that it should be detectable in American beers although it is more common in English beers. In the UK, commercial brewers add it to beers as a preservative. It is able to mop up excess oxygen in solution and that is the primary mechanism, but it can also react with, and bind to, compounds that may eventually create stale flavors.
Sulfur dioxide can combine with aldehydes in wort to form compounds that survive processing. This prevents the aldehyde oxidizing further to stale flavors. However, if this comes into contact with air during storage it can oxidize back to sulfate and the aldehyde, which in turn reacts with a higher alcohol to produce trans-2-nonenal, the compound responsible for the cardboard taste in stale beer.
Sulfur dioxide can also be liberated from yeast cells when the growth cycle goes awry. It can be added to beer inadvertently along with isinglass finings when these finings are used as a preservative.
Dimethyl sulfide (DMS) is a troublesome flavor in beer. It can vary in intensity from cooked corn to cooked vegetables, cabbage, onion, even garlic. In Pilsner lagers, it adds a fullness and roundness to the flavor, and some pale American pilsners benefit from a fresh corn-like aroma. It has its origins in malt and is removed from beer by aggressive and volatile wort boiling.
Wort spoiling bacteria can produce DMS should there be a delay in starting primary fermentation. Once it is in the wort, though, it cannot be removed by yeast. Some may be scrubbed out of the beer with escaping CO2 during fermentation. DMS may be oxidized to the non-volatile DMSO during kilning of the malt and this can be reduced by yeast back to DMS in the fermenter.
Mercaptans are an extremely volatile class of compound with flavor thresholds measured in parts per trillion. They are often only detectable by the human senses, being below the sensitivity of some laboratory instruments. They are thio-alcohols, which is a compound similar to an alcohol but with an active sulfur group. They may resemble cabbage, stewed vegetables, drains and rotting vegetables, and are produced by yeast during the production of amino acids and also released into beer when yeast cells autolyze.
Sulfur compounds are an area of brewing chemistry in which a great deal of work still needs to be done. As they are so difficult to detect and measure it may be some time before we are more aware of the mechanisms of their formation. Luckily, experience has shown us how to deal with them. Their volatility enables us to remove them using purging with CO2 gas.
