Brewer’s Yeast & Brett Fermentation Flavors
Yeast cultures have flavor. Ever tried one? An unfiltered beer with yeast in it therefore will have yeast flavor. But what about filtered beers? Yes, those beers have yeast-derived flavors too. Yeasts synthesize flavors during fermentation, and many of these flavors stay in the beer, even when the yeast is removed.
Yeasts do this in all fermentations, whether it is bread, wine, beer, or even other fermented foods such as chocolate. But beer is special! The background flavors of most beers are low, the alcohol is low, and the contact time is long so that beer expresses yeast flavor more than any other fermented food or beverage. Yeast make more than 500 flavor and aroma compounds during beer fermentation.
They don’t really do this on purpose; the yeast cells are simply reproducing in the medium they are growing. And in the case of beer the medium is brewer’s wort. Yeast grow very similarly to human cells; they absorb simple sugars, break them down into smaller carbon-based pieces and release energy the cell needs to make new cells. This is called carbohydrate metabolism. Yeast has two other metabolic pathways that lead to the formation of flavor compounds; amino acid metabolism and fatty acid metabolism.
Yeasts synthesize fatty acids and sterols, compounds that surround cells and each organelle within cells. Fatty acids are arranged in a bi-layer to create a lipid membrane. These membranes allow cells to remain intact in high water environments. Unsaturated fatty acids (fatty acids containing one or more double bonds) and sterols are made by using Acetyl CoA and oxygen. In order to synthesize new cells, yeasts first need to synthesize fatty acids and sterols.
Sterols give fluidity to the lipid membranes, which is very important to brewer’s yeast because they are in a high alcohol environment, and the membranes need extra fluidity to keep the cells from breaking open. Oxygen is required for sterol production and becomes part of new sterol molecules. During a typical fermentation, the yeast population increases five-fold and the oxygen added to wort during aeration is critical for sterol production. Many homebrewers have heard of supplying fatty acids in the form of olive oil, instead of oxygen, and some reading this are experimenting with this method. Although this has worked for many brewers, there has been little research outside of the New Belgium Brewery work published in 2005, and most brewers using this method are not using the procedure described by New Belgium, such as dissolving the olive oil in ethanol and incubating with yeast for 24 hours prior to pitching. More interestingly, olive oil does NOT contain sterols. More research needs to be done on the long-term consequences of fermentations fed with olive oil only and no oxygen.
Fatty acid metabolism adds soapy flavors to beer because oxygen drives unsaturated fatty acid and sterol production. This keeps ester synthesis in check. Fatty acid metabolism also produces acids produced during fatty acid metabolism that become esters in the final beer.
In nature, yeasts also use oxygen for aerobic carbohydrate metabolism, where carbohydrates are completely broken down to carbon dioxide and water, thereby gaining the maximum energy possible. This is commonly called respiration. In beer fermentation, yeasts are starved from oxygen in two ways; from the lack of its presence, and from the high sugar concentration preventing respiration (called the Crabtree Effect), even when oxygen is present. When the cells cannot respire they limp along creating ethanol by fermentation, and only generate about 10% of the total possible. The remaining 90+% ends locked up in other carbon compounds, some that will be flavor and aroma active.
When brewers keep oxygen out of wort (by not actively adding it) they get two important parts to beer — ethanol and flavor.
But wait — brewer’s yeast is not strictly anaerobic! That means brewer’s yeast cannot survive without some oxygen. Brewers have learned that they need to supply oxygen when yeast is pitched into wort. And this oxygen is used in fatty acid metabolism as described previously.
In carbohydrate metabolism, yeast break down sugar first to pyruvate, a process called glycolysis, then pyruvate can go to two different pathways (of course there are more options – it can also go into fatty acid metabolism and amino acid metabolism). When oxygen is present pyruvate flows through the tricarboxylic acid (TCA) cycle. Without oxygen, pyruvate is broken down first into acetaldeyde, and then acetaldehyde is reduced to ethanol (see this figure). NAD+ is also regenerated in creating ethanol, which the yeast retain in order to keep the process of glyolcysis going, and they dispose of the ethanol by excreting it.
Since acetaldehyde is made in this way, it is not surprising that this is a major flavor compound in beer. Acetaldehyde has a flavor threshold of 10 ppm and tastes of green apples over that level. If you taste it in significant quantities, it becomes dominant and a fault in beer. A lot of homebrewed beer has significant levels of acetaldehyde, and it is rarely talked about, so I will expand on this topic here. The enzyme that makes ethanol, alcohol dehydrogenase, will also convert ethanol back into acetaldehyde under the right conditions (this is the same enzyme in the human liver that breaks down ethanol humans consume). Oxygen promotes acetaldehyde formation. If you continually add oxygen during beer fermentation, rather than just in the beginning, you will promote a lot of acetaldehyde production. High temperatures during fermentation also promote acetaldehyde production, so it is important to not let ales get into the 70s °F (21 °C), and to keep lagers in the 50–55 °F (10–13 °C) range if acetaldehyde production is to be limited. The alcohol dehydrogenase enzyme is a zinc dependent enzyme, meaning it needs zinc in its active site to convert acetaldehyde to ethanol. If your wort has a low availability of zinc, the beer can have high acetaldehyde levels. Many breweries make it a practice of adding food grade zinc sulfate, zinc chloride, or Servomyces (dead yeast loaded with zinc) to their wort at knock out in order to prevent acetaldehyde off-flavors.
Another kind of yeast metabolism that builds flavor compounds is amino acid metabolism. In fact, this might be more important to flavor than carbohydrate metabolism. Yeasts have to synthesize proteins from amino acids; some of these proteins will be enzymes and others will be cellular constituents. Yeasts are reproducing during the early stages of fermentation, so they need to manufacture a new complement of proteins for each new cell. There are 20 different amino acids, and proteins are comprised from a variety of combinations of these building blocks. Yeasts can synthesize some amino acids, and they also absorb and metabolize the amino acids supplied from brewer’s wort. The fact is they get the amino acids they need by a combination of manners. Many flavor compounds such as fusel alcohols, esters, sulfur compounds, and diacetyl come from amino acid metabolism, and the amount supplied from the wort is a big factor in flavor. The acronym brewers use for the available amino acids is FAN or free amino nitrogen. FAN can be measured, and the minimum target for beer is 160 ppm FAN. Most all malt wort contains the minimum, but the number does not tell us which amino acids are supplied. This information is more difficult to determine and a sophisticated lab is needed, so it is not something most brewers will ever know. This is one reason that FAN and its composition is difficult for brewers to control. Brewers who use starchy adjuncts like rice and corn or sugar adjuncts are intentionally diluting FAN and by doing so are reducing flavor compounds associated with amino acid metabolism.
Diacetyl formation in beer is a classic example of a flavor compound derived from amino acid metabolism. Valine is an amino acid that yeast need to synthesize. In order to make valine, yeast convert pyruvate to acetolactate, then acetolactate is converted to valine. As happens throughout metabolism, this is not 100% efficient. Some acetolactate is not converted to valine, but instead goes outside of the cell. Acetolactate is flavorless in the quantities found in beer. However, acetolactate can be converted to diacetyl in a non-enzymatic reaction. If the yeast is still present, diacetyl can go back inside the cell and be converted to acetoin and 2,3 butanediol, both flavorless compounds. This reaction also regenerates NAD+ which, like NAD+ regenerated in ethanol formation, the cell can use to create energy for growth. The problem is if the yeast is no longer present when diacetyl is made, the beer will have a permanent butterscotch flavor if over 100 parts per billion. The brewer can easily be fooled when tasting young beer, because if acetolacate is present, they cannot taste it.
As seen in this figure, the diacetyl peak is highest days after fermentation is complete, so if the brewer is not patient and moves the beer off the yeast too quickly, the beer can have high potential for diacetyl. The diacetyl peak will only go down as shown here if yeast is still present.
Sulfur compounds are also made from amino acid metabolism. Two of the amino acids yeast make, cysteine and methionine, contain sulfur. During metabolism, the sulfur intended for these two amino acids can get incorporated into other compounds. Depending on the strain, they make more or less sulfur containing compounds, examples being H2S and low level mercaptans. Lager yeast (Saccharomyces pastorianus) is a different species of yeast from ale yeast (Saccharomyces cerevisiae) and usually produces more sulfur compounds than ale yeast. Anyone who has made lager beers remembers the first time they did and the sulfur (H2S) emanating from the fermenter. This is normal. The only thing the brewer needs to do is to raise the temperature at the end of the fermentation (called the diacetyl rest to remove diacetyl potential), and sulfur (if H2S) will volatize into the air.
The main flavor compounds brewers speak of in beer are fusel alcohols and esters. Fusel alcohols, all alcohols other than ethanol, are also produced from amino acid metabolism. An example is isoamyl alcohol, which by itself, like ethanol, is relatively tasteless. But it can be converted to a more flavor active ester, or it can stay as isoamyl alcohol and in combination with other fusel alcohols in the beer, the beer can have a hot, solvent-like taste and aroma. Isoamyl alcohol can come from the amino acid leucine. When wort is low in amino acids, this triggers more amino acid production by yeast, making more fusel alcohols. Fusel alcohols also increase with higher fermentation temperature and higher oxygen levels, because these and other factors increase yeast growth, which in turn increases amino acid syntheses.
Esters are wanted by many brewers and beer drinkers, and even the lighter-tasting American lagers have some esters. Esters give the fruitiness to beers. Fusel alcohols are required to make esters, because esters are made from a combination of a fusel alcohol and an organic acid. Many organic acids are produced during fermentation, and are responsible for the decrease in pH that is observed during fermentation. Fatty acids are the acids that form esters. An example of an ester produced is isoamyl acetate, giving a banana like flavor to beer, most commonly associated with hefeweizen beers. Isoamyl acetate is formed from a combination of Acetyl CoA and isoamyl alcohol. The more oxygen added at the beginning of fermentation, the more yeast growth obtained, and the less esters formed because yeast are using the Acetyl CoA to build lipid membranes instead of making esters such as isoamyl acetate. However, that can be the opposite at times! When you add more oxygen, you are also creating more of the other substrate for esters, fusel alcohols. It is important to keep ester formation in check, because yes we want esters, but too much will make the beer become juicy fruity.
That explanation helps to describe how ale and lager yeast strains make esters, fusel alcohols, sulfur, diacetyl, and acetolacate. But what about Belgian-type strains? The Belgian-type strains I refer to are the ones used to make Trappist style beers and Belgian pale ales. These beer styles are yeast flavor dominant. Belgian style yeast strains are technically ale strains, but are more ‘wild-type.’ They make more esters and fusel alcohols than other ale strains under the same conditions. Most are also phenol negative. But some strains are phenol positive. Phenol is a type of compound most yeast in the wild make.
They use the basic phenol structure to make many different phenol containing compounds, including 4-vinyl guaiacol (4VG) and 4-ethyl phenol (4EP). Most brewer’s yeasts contain mutations, selected by brewers long ago, to inactivate phenol production. Phenol gives a strong antiseptic, Band-Aid like flavor and aroma to beer. Belgian wit style strains, most Saison strains, German weizen style strains, Brettanomyces, and a sprinkling of other brewer’s yeast, produce noticeable phenolic flavors. We call these strains phenol off flavor positive, POF+. The wheat and wit beer strains make 4VG, and Brettanomyces make 4EP.
The genetics of yeast mostly determines how yeast does this metabolism, and each strain of yeast has a similar, but different, complement of DNA. When a strain of yeast ferments a beer, it clones itself in the fermentation, which is why we can get consistent results from yeast. Brewers and yeast labs of the world work hard to keep yeast happy and free from mutations.
The environment a yeast is in will also impact metabolism and the flavors produced. When the environment changes with beer fermentation, i.e. we make different styles of beer, the yeast can behave differently. The main job for a brewer — hobby or commercial — is to control this environment by giving yeast the right amount of oxygen, the right fermentation temperature, and lots of love.
