by Jim Busch (Brewing Techniques)
Brewers make wort; yeast make beer. To make a great beer takes a well-composed wort, proper wort handling, and optimal yeast preparation — some of the finer points of the brewer’s art.
Making beer is essentially a two-part process: creating wort and fermenting it. Brewers have a huge degree of control over the creation and composition of the wort, but we can only influence the fermentation of wort — the yeast are the ones that produce the beer from our wort. Therefore, it is essential that we understand what initial conditions can be provided so that the yeast will have the best chance of producing the type of beer we desire. The main factors that affect proper fermentation are:
One often hears brewers lamenting how they shook the carboy for 30 minutes, and the fermentation still ended at 1.024 or higher. What went wrong?
In my opinion, the principal causes of high terminal gravity are inadequate aeration, pitching too little yeast, and, if brewing from extract, inadequate FAN levels. It is critical that you pitch enough yeast, but how much is enough? This article provides some guidelines not only on the quantity but also on the quality of the yeast pitched.
But before we get that far, bitter wort must be prepared. Wort production is really the area over which brewers have direct control, and it is critical that the wort contain nutrients and other characteristics favorable to yeast growth. So first let’s delve into the importance of wort composition.
The Importance of Amino Acids
Wort contains numerous amino acids that are essential to proper yeast growth and hence healthy fermentation. Amino acids originate in barley, are liberated during malting and mashing, and survive into the finished beer. Amino acids viewed as a collective quantity are referred to as free amino nitrogen or FAN.
Nature was very kind to brewers when it comes to amino acids in that most all-grain worts usually have FAN levels between 200 and 400 mg/L; a minimum of 180 mg/L is desirable (1). High FAN levels are obtained in higher gravity worts, because more malt means more amino acids. Levels below 180 mg/L are typically the result of the mash containing a large percentage of nonmalt adjuncts. Low FAN levels are also typical of some extracts, no doubt the result of processing and the use of nonmalt adjuncts in their production (3). Worts with low FAN content may exhibit high final gravities, depending on the yeast strain used.
Amino Acids Classifications
Class I: Aspartic acid, asparagine, glutamic acid, glutamine, threonine, serine, methionine.
Class II: Isoleucine, valine, phenylalanine, glycine, alanine, tyrosine.
Class III: Leucine, lysine, histidine, arginine.
Group A: Aspartic acid, asparagine, glutamine, glutamic acid, threonine, serine, lysine, arginine.
Group B: Valine, methionine, leucine, isoleucine, histidine.
Group C: Glycine, phenylalanine, tyrosine, tryptophan, alanine, ammonia.
Group D: Proline.
*Source: Reference 5.
†Source: Reference 2.
Different malted barleys will have different amino acid compositions, while unmalted barley has virtually no amino acids. The use of adjuncts like rice, corn, or unmalted barley or sugar will result in lower total FAN levels. Mashing programs can influence the concentration of amino acids, because amino acids are a product of the enzymatic degradation of proteins. Since most of the proteolytic breakdown occurs during the malting process, the bulk of the amino acid concentration is fixed by the maltster, the choice of raw barley, and seasonal variations in the production of this barley.
In general, more intensive mashing programs that include a protein rest may result in increased amino acid contents, but this is also very dependent on the barley malt and malting process, and caution should be exercised in low-temperature mashing so as not to negatively affect head retention.
Wort contains a large number of individual amino acids. In the literature, they are normally grouped into three classes and four groups. The classes (I–III) refer to the degree of essential importance to yeast metabolism (see box). The groups (A–D) categorize them according to the rate at which they are absorbed from the wort by yeast.
Amino acids and fermentation: Amino acids are essential nutrients that yeast assimilate during fermentation (two good discussions of this process are found in references 3 and 4). The reason amino acids are crucial to proper fermentation is that they are a primary source of nitrogen. It is this amino nitrogen that the yeast use to synthesize more amino acids and, in turn, to synthesize proteins (1). Class I amino acids are readily synthesized by the yeast cells, which use products of normal sugar catabolism as carbon sources, and thus their presence or absence in the wort is immaterial. Conversely, it is essential that the Class III amino acids be present in the wort because the yeast cells are unable to synthesize them. Class II acids can be synthesized by yeast, but their presence or absence in the wort can have marked effects on the flavor of the beer.
Valine and leucine are particularly significant in this regard. Valine is synthesized from pyruvate, which is a product of glycolysis. In the first step, mediated by the enzyme acetolactate synthase (and the cofactor hydroxyethyl TPP), the pyruvate is decarboxylated to α-acetolactate, which is rearranged, dehydrated, and aminated (all by other enzymes) to form valine. But not all the α-acetolactic acid is converted to valine. Some of it leaks through the cell wall and into the wort, where it is oxidized to diacetyl, a vicinal diketone (VDK) responsible for a buttery flavor in the finished beer.
The activity of acetolactate synthase is a function of the concentration of valine: the more valine the less activity. Thus, when valine is present, the cell senses its presence and produces less acetolactic acid. This negative feedback regulatory mechanism is found in many biochemical reaction systems. The significance of it to the brewer is that high valine levels in the wort result in reduced production of acetolactic acid and thus less diacetyl.
Isoleucine behaves in a similar way, regulating the first step in the synthesis of additional isoleucine from threonine, another amino acid. In this case, the intermediate product is α-ketobutyric acid, which is oxidized outside the cell to 2,3-pentanedione, a VDK that gives a honey-like flavor to beer. Thus we can minimize 2,3-pentanedione production by supplying lots of isoleucine and eliminating threonine (but note that threonine is a Class I amino acid, which will be synthesized by the yeast at levels set by other regulatory mechanisms).
Finally, amino acids play a significant role in the production of fusel alcohols, which impart a harshness to beer. Leucine, valine, threonine, and isoleucine are each involved in the biochemical formation of 3-methyl-1-butanol, isobutanol, n-propanol, and 2-methyl-1-butanol (isoamyl alcohol), respectively. In these reactions, an amino acid is broken into amino nitrogen and its respective α-ketoacid through a process called transamination, which occurs inside the yeast cell. The resultant α-ketoacid is then decarboxylated, yielding an aldehyde. The aldehyde is then available to be reduced to a fusel alcohol by nicotinamide adenine dinucleotide (NAD) as catalyzed by alcohol dehydrogenase. This is the same mechanism by which acetaldehyde gets reduced to ethanol.
Worts that are high in FAN and properly aerated will ferment quickly and cleanly. Yeast pitched in this environment will rapidly transition through the initial stages of aerobic activity, consuming internal glycogen and oxygen to form sterols, which result in strong cell walls. Strong cell walls enable the ready transport of amino acids to the interior of the cell.
Healthy fermentations will consume many of the primary amino acids (Class I) within the first 20 hours. This will allow rapid reproduction of the yeast through budding, thereby ensuring the proper density of yeast cells required during the anaerobic stage of fermentation. The end result is clean beer of low ester and diacetyl content that is fermented in 3–4 days for ales and 7–10 days for lagers.
Preparing Yeast for Pitching
Two methods are used to obtain proper quantities of yeast for fermentation — reusing yeast taken from the slurry of a previous fermentation, or stepping up a small amount of yeast into a “yeast starter.”
Reusing yeast from the slurry of a previous batch of homebrew, or from a local brewery, is probably the best way to ensure a healthy and quick fermentation. You can collect the yeast slurry from the bottom of the secondary or primary fermentor and store it in a sanitized container in a refrigerator set close to 32 °F (0 °C). It will remain viable for about two weeks, depending on the yeast strain. During storage, the still beer will separate from the yeast slurry and rise to the top of the container. This should be decanted and discarded because the yeast stores better when removed from the beer.
When choosing yeast for repitching, make sure that the previous fermentation finished out properly, was adequately attenuated, and that the still beer from your refrigerator storage tasted and smelled good. Never reuse yeast that did not fully ferment the previous batch. If open fermentation and a true top-fermenting yeast are used, the yeast may be skimmed with a sanitized spoon from the top of the fermentor. This is a particularly good method for weizen and English yeast strains. When reusing yeast, pitch approximately 0.5 fl oz of slurry/gal of wort. Professionally, the amount is between 0.5 and 1 lb/bbl, if measured by weight.
One issue that arises in harvesting yeast from fresh fermentations is the question of which layer of the yeast cake to harvest from. The bottom layer has mostly trub and dead cells (those that fell out first were present in the original pitching slurry). The top layer largely consists of more slowly flocculating cells. Thus the middle layer is best, but it is difficult for home brewers to isolate. Small-scale professional brewers using cylindroconical tanks can discard the first rush of yeast and trub, harvesting the better yeast more easily.
Unfortunately, many of us don’t have easy access to a local brewery and it is problematic to store yeast slurry beyond two weeks, so many home brewers are forced to repropagate yeast from small amounts. To build up a yeast starter, you can begin from slants of yeast or from the popular yeast smack packs. In either case, a yeast starter should always be prepared several days in advance of brewing. If using a smack pack, break the pack to mix the yeast and food and allow to swell for one or two days. Sanitize your scissors and the outside of the package by wiping with chlorox, isopropyl alcohol, or iodophor solution or by flaming the outside of the package. Add the active yeast to a sanitized and cooled container of wort. Typical yeast-to-wort ratios are 1:10 or 1:20, so a 50-mL package of yeast may be pitched directly into 500–1000 mL of wort. Many brewers discard the fluid used to build up the yeast at each step and replace it with freshly aerated wort. Doing this for several steps will produce a thick paste of yeast.
Be sure to aerate the wort! Wort should be aerated at each feeding because oxygen is required for the synthesis of sterols, which are in turn required for strong yeast cell walls and rapid growth.
The wort for starters can be produced using extracts or, even better, can be saved from a previous brew. If you brew from extract, it is a good idea to use some yeast nutrient because extracts are notoriously deficient in FAN. If saving wort from a previous brew, be sure to store it in mason jars and follow proper canning procedures.
When using slants, first flame a wire loop to sanitize the tip. Insert the sanitized tip in the slant to cool before dipping into the yeast culture. You can then either use this loop to plate the yeast or place it into a 1–2 mL wort vial to grow. After one day, this can be stepped into 10–40 mL of wort. After another day or two, step up to 500 mL of wort. I prefer to allow my starters to completely ferment, decanting the still beer from the slurry and using only the slurry. By waiting for the yeast to ferment completely, the yeast cells are high in energy stores in the form of glycogen. Glycogen is consumed (catabolized to acetyl coenzyme A through the same pathway as glucose) during sterol synthesis. Sterol synthesis is important because it prepares the yeast’s cell walls so that wort nutrients can be readily assimilated. Yeast that is pitched during high krausen (fermentation) are at their lowest glycogen levels, and this may be a problem in some fermentations. Stored yeast should be fed wort so they can maintain adequate glycogen levels.
How much yeast should you pitch? Many home brewers underpitch to the extreme. In professional breweries, the yeast cells are counted under a microscope to determine whether enough viable cells are present (see reference 5 for details on how to perform cell counts). The cell count is used to estimate the density of cells so that the correct amount of cells are pitched. A typical rule of thumb is to pitch 1 million cells/°Plato/mL of wort. For example, a 12 °P wort would require 12 million cells per mL for optimum fermentations. This level will never be reached by home brewers who grow yeast in pint or quart amounts for 5-gal batches. Only by repitching slurry can home brewers realize this cell density.
To make matters worse, many lagers need even more yeast, and certainly all strong ales should be pitched with as much yeast as possible, within reason. When growing starters for fermentations, I recommend using the slurry from 2 L of starter for ales and 4 L for lagers per 5 gal of wort.
When propagating yeast, bring the temperature of the starter closer to the target temperature of the main fermentation as the quantity of the starter increases. For example, when aiming to brew a pale ale at 65 °F (18 °C), the last stages of the starter should be grown at around 65 °F (18 °C). More important is the case of lagers, where the temperature has a profound impact on the flavor of the finished beer. Lager starters should be chilled to near 50 °F (10 °C) for the final stages of the stepping process and, for pitching, to the temperature of the wort into which it is to be pitched (often in the 44–50 °F [7–10 °C] range).
The Importance of Temperature
Increased temperature has several effects on yeast metabolism. It increases fusel alcohol production and the level of intracellular acetyl coenzyme A. As this is the starting point for lipid and sterol synthesis, lipid anabolism increases, which results in increased availability of acyl coenzyme A products. These products can combine with fusel alcohols to form the higher esters. Acetyl coenzyme A is itself an acyl coenzyme A molecule; because ethanol is the alcohol in highest concentration, ethyl acetate is the predominant ester in beer.
Note that when the wort has been insufficiently aerated some of the acetyl coenzyme A that would ordinarily go into sterol synthesis does not do so because of the lack of oxygen. The acetyl coenzyme A can then flow into the lipid synthesis pathway, producing extra acyl coenzyme A molecules and thus higher ester levels.
Higher temperatures produce more yeast faster, and many esters are formed by yeast metabolism of lipids and oxidative decarboxylation of pyruvate to acetyl coenzyme A. Higher temperatures are also well-known for causing increased fusel alcohols. Higher temperatures may also lead to greater initial diacetyl levels, but many yeasts reduce this diacetyl more effectively at elevated temperatures — hence the popular use of a “diacetyl rest” in some breweries.
It should be emphasized that many of the reactions and by-products mentioned here are vastly influenced by the strain of yeast. Numerous studies have demonstrated the great variance in by-products — such as ketones and fusel alcohols — depending on such variables as yeast strain, pitching rate, wort composition, and aeration, among others.
Making the Odds Favor Great Beer
The best brewers in the world under the best of conditions can control only the composition of the bitter wort. Making good wort is not that difficult; consistently transforming this wort into great beer is the magic of yeast. The challenge to consistently produce high-quality beer depends on both the composition of the wort and on the health of the pitching yeast. By pitching the correct amount of yeast into properly aerated wort, you can make great strides in ensuring that the finished beer is of the highest quality.
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