Brewing beer is an intricate process that requires patience and care at each step to produce a good quality beverage. One of the main concerns in the entire brewing practice is ensuring the appropriate levels of nitrogenous compounds in the mixture by proper management of the processes from malting until the end of fermentation. Because nitrogen is a nutrient that contributes to cellular structures and metabolic enzymes, a number of negative effects can result from too little nitrogen available at the various stages of the brewing process. On the other hand, excessive nitrogen may lead to off-flavors and overall poor quality of beer.
Nitrogen in beer comes from amino acids, peptides, proteins, nucleic acids, and other malt endosperm degradation products (Lekkas et al., 2005). Nitrogen compounds that can be metabolized by yeast (Saccharomyces cerevisiae or Saccharomyces pastorianus) during fermentation are referred to as free amino nitrogen (FAN). Several factors affect the FAN content of the wort, from the choice of barley variety, conditions during malting, mashing, and wort boiling. But to understand where the nitrogen compounds, including FAN in wort comes from, we need to take a closer look at its ultimate source — barley cereal.
Nitrogen in malted barley is mainly composed of proteins, with minor additions from other amino acids and nucleic acids. The greatest amount of proteins are found in the endosperm, the innermost part of the grain. There are two major types of proteins based on their roles in the barley seed: storage proteins and cytoplasmic (or metabolically active) proteins. The former include hordein (35-45% of total proteins) and glutelin (35-45%), while the latter include albumins (3-5%) and globulins (10-20%) (Lasztity, 1995). Based on where they are found in the cereal, proteins can also be classified into endosperm proteins, aleurone proteins, and germ (embryo) proteins.
Generally speaking, barley that is ideal for malting should have low grain protein (or crude protein) content (Qi et al., 2005). High amounts of protein in the grains will decrease malt extract and negatively affect beer quality (Briggs & Hough, 1981). However, high protein content also means greater diastatic power (amount of starch-converting enzymes). The goal is to produce barley seeds with the minimum amount of crude protein that will still yield sufficient diastatic power.
The protein content of barley is evidently influenced by the barley variety and the conditions under which it was grown, including the seeding rate and soil fertility. High seeding rates and less nitrogen fertilization of soil produce barley with smaller, uniformly sized kernels with less grain protein (Edney et al., 2012). The small and uniform size of the kernels allow more complete modification during malting, leading to malt with higher extract and lower wort β-glucan. Beta glucans are polysaccharides from the cell walls that envelope starch granules in a seed. If they are not broken down, problems may arise during lautering due to their gummy properties.
Proteins are degraded by enzymes called proteases. These enzymes are produced during the malting process, and do most of their work of breaking down proteins during mashing. An important step in mashing is the protease rest, and it is when most of the FAN is produced.
Components of FAN
Protein degradation from malted barley results in FAN, which is defined as the sum of individual wort amino acids, small peptides, and ammonium ions.
Amino acids are the basic building blocks of proteins. There are about 20 amino acids that normally occur in yeasts, and the types and relative amounts of each are influenced by the nutritional conditions of the wort. Amino acids can be classified into four groups, depending on the rates at which yeast cells absorb them — fast, intermediate, slow, and little/no absorption (often designated as Groups A, B, C, and D, respectively). The differences in uptake rates of amino acids depend on cell membrane components generally called permeases. These are proteins themselves that act like channels for amino acids. Each type of permease has different affinities and specificities for the different amino acids. Only when their preferred amino acids (Group A) occur in very low concentrations do these protein channels allow passage of the less preferred ones (Groups B and C).
Linking two or more amino acids produces the second component of FAN, peptides. Short peptides (oligopeptides) typically consist of two (dipeptide) or three (tripeptide) amino acids. They can be used by yeast for growth. Yeast show a much slower growth, however, when peptides are the sole nitrogen source available. Yeast can break down peptides by using proteolytic enzymes, and up to 40% of peptides are used up by yeast during fermentation (Lekkas et al., 2009). As a result, the peptide profile of wort is vastly different than that of beer.
The third component of FAN are ammonium ions, which form a mildly acidic compound that results from the protonation (addition of a hydrogen atom) of ammonia, a weak base. Yeast growth with ammonium ions as the sole source of nitrogen is better than when any single amino acid is used (Hough et al., 2012). However, it’s still inferior to growth in media that blend several amino acids. In wort, yeast cells obtain most of their required nitrogen from amino acids and peptides rather than ammonium ions.
Release of FAN During Malting and Mashing
Yeast is unable to metabolize the macromolecules in unmalted barley. Therefore, the malting process is necessary to produce enzymes that can break down the complex carbohydrates and proteins in barley cereal. The nitrogenous components of the endosperm undergo drastic changes during malting. The first goal of malting is essentially seed germination, and the requirements of a developing embryo drive the changes in the endosperm. Nitrogenous compounds are taken up by the embryo for the synthesis of new tissues, especially the roots, which are the first to grow. Since the embryo is eventually removed at the end of malting, the uptake of nitrogenous compounds by the embryo results in reduced nitrogen levels in the finished malt (Briggs & Hough, 1981).
The enzymes produced during malting are put to work during the enzymatic rests in mashing. The rests are β-glucanase rest (to degrade cell walls and let starch granules out), protease rest (to break down proteins), and amylase rest (to hydrolyze starch). In addition to releasing FAN into the wort, the protease rest also frees small proteins from large ones, thereby improving foam stability in beer.
FAN Content and Beer Quality
Since the sugar content of wort is the substrate that is eventually converted to alcohol, it is intuitive that it be considered the best indicator of yeast performance during fermentation. However, even if the yeast exhibits the same carbohydrate attenuation (i.e., same amounts of alcohol are produced), two batches of beer may still differ significantly in quality. This has led to the popular recognition of FAN as a better indicator of beer quality and stability than the sugar content in wort. It is a better predictor of healthy yeast growth, viability, vitality, and fermentation efficiency (Stewart et al., 2013). Too little or too much FAN in the wort may result in several problems.
To increase the alcoholic content of beer, many breweries raise the carbohydrate levels by using adjuncts. Adjuncts, like rice and corn grits, are added to the wort before fermentation, which significantly dilutes its FAN content. Insufficient FAN itself leads to slower and longer fermentation because the yeast is required to generate its own amino acids, leading to a decrease in growth rates. The activation of cellular de novo amino acid synthesis may also produce undesirable by-products that contribute to off-flavors or a decrease in foam formation and stability (dos Santos Mathias & de Mello, 2014).
For example, low valine concentrations in wort activate the intracellular production of the amino acid. Unfortunately, an intermediate in the pathway for valine synthesis called α-acetolactate is also the direct precursor of diacetyl. Considered an off-flavor, diacetyl gives a butter or butterscotch taste to beer. Furthermore, poor attenuation due to poor yeast health also results in incomplete fermentation of sugars, producing beer with sweetness levels that are too high and are unacceptable in most beer types (Barnes, 2011).
From the point of view of yeast growth and fermentation, excessive FAN wort content is a good thing. It means that yeast grows faster, and can perform fermentation at a faster rate, increasing conversion of sugars into ethanol and carbon dioxide. This is on account of the decreased use of the the products of glucose metabolism (e.g. pyruvate) for the synthesis of amino acids (Krogerus & Gibson, 2015). The carbon flow through the fermentation process is then maximized.
On the other hand, higher FAN wort may also lead to the formation of higher alcohols (also called fusel alcohols), which can arise from the intermediate products of the metabolism of amino acids (Krogerus & Gibson, 2015). For example, isobutanol, a compound that can give beer an undesirable paint thinner or solvent taste, is formed when there is an over-supplementation of valine in the wort. High FAN wort content has also been shown to result in an increased production and higher final levels of esters, such as isoamyl acetate. This compound gives beer a banana-like flavor, reminiscent of the candy banana runts. Although it’s a characteristic of certain beer styles, isoamyl acetate is generally considered a source of fruity off-flavors in beer.
The most popular method to measure FAN content in beverages is the Ninhydrin method (ASBC, 2014). It is basically a colorimetric assay that
uses a ninhydrin solution which takes on a blue or purple color in the presence of free amines (vlab.amrita.edu, 2016). The degree of color change is proportional to the amount of FAN in beer. A spectrophotometer is then used to measure this color change by passing light through the sample and detecting how much light it absorbs or transmits. The results from the spectrophotometer can then be used to calculate the FAN concentration using this spreadsheet (it may be required to copy or download the spreadsheet in order to edit and use it).
Controlling Levels of FAN in Wort
Beginning with barley selection, the amount of protein in the wort can already be predicted, and hence managed. Smaller barley grains mean fewer proteins, while larger ones mean greater protein content (Edney et al., 2012). Barley kernels that are small and uniformly sized have higher germination rates and greater enzymatic power, leading to higher malt extract yields that have low protein content. During malting, addition of exogenous gibberellic acid (GA) increases the fraction of soluble nitrogen. GA is a plant hormone that is responsible for the mobilization of the enzyme α-amylase in the aleurone layer of cereal, leading to hydrolysis or breakdown of starch and growth of the embryo (dos Santos Mathias & de Mello, 2014). The drying and kilning of malted barley may denature some proteases, which can result to less breakdown of large proteins. Therefore, kilning at higher temperatures results to decrease in the protease activity and FAN levels in wort, while kilning at lower temperatures have the opposite effect (Barrett et al., 1967).
To decrease the relative amount of FAN in the wort, adjuncts can be added during mashing. The FAN levels are diluted since the adjuncts supply mostly carbohydrates only, and they typically have much lower protein content and enzymatic power. To obtain higher FAN levels with adjuncts, exogenous proteases should be added. Most of the FAN in wort is produced during the protease rest of the mashing process. Prolonging this rest increases FAN levels, which can lead to off-flavors. But it will also lower the chances of producing beer haze.
Free amino nitrogen is an important component of wort that directly affects the performance of yeast cells, and hence the quality and stability of the final beer product. The concentration and composition of FAN influences the metabolic processes whose products and by-products determine the flavor detectable in beer. Because too little or too much FAN may have detrimental effects on the beer, measurement and control of FAN levels are imperative in brewing.
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