Editing Protein

{{In progress}}
[[File:2xfr b amylase.png|thumb|3D representation of β-amylase enzyme structure]]

Proteins are a versatile substance required for all forms of life. They consist of long chains built from an assortment of different [[amino acids]] joined by "peptide links". When a protein is being built, the chain is folded into a particular 3-dimensional structure, and this structure is the basis for its function. Examples of function include building, degrading, or modifying other molecules (i.e. [[enzymes]]), maintaining cellular structure, or transporting molecules across the cell membrane. Proteins may be combined with various other molecules, such as [[sugars]] in the case of [[glycoproteins]], or various other groups (such as [[iron]]) in the cases of some enzymes.<ref name=bsp/>

The cereal [[grain]]s used in [[brewing]] contain a substantial amount of protein, second only to [[starch]].<ref name=yu>Yu W, Zhai H, Xia G, et al. [https://www.sciencedirect.com/science/article/abs/pii/S0924224420306002 Starch fine molecular structures as a significant controller of the malting, mashing, and fermentation performance during beer production.] ''Trends Food Sci Technol.'' 2020;105:296–307.</ref> Thousands of different proteins have been detected in wort, and these proteins and their degradation products are important factors in beer quality. They are influenced, modified, and aggregated throughout the whole [[malting]] and brewing process.<ref name=steiner/><ref name=kerr/> Arguably the most important proteins in the [[malt]] are the [[enzymes]] responsible for various functions during [[mashing]]. Some of the malt protein is broken down by proteolytic enzymes, and the protein degradation products are responsible for beer [[foam]] and mouthfeel while others are utilized by the [[yeast]] as a source of nitrogen (to build their own proteins).<ref name=bsp/><ref name=kunze>Kunze W, Hendel O, eds. [[Library|''Technology Brewing & Malting.'']] 6th ed. VLB Berlin; 2019.</ref><ref name=fix/> Indirectly, the proportions of protein components affect the flavors produced by yeast during fermentation. The color of the beer is influenced by [[Maillard reaction]]s between the sugars and protein components during the [[boiling|boil]], which give rise to color and flavor compounds. Proteins also serve as a natural layer of protection against [[oxidation]]. Not all proteins are beneficial however; some enzymes cause unwanted effects, other proteins may contribute to beer [[haze]] (in combination with [[phenolic compounds]]), some may cause [[lautering]] problems, and some proteins can have negative health effects on certain beer drinkers (e.g. hordein, a "[[gluten-free beer|gluten]]" protein from barley). Stemming from protein degradation, the nitrogen level in the final beer can also affect its susceptibility to the growth of contaminating organisms. 

The structures of proteins are somewhat delicate. At high temperature, protein molecules become "denatured"—they unfold, losing their shape and therefore their function. Protein function can also be damaged by [[shear force]]s or inactivated by high or low [[pH testing|pH levels]]. Large proteins typically are either broken down by enzymes in the mash or they eventually precipitate (coagulate) during [[mashing]], [[boiling]], [[wort chilling|chilling]], or [[yeast|fermentation]]. Medium and small proteins, protein fragments, and amino acids are typically what pass to the final beer, affecting its sensory characteristics. However some malt proteins or modified products partly survive mashing and boiling and appear in the final beer relatively unchanged.<ref name=adb>Narziss L, Back W, Gastl M, Zarnkow M. [[Library|''Abriss der Bierbrauerei.'']] 8th ed. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA; 2017.</ref><ref name=bsp/>

The words '''peptides''' or polypeptides refer to fragments of proteins consisting of smaller chains of amino acids, generally without a defined 3-dimensional structure.<ref name=fix/>

==Proteins in grain==
The mature [[barley]] grain contains a spectrum of proteins that differ in function, location, structure, and other physical and chemical characteristics. The level of protein is a critical aspect of the quality of [[malt]] and [[beer]], and it is influenced by [[grain]] variety, soil conditions, crop rotation, fertilization, and weather conditions.<ref name=steiner/><ref name=picariello/><ref name=mahalingam/> Generally, the protein content in barley grain represents approximately 8–16% of its total mass.<ref name=silva>Silva F, Nogueira LC, Goncalves C, Ferreira AA, Ferreira IMPLVO, Teixeira N. [https://www.sciencedirect.com/science/article/abs/pii/S0308814607006085 Electrophoretic and HPLC methods for comparative study of the protein fractions of malts, worts and beers produced from Scarlett and Prestige barley (''Hordeum vulgare'' L.) varieties.] ''Food Chem.'' 2008;106(2):820–829.</ref><ref name=mahalingam>Mahalingam R. [https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-016-3408-5 Shotgun proteomics of the barley seed proteome.] ''BMC Genomics.'' 2017;18(44).</ref> Brewers prefer lower protein levels as long as there is plenty of soluble nitrogen for good yeast nutrition and beer foam potential.<ref name=crit>Bamforth CW, Fox GP. [https://www.brewingscience.de/index.php?tpl=table_of_contents&year=2020&edition=0009%2F0010&article=92781 Critical aspects of starch in brewing.] ''BrewingScience.'' 2020;73(9/10):126–139.</ref> This is because excessive protein content decreases the relative amount of [[carbohydrates]] (mainly [[starch]]) and also has other negative influences on the [[brewing]] process.<ref name=yu/> The barley used for [[malting]] should generally fall at the lower end of this range, with about 9–11% protein.<ref name=fix/><ref>Yu W, Tao K, Gidley MJ, Fox GP, Gilbert RG. [https://www.researchgate.net/publication/328971139_Molecular_brewing_Molecular_structural_effects_involved_in_barley_malting_and_mashing Molecular brewing: Molecular structural effects involved in barley malting and mashing.] ''Carbohydr Polym.'' 2019;206:583–592.</ref> Six-row barley tends to have more protein content and thus less starch than two-row.<ref name=mahalingam/> Hordeins (storage proteins) are the most abundant proteins found in a barley grain, and they form a matrix around the [[starch]] granules, increasing grain hardness.<ref name=celus>Celus I, Brijs K, Delcour JA. [https://www.sciencedirect.com/science/article/abs/pii/S0733521006000762 The effects of malting and mashing on barley protein extractability.] ''J Cereal Sci.'' 2006;44(2):203–211.</ref><ref name=iimure/> These storage proteins are created specifically to be broken down and used as a source of amino acids for building other proteins during seed germination, facilitating the plant's growth. A large variety of other proteins are present in barley, with over a thousand unique proteins identified.<ref name=kerr>Kerr ED, Fox GP, Schulz BL. [https://www.sciencedirect.com/science/article/pii/B9780081005965228692 Grass to glass: Better beer through proteomics.] In: Cifuentes A, ed. ''Comprehensive Foodomics.'' Elsevier; 2020:407–416.</ref> Notable among these are [[lipid transfer protein]]s (LTP) and [[protein Z]], which are the two major proteins in finished beer.

Although there are some differences in protein structure, the overall protein profile of beers produced from other cereal grains used for brewing tends to be similar, including [[wheat]] and [[oats]].<ref name=picariello/><ref name=klose>Klose C, Thiele F, Arendt EK. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2010-0312-01 Changes in the protein profile of oats and barley during brewing and fermentation.] ''J Am Soc Brew Chem.'' 2010;68(2):119–124.</ref>

===Malt===
During [[malting]], the protein content of the grain is "modified". In particular, the term "modification" largely refers to the complex process whereby hordeins are partially degraded by protease enzymes into [[amino acids]] and peptides.<ref name=picariello/><ref name=benesova/><ref name=zhang>Zhang N, Jones BL. [https://www.sciencedirect.com/science/article/abs/pii/0733521095900306 Characterization of germinated barley endoproteolytic enzymes using two-dimensional gels.] ''J Cereal Sci.'' 1995;21(2):145–153.</ref><ref name=steiner/><ref>Jones BL. [https://www.sciencedirect.com/science/article/abs/pii/S0733521005000743 The endogenous endoprotease inhibitors of barley and malt and their roles in malting and brewing.] ''J Cereal Sci.'' 2005;42(3):271–280.</ref> Well-modified malt contains less than half the amount of hordeins present in the original barley.<ref name=celus/> The level of modification directly impacts the brewing process by providing sources of nitrogen and making the starch more accessible to enzymes (alpha-amylase in particular), and therefore modification is closely tied to overall malt quality.<ref name=osman03>Osman AM, Coverdale SM, Onley-Watson K, Bell D, Healy P. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.2003.tb00592.x The gel filtration chromatographic-profiles of proteins and peptides of wort and beer: effects of processing—malting, mashing, kettle boiling, fermentation and filtering.] ''J Inst Brew.'' 2003;109(1):41–50.</ref><ref name=jin>Jin B, Li L, Liu GQ, Li B, Zhu YK, Liao LN. [https://www.mdpi.com/1420-3049/14/3/1081/pdf Structural changes of malt protein during boiling.] ''Molecules.'' 2009;14(3):1081–1097.</ref><ref name=slack/> Malting also creates or activates various [[enzymes]] necessary for the brewing process. Differences among malting practices contribute to diversify the range of malts used for brewing.<ref name=picariello/> The level of protein modification during malting is conventionally measured in the brewing industry as the Kolbach index (soluble nitrogen/total nitrogen * 100).<ref name=steiner/>

The malting process also includes kilning. It is suggested that the "browning" of proteins ([[glycoproteins|glycation]] via the [[Maillard reaction]]) is the primary effect that occurs due to kilning.<ref name=jegou/> In general, proteins show resistance to denaturation from this heat treatment because they are in the dry state.<ref name=jegou/> For example, kilning appears not to affect the overall proteolytic activity of the malt.<ref>Jones BL, Marinac LA, Fontanini D. [https://pubs.acs.org/doi/abs/10.1021/jf000458k Quantitative study of the formation of endoproteolytic activities during malting and their stabilities to kilning.] ''J Agric Food Chem.'' 2000;48(9)3898–3905.</ref><ref name=benesova/><ref name=picariello/><ref name=osman/><ref name=jonesbudde>Jones BL, Budde AD. [https://www.sciencedirect.com/science/article/abs/pii/S0733521004001067 How various malt endoproteinase classes affect wort soluble protein levels.] ''J Cereal Sci.'' 2005;41(1):95–106.</ref> Amylases also largely survive the heat of kilning.<ref name=picariello/> However, kilning does reduce the amount of certain proteins to some extent such as protein Z and LTP1.<ref name=steiner/>

FYI, some references report the malt protein content as the nitrogen content of a material multiplied by 6.25 (or some other factor). This is incorrect and misleading, since many substances besides proteins contain nitrogen.<ref name=bsp/>

Interestingly, barley naturally contains proteins that inhibit fungal (e.g. yeast) growth, such as barwin and Thaumatin-like protein (active only in unmalted grain).<ref name=iimure/> These proteins can pass to the final beer, although the yeast inhibition by these proteins is obviously not a significant concern for brewers.

===Adjuncts===
[[Adjuncts]] generally do not contain proteins that are soluble in wort, so their use will directly dilute the protein content of the wort (which may cause negative effects).<ref name=fix>Fix G. [[Library|''Principles of Brewing Science.'']] 2nd ed. Brewers Publications; 1999.</ref> Furthermore, some raw grains including barley and wheat contain proteins that inhibit proteases from malt.<ref name=bsp/><ref name=jonesbudde/> Therefore, adding these adjuncts to a mash can reduce the level of wort-soluble nitrogen to a disproportionately greater extent.

==Proteins in the mash==
Complex protein biochemistry occurs during the mash.<ref name=kerr/> The activity of enzymes is the prime example, and they have numerous important effects (see [[Enzymes]]). Beyond this, certain proteins combine with other substances such as sugars or [[phenolic compounds|polyphenols]], storage proteins are partly degraded and/or solubilized and transferred into the produced wort, and some proteins can help buffer against [[oxidation]].<ref name=steiner/> Negative effects on the brewing process can also occur as a result of protein chemistry. The protein matrix (hordeins) of poorly-modified [[malt]] may inhibit [[saccharification|starch degradation]] during mashing by physically preventing access by α-amylase, which can potentially reduce the amount of [[extract]] obtained.<ref name=yu/> Protein aggregation may reduce or prevent wort flow during [[lautering]] or recirculation (i.e. a "[[stuck mash]]") by forming a gel.<ref name=slack>Slack PT, Baxter ED, Wainwright T. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1979.tb06837.x Inhibition by hordein of starch degradation.] ''J Inst Brew.'' 1979;85(2):112–114.</ref> Despite all this activity, much of the protein is insoluble and therefore discarded with the [[spent grains]]. The intact proteins present in the wort at the end of mashing (at least 20 different types) are generally those that are resistant to degradation by malt proteinases.<ref name=iimure/> Proteins are not significantly modified via [[glycoproteins|glycation]] during the mashing process. However, mashing at higher temperature increases the level of [[glycoproteins]], perhaps due to greater extraction.<ref name=jegou/>

===Oxidation===
Oxygen introduced into the mash causes the formation of larger polypeptide aggregates (linked via disulfide bridges, see below), which become insoluble at higher mash temperatures and which settle as a doughy layer on top of the spent grains in a traditional lauter tun.<ref name=adb/><ref name=poyri>Pöyri S, Mikola M, Sontag-Strohm T, Kaukovirta-Norja A, Home S. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.2002.tb00550.x The formation and hydrolysis of barley malt gel-protein under different mashing conditions.] ''J Inst Brew.'' 2002;108(2):261–267.</ref><ref name=celus/> This layer is often called the ''Oberteig'' (which means "upper dough" in German), and it can prevent clarification, and slow down or stop lautering and/or recirculation. Similar behavior is seen by the so-called "gel proteins", which are certain types of hordeins dissolved during malting. During mashing, they too can result in groups of a protein/polypeptide/lipid aggregates resulting from oxidation. Small starch granules, β-glucans, and pentosans, which in turn are linked to proteins, also take part in the formation of complexes, which lead to increased dough formation. They can also hinder the release of starch granules and thus the effect of the amylases. The formation of gel-protein is minimal at low temperature (e.g. 118°F, 48°C) possibly due to the action of proteases, but during and after the saccharification rests (e.g. 145°F, 63°C), the amount of gel-protein rises rapidly when the mash is oxidized. The ''Oberteig'' tends to become especially prominent if the mash is recirculated, or it can also form during vorlauf.

All of these negative effects can be prevented by limiting oxygen in the mash, especially when oxygen-scavengers are also used.<ref name=poyri/> In other words, [[low oxygen brewing]] improves lautering and recirculation speed due to the prevention of ''Oberteig'' and gel protein aggregate formation. However, low-oxygen mashing actually encourages effective coagulation of larger proteins such that they do not contribute to haze.<ref name=derouck>De Rouck G, Jaskula-Goiris B, De Causmaecker B, et al. [https://www.brewingscience.de/index.php?tpl=table_of_contents&year=2013&edition=0001%252F0002&article=82374 The impact of wort production on the flavour quality and stability of pale lager beer.] ''BrewingScience.'' 2013;66(1/2):1–11.</ref> They are retained in the spent grains after lautering, but not in a form that reduces wort flow.

To delve further into the chemistry, many proteins contain "thiol" groups, each made up of a sulfur and hydrogen (–SH) branch from an amino acid (especially cysteine) in the protein or polypeptide. These thiol groups can bond to each other, forming a "disulfide bridge" by linking the sulfur atoms together, removing the hydrogen atoms. This bonding occurs very rapidly under oxidative conditions; oxygen in the mash oxidizes the free thiols, creating links between molecules, thereby forming aggregates.<ref name=lund/> Certain enzymes in grain catalyze the oxidation of thiols, and these may include sulphydryl oxidase, glutathione oxidase, glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase (but neither peroxidases nor lipoxygenases).<ref name=stephenson>Stephenson WH, Biawa JP, Miracle RE, Bamforth CW. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.2003.tb00168.x Laboratory-scale studies of the impact of oxygen on mashing.] ''J Inst Brew.'' 2003;109(3):273–283.</ref> It's interesting that thiol oxidation is actually used in many scientific studies as a measure of wort oxidation, which is of particular interest to many breweries due to its correlation with gel formation.<ref name=celus/> Disulfide bonds may be broken by reducing (oxygen-scavenging) compounds such as [[sulfite]], resulting in the regeneration of the original protein thiol groups. Regenerated thiol groups can participate in new reaction cycles with reactive oxygen species (ROS) and, taken as a whole, act as catalysts for the removal of ROS by sulfite.<ref name=lundmn>Lund MN, Andersen ML. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2011-0620-01 Detection of Thiol Groups in Beer and Their Correlation with Oxidative Stability.] ''J Am Soc Brew Chem.'' 2011;69(3):163–169.</ref> This antioxidant mechanism includes [[lipid transfer protein]] 1 (LTP1) as an important thiol-containing protein. See [[Protein#Influence on flavor stability|Influence on flavor stability]] below, and [[Oxidation]] for more information.

===Protein degradation===
During [[malting]] and [[mashing]], proteolytic [[enzymes]] (proteases) break proteins into smaller peptides or [[amino acids]] that are critical for brewing high-quality beer. Approximately 75% of wort protein is solubilized as a result of protein degradation during malting, and the remaining 25% of the final wort protein solubilized during mashing.<ref name=jones/><ref name=aldred/><ref name=jonesbudde/><ref name=kuhbeck/> As such, proteolytic activity during mashing is especially important when unmalted cereal [[adjuncts]] are used. Overall, efficient degradation of the hordein storage proteins of barley in malting and brewing is essential for several reasons:
* Hordein breakdown allows the release of starch into a form accessible by amylases.<ref name=aldred>Aldred P, Kanauchi M, Bamforth CW. [https://onlinelibrary.wiley.com/doi/abs/10.1002/jib.635 An investigation into proteolysis in mashing.] ''J Inst Brew.'' Awaiting publication. Accessed January 2021.</ref> Furthermore, proteinase activity directly increases the activity of the starch-degrading enzymes.<ref>Hu S, Dong J, Fan W, et al. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/jib.172 The influence of proteolytic and cytolytic enzymes on starch degradation during mashing.] ''J Inst Brew.'' 2014;120(4):379–384.</ref> Protease activity is actually the parameter most closely tied to the amount of malt [[extract]] obtained from mashing.<ref name=osman>Osman AM, Coverdale SM, Cole N, Hamilton SE, de Jersey J, Inkerman PA. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.2002.tb00125.x Characterisation and assessment of the role of barley malt endoproteases during malting and mashing.] ''J Inst Brew.'' 2002;108(1)62–67.</ref>
* The liberated amino acids are required for yeast fermentation and they also contribute to color and flavor formation.<ref name=aldred/><ref name=osman/> With well-modified malt, the desired concentration of free amino acids is typically achieved as a result of protein degradation during malting.<ref name=kuhbeck/>
* Solubilized proteins contribute to beer foam (see [[Foam]]).<ref name=osman/>
* Protein degradation lowers the amount of haze-forming materials (see [[Haze]]).<ref name=aldred/>

A wide variety of protease [[enzymes]] are involved in the protein degradation process, and besides amylases, they are the key degradation enzymes.<ref name=steiner/> Grain proteins are initially solubilized by '''endo'''proteases that break bonds in the middle of protein chains (endo- means inner), similar to the action of α-amylase on starch.<ref name=lund/><ref name=osman/><ref name=benesova/> Then they are further degraded by '''exo'''proteases that break off single amino acids from the ends of polypeptides (exo- means outer), similar to the action of β-amylase on starch. Besides degrading proteins, many of these enzymes also have other activities — ester modification, coagulation, and/or transpeptidase activity.<ref name=benesova>Benešová K, Běláková S, Mikulíková R, Svoboda Z. [http://kvasnyprumysl.eu/index.php/kp/article/download/95/74 Activity of proteolytic enzymes during malting and brewing.] ''Kvasný Prům.'' 2017;63(1):2–7.</ref> Proteolytic enzyme activity appears to be fairly consistent across widely different malt varieties, regardless of the breed or growing conditions.<ref name=osman/><ref name=jonesbl>Jones BL. [https://www.sciencedirect.com/science/article/abs/pii/S073352100500055X Endoproteases of barley and malt.] ''J Cereal Sci.'' 2005;42(2):139–156.</ref> Enzyme thermostability also appears consistent across barley types.<ref name=jones/>

Malt '''endo'''proteases are complex and diverse, and are created in multiple forms during the germination of barley. They exhibit different optimal activity levels for pH, reaction temperature, and thermostability.<ref name=osman/> Barley malt contains over 40 endoproteinases, grouped into four classes: cysteine proteases, metalloproteases, aspartic proteases, and serine proteases.<ref name=zhang/><ref name=jones>Jones BL, Marinac L. [https://pubs.acs.org/doi/abs/10.1021/jf0109672 The effect of mashing on malt endoproteolytic activities.] ''J Agric Food Chem.'' 2002;50(4):858–864.</ref> The aspartic-, cysteine- and metalloproteinases likely play roles in solubilizing the storage proteins of barley and malt, whereas the serine proteinases apparently do not.<ref name=steiner/><ref name=jonesbudde/>

Two types of '''exo'''proteases exist, aminopeptidases and carboxypeptidases, which work from opposite ends of the amino acid chains. Five carboxypeptidases are present in barley malt.<ref name=steiner/> Of all proteolytic malt enzymes, these carboxypeptidases are the most thermally resistant and also their optimal pH (4.8–5.6) is close to that of mashes. As a result, about 80% of the released amino acids are liberated during mashing by these enzymes.<ref name=benesova/><ref name=jonesbudde/> In contrast, the aminopeptidases (of which there are at least six forms)<ref>Strelec I, Vukelic B, Vitale L. [https://hrcak.srce.hr/file/62486 Aminopeptidases of germinated and non-germinated barley.] ''Food Technol Biotechnol.'' 2009;47(3):296–303.</ref> and also the dipeptidases (which cleaves dipeptides to amino acids) present in malt are generally inactive and have no effect on the level of amino acids in wort.<ref name=benesova/>

Excessive protein degradation is unnecessary and undesirable.<ref name=kunze/> Too much proteolysis may cause the development of undesirable flavors and color, reduced foam, and a thinner mouth feel.<ref name=osman/><ref name=lund/> Brewers can manipulate the amount of protein degradation mainly through control of the mash temperature and duration. At mash temperatures around 113–131°F (45–55°C), proteins are most thoroughly degraded, producing small protein fragments and amino acids. A rest in this temperature range (i.e. a "protein rest") should be avoided ''except'' when using poorly-modified malts.<ref name=kunze/> At higher mash temperatures such as 140–158°F (60–70°C), mostly larger protein fragments are formed, which are responsible for [[foam]] stability and mouth feel. Due to their heat-sensitivity, proteinases generally begin to inactivate at temperatures above 140°F (60°C). Above 158°F (70°C), they are inactivated more quickly, although they retain activity for about 15–20 minutes or possibly longer.<ref name=jones/><ref name=klose/><ref name=poyri/> The Hoch-Kurz [[mashing]] process can be implemented in order to restrict the activity of the endopeptidases and thus increase the percentage of larger peptides while retaining sufficient amino acid formation through the activity of the more thermostable carboxypeptidases.<ref name=sacher/><ref name=derouck/> The proteases are stable enough that even with these relatively high mashing temperatures, the total soluble protein level cannot drop below a value that was already predetermined by the malt quality.<ref name=adb/> Proteolysis doesn't fully stop until around 176°F (80°C), well above mash temperature.

To a lesser extent, [[brewing pH|mash pH]], grist to liquor ratio, and other factors can influence the activity of proteases and solubility of polypeptides.<ref name=picariello/> The activity of cysteine proteases (and therefore protein degradation) is inhibited by mash oxidation and stimulated by reducing agents.<ref name=benesova/><ref name=poyri/><ref name=sacher>Sacher B, Becker T, Narziss L. [http://www.lowoxygenbrewing.com/wp-content/uploads/2017/04/pkjdf.pdf Some reflections on mashing – Part 1.] ''Brauwelt International.'' 2016;5:309-311.</ref><ref name=jonesbl>Jones BL. [https://www.sciencedirect.com/science/article/abs/pii/S073352100500055X Endoproteases of barley and malt.] ''J Cereal Sci.'' 2005;42(2):139–156.</ref> Metalloproteases are inhibited by chelators (especially those with affinity for [[zinc]]).<ref name=benesova/><ref>Rizvi SMH, Beattie A, Rossnagel B, Scoles G. [https://www.researchgate.net/publication/215493909_Thermostability_of_Barley_Malt_Proteases_in_Western_Canadian_Two-Row_Malting_Barley Thermostability of barley malt proteases in western Canadian two-row malting barley] ''Cereal Chem.'' 2011;88(6):609–613</ref> Protease activity generally increases as the mash pH approaches 5.0 (see [[Brewing pH]]).<ref name=adb/> Protein degradation is greater in thicker mashes because enzyme activity is protected from thermal inactivation due to a protective colloid effect.<ref name=adb/> Finer grist appears to release amino acids into the wort more quickly, but ultimately it does not affect the level of protein degradation.<ref name=kuhbeck>Kühbeck F, Dickel T, Krottenthaler M, et al. 
[https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.2005.tb00690.x Effects of mashing parameters on mash β-glucan, FAN and soluble extract levels.] ''J Inst Brew.'' 2005;111(3):316–327.</ref>

Throughout the mashing process, high molecular weight (large) proteins and protein fragments tend to precipitate, especially at higher temperatures.<ref name=kunze/><ref name=kerr/> Therefore a step mash produces a more clear wort going into the kettle.<ref name=bsp/> Regardless of the mashing method used, significant amounts of malt protein remain in the spent grains following lautering.<ref name=klose/>

===Exogenous enzymes===
Treating wort with commercially-available proteases may cause the following effects:
* Increased free amino nitrogen.<ref name=lund/><ref name=lundm/>
* Variable effect on fermentation, based on the particular product used and other factors such as wort gravity. Treatment may cause increased or decreased fermentation performance and either increased or decreased higher alcohol and ester formation during fermentation (yeast flavor compounds).<ref name=lei/>
* Slightly increased extract yield, although probably not significant.<ref name=lund/><ref name=lundm/><ref name=lei/>
* Increased presence of free thiols (only if oxidation is avoided).<ref name=lund/><ref name=lundm/>
* Increased concentration of sulfite in the beer (only if oxidation is avoided). This can be explained by the increase in free amino acids, which results in increased sulfite production during fermentation.<ref name=lundm/>
* Decreased [[copper]] in the beer.<ref name=lundm/>
* Increased wort turbidity (haze), likely from increased peptides.<ref name=lund/>
* Slower wort flow during lautering.<ref name=aldred/>
* Increased staleness on sensory evaluation.<ref name=lundm/> This is likely due to the increased content of free amino acids increasing furfural via the Maillard reaction, a compound causing "biscuits", "caramel", and "burnt", and fruity aged or vinous character.

Mash pH appears to be unaffected.<ref name=lund/> Also of note, LTP1 is not degraded by added protease. Added protease does not improve starch solubilization, although effects on starch degradation are somewhat complicated (possibly increased degradation of small starches but decreased degrading of larger starch).<ref>Yu W, Gilbert RG, Fox GP. [https://escholarship.org/content/qt4v9546mx/qt4v9546mx.pdf Malt protein inhibition of β-amylase alters starch molecular structure during barley mashing.] ''Food Hydrocoll.'' 2020;100(105423).</ref>

Given the potential for a variety of negative effects, and lack of clear benefits, we generally recommend against the use of exogenous proteases.

===Wort nitrogen levels===
Nitrogen is present in many forms in wort. All-malt wort contains about 650–1000 mg/L nitrogen, of which about 20% is proteins, 30–40% is polypeptides, 30–40% is free amino acids, and 10% is nucleotides and other nitrogenous compounds.<ref name=lei>Lei H, Zheng L, Wang C, Zhao H, Zhao M. [https://www.sciencedirect.com/science/article/abs/pii/S0168160512006150 Effects of worts treated with proteases on the assimilation of free amino acids and fermentation performance of lager yeast.] ''Int J Food Microbiol.'' 2013;161(2):76–83.</ref>

Free amino nitrogen (FAN) is a measure of the low molecular weight substances, mainly amino acids, which are needed to support yeast growth and metabolism.<ref name=bsp>Briggs DE, Boulton CA, Brookes PA, Stevens R. [[Library|''Brewing Science and Practice.'']] Woodhead Publishing Limited and CRC Press LLC; 2004.</ref> FAN has long been regarded as a predictor of healthy yeast growth, viability, vitality, fermentation efficiency, and beer quality and stability.<ref name=lekkas>Lekkas C, Hill AE, Stewart GG. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2014-0113-01 Extraction of FAN from malting barley during malting and mashing.] ''J Am Soc Brew Chem.'' 2014;72(1):6–11.</ref> This is because FAN is used to provide not only nitrogen to the yeast cells for growth but also the wort nitrogen content or its metabolic products which affect beer flavor compounds and overall stability. The desired concentration of 200–250 mg/L free amino acids is typically available even without the proteolytic activity during mashing.<ref name=kuhbeck/> Around 88% of the total yeast utilizable nitrogen is produced during malting and 12% is produced during mashing (on average, with some variation between malts). This means that brewers producing all-malt wort generally don't need to worry about supplementing yeast nutrition as long as good practices are used for brewing and pitching. The FAN level of wort is fairly consistent (when produced within the standard pH range, which is optimal for FAN production) regardless of whether a protein rest is performed.<ref>De Rouck G, Jaskula B, De Causmaecker B, et al. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2013-0113-01 The influence of very thick and fast mashing conditions on wort composition.] ''J Am Soc Brew Chem.'' 2013;71(1):1–14.</ref><ref name=kuhbeck/><ref name=schwarz>Schwarz KJ, Boitz LI, Methner FJ. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2012-1011-02 Release of phenolic acids and amino acids during mashing dependent on temperature, pH, time, and raw materials.] ''J Am Soc Brew Chem.'' 2012;70(4):290–295.</ref>

The amount of yeast-assimilable nitrogen (YAN; the amount of usable amino acids and ammonia in the wort) can be approximately measured by using a formal titration, although it's generally not a worthwhile practice for brewers.<ref name=adb/><ref name=bsp/> See [[YAN testing]].

==Proteins in the boil==
This section is in progress.

The complex array of unique proteins brought into solution during the mash continue on into the [[boiling|boil]], and their quantities and structures are further changed during this step of the brewing process.<ref name=kerr/> The most studied protein changes during wort boiling relate to [[lipid transfer protein|LTP1]] and [[Protein Z]], due to their potential role in beer [[foam]] stability and [[haze]] formation. Other proteins affecting foam and haze are known to exist as well, but are less studied.<ref name=jin/><ref name=iimure/> Structural changes during boiling are are the same across all malt varieties.<ref name=steiner/>

===Wort protein changes===
Wort boiling leads to an important unfolding of LTP1, giving it a foam-promoting effect.<ref name=jegou/><ref name=jin/> LTP1 may then be further modified by glycation (via a Maillard reaction), which further increases its foam effect and may prevent its precipitation (glycation of LTP1 also occurs during the malting process).<ref name=iimure/><ref name=steiner/><ref name=jegou>Jégou S, Douliez PJ, Mollé D, Boivin P, Marion D. [https://pubs.acs.org/doi/abs/10.1021/jf010487a Evidence of the glycation and denaturation of LTP1 malting and brewing process.] ''J Agric Food Chem.'' 2001;49(10):4942–4949.</ref><ref name=jin/> Interestingly, wort boiling temperature is a key factor controlling this modification of LTP1.<ref name=iimure/> A higher wort boiling temperature (about 102 °C), resulting from the low altitude at sea level, reduces the LTP1 level of beer to 2~3 μg/mL, whereas lower wort boiling temperatures (about 96 °C), resulting from higher altitudes, leads to a LTP1 level of 17~35 μg/mL.<ref name=jin/> This may affect several beer quality traits such as beer foam, gushing and allergenic properties.<ref name=iimure/> Because boiling temperature varies by elevation, brewing in different locations can give different resulting wort compositions even if all other factors are exactly the same. Modification of Protein Z also occurs during wort boiling, possibly in a similar manner as LTP1 although it is less studied.<ref name=iimure/>

===Protein from hops===
Hops contain a vast array of different proteins — over 1000 unique proteins have been identified.<ref name=kerr/>  Despite this, no hop proteins have been identified in beer.<ref name=picariello/>

===Protein removal (trub formation)===
During the boil, some of the large proteins break into smaller ones, and some small proteins form larger molecules via aggregation or bonding with other wort components or hop compounds (e.g. via acylation or glycation).<ref name=jin/> High temperature also results in the unfolding (denaturation) of many proteins.  This denaturation generally causes the larger proteins to precipitate (coagulate) into a solid form called "trub" (also referred to as "hot break") as the wort is heated and throughout the boil.<ref name=steiner/><ref name=iimure/><ref name=kerr/><ref name=bsp/><ref name=jin/> The trub can be removed from the wort during the chilling, settling, and transfer steps, since the trub is undesirable in the fermenting beer.<ref name=steiner/> Despite the precipitation of various proteins during mashing, boiling, and fermentation, much of the overall wort protein remains soluble and is passed to the finished beer.<ref name=fix/><ref name=iimure/> This includes some amount of soluble protein aggregates.<ref name=jin/> The effect of wort boiling on protein properties is independent of malt variety.<ref name=jin/>

Glycation might prevent protein precipitation during the wort boiling step, notable in the case of LTP1.<ref name=jegou/>

It's interesting to note that some studies have demonstrated that effective protein precipitation during the boil is not always achieved,<ref name=osman03/><ref name=jin/> likely indicating that protein removal may be greatly influenced by the brewing process utilized, including factors such as pH. The amount of amino acids (FAN) has also been demonstrated to decrease during boiling in some cases but not others,<ref name=jin/> possibly related to boil intensity.

Boiling wort for a longer time leads to decreasing foam stability, possibly due to removal of Protein Z, which slowly binds with other proteins and precipitates.<ref name=iimure/>

Science FYI: Precipitation of the proteins occurs via cross-linking of sulfhydryl groups.<ref name=mullerr>Muller R. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-53-0053 Use of 5,5’-dithiobis (2-nitrobenzoic acid) as a measure of oxidation during mashing.] ''J Am Soc Brew Chem.'' 1995;53(2):53–58.</ref><ref name=jin/>

==Proteins during fermentation==
This section is in progress.

During fermentation the pH decreases, which causes some additional proteins to aggregate and precipitate, removing them from solution.<ref name=steiner>Steiner E, Gastl M, Becker T. [https://www.researchgate.net/profile/Elisabeth_Wiesen/publication/226773564_Protein_changes_during_malting_and_brewing_with_focus_on_haze_and_foam_formation_A_review/links/56bc4bc508ae2481ab6aeeba.pdf Protein changes during malting and brewing with focus on haze and foam formation: a review.] ''Eur Food Res Technol.'' 2011;232:191–204.</ref> Furthermore, some proteins adhere on the yeast and can be discarded with the sediment.

The protein structure of native barley LTP1 is stabilized by four disulfide bonds, but during fermentation these disulfides are reduced to thiols.<ref name=lund/>

Very little proteomic research has investigated yeast fermentation in beer brewing.<ref name=kerr/> Comparing the start and end of fermentation, it was shown that most proteomic changes were related to carbohydrate metabolism, respiration, and amino acid and protein biosynthesis.

Certain proteins such as LTP1 can help promote yeast growth via their ROS-scavenging mechanism (only if oxidation is prevented during the mash).<ref name=wu>Wu MJ, Rogers PJ, Clarke FM. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/jib.17 125<sup>th</sup> anniversary review: The role of proteins in beer redox stability.] ''J Inst Brew.'' 2012;118(1):1–11.</ref>

*[https://www.sciencedirect.com/science/article/abs/pii/S1567135604000960 Two-dimensional protein map of an “ale”-brewing yeast strain: proteome dynamics during fermentation.]

==Proteins in beer==
This section is in progress.

As described above, grain proteins are solubilized during the mash.  Much of this protein is then removed during the mash, the boil, and during fermentation. Yet, approximately one-third of the protein content is resistant to degradation and precipitation and hence passes through the processing steps, intact or somewhat modified, to the final beer.<ref name=steiner/><ref name=jin/><ref name=lund/><ref name=han>Han Y, Wang J, Li Y, Hang Y, Yin X, Li Q. [https://www.sciencedirect.com/science/article/abs/pii/S0308814615005993 Circular dichroism and infrared spectroscopic characterization of secondary structure components of protein Z during mashing and boiling processes.] ''Food Chem.'' 2015;188:201–209.</ref> A total of ~1900 unique proteins have been identified in beer.<ref name=kerr/> Almost all beer protein is derived from barley, with yeast proteins as minor constituents.<ref name=steiner/><ref name=wu/><ref name=picariello/> These proteins are well-known to influence beer characteristics such as foam, haze, color, flavor, flavor stability, and mouthfeel of beer.<ref name=steiner/><ref name=lund/><ref>Langstaff SA, Lewis MJ. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1993.tb01143.x The mouthfeel of beer—a review.] ''J Inst Brew.'' 1993;99(1):31–37.</ref> The amount of protein in beer is expected to vary enormously according to the malt used and the brewing process employed. However, the main protein components are similar in practically all beer produced.<ref name=picariello/> The "average" beer should contain somewhere in the neighborhood of 0.5–1 g/L proteins.<ref name=didier>Didier M, Bénédicte B. [https://www.sciencedirect.com/science/article/pii/B9780123738912000249 Soluble proteins of beer.] In: Preedy VR, ed. ''Beer in Health and Disease Prevention.'' Academic Press; 2009:265–271.</ref> 

Three major protein components are present in beer:<ref name=jin/><ref name=steiner/><ref name=wu/>
# [[Protein Z]] - a polypeptide with a molecular mass of 40 kDa. It's role is unclear but it may be involved with beer foam formation and/or stability, or possibly haze.<ref name=jin/><ref name=silva/><ref name=steiner/><ref name=han/> Protein Z is a member of the serine protease inhibitor (serpin) family.<ref name=wu/>
# [[Lipid transfer protein]] 1 (LTP1) - a 9.7 kDa polypeptide, which is responsible for foam stability and serves as an antioxidant.<ref name=jin/><ref name=steiner/><ref name=han/><ref name=wu/>
# A group of proline-rich hordein-derived polypeptides (with sizes ranging from 10 kDa to 30 kDa) that are involved in haze formation and possibly promote foam.<ref name=jin/><ref name=steiner/><ref name=iimure/><ref name=kerr/>

While the main focus of research has been on foam or haze, some beer proteins appear to have no function in beer except their contribution to mouthfeel, flavor, texture, body, color, and nutritional value.<ref name=silva/> In addition, some barley malt proteins are suggested to be allergens, and fungal contamination can modify LTP1 to result in beer gushing (over foaming at the bottle opening).<ref name=iimure>Iimure T, Nankaku N, Kihara M, Yamada S, Sato K. [https://www.sciencedirect.com/science/article/abs/pii/S0963996911006144 Proteome analysis of the wort boiling process.] ''Food Res Int.'' 2012;45(1):262–271.</ref>

Proteins of beer can elicit IgE-mediated allergic reactions, although the prevalence of allergy to beer is relatively low.<ref name=picariello>Picariello G, Mamone G, Nitride C, Ferranti P. [https://www.sciencedirect.com/science/article/pii/B9780128040072000230 Proteomic analysis of beer.] In: Colgrave ML, ed. ''Proteomics in Food Science.'' 2017:383–403.</ref> On the other hand, the (poly)peptides derived from hordein (AKA gluten) can trigger an autoimmune reaction in subjects suffering from celiac disease. Intact hordeins occur at low concentrations in beer because of their scarce solubility in the low-alcohol solutions. See [[Health and safety]].

===Influence on flavor stability===
Flavor stability is the ability of the beverage to resist changes to flavor, mainly due to oxidation. Proteins in the beer have a significant influence on flavor stability.

Protein thiols, which are present on cysteine residues in proteins (as discussed above), possess antioxidative capacity in beer and wort.<ref name=lund>Lund MN, Lametsch R, Sørensen MB. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/jib.155 Increased protein–thiol solubilization in sweet wort by addition of proteases during mashing.] ''J Inst Brew.'' 2014;120(4):467–473.</ref><ref name=wumj/> The concentration of free thiols correlates with the oxidative stability of beer because thiols remove reactive oxygen species (ROS).<ref name=lundm>Lund MN, Petersen MA, Andersen ML, Lunde C. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2015-0602-01 Effect of protease treatment during mashing on protein-derived thiol content and flavor stability of beer during storage.] ''J Am Soc Brew Chem.'' 2015;73(3):287–295.</ref> The major contribution to the protein thiol concentration in beer comes mainly from LTP1 because it is rich in cysteine and is a major component of beer protein.<ref name=lund/><ref name=lundm/><ref name=wumj>Wu MJ, Clarke FM, Rogers PJ, et al. [https://www.mdpi.com/1422-0067/12/9/6089/pdf Identification of a protein with antioxidant activity that is important for the protection against beer ageing.] ''Int J Mol Sci.'' 2011;12(9):6089–6103.</ref> In fact, beer LTP1 has been shown to scavenge one of the dominating radical compounds in beer, the 1-hydroxyethyl radical, at a rate similar to other reactive compounds in beer such as hop bitter acids.<ref name=lundm/> Thiols will react with almost all of the reactive oxygen species, which makes them especially important as far as beer aging is concerned.<ref name=wu/> A number of other proteins have also been identified in beer that contain several cysteine residues, so they could also contribute significantly to the thiol concentration in the beer.<ref name=lund/> In particular, yeast thioredoxin (TRX) is another thiol-rich protein suggested to provide defense against ROS.<ref name=wu/>

Note that the free thiol concentration diminishes as they are exposed to oxygen, removing the antioxidative ability of LTP1 and the other cysteine-rich proteins.<ref name=lund/><ref name=lundm/><ref name=wu>Wu MJ, Rogers PJ, Clarke FM. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/jib.17 125<sup>th</sup> anniversary review: The role of proteins in beer redox stability.] ''J Inst Brew.'' 2012;118(1):1–11.</ref> However, even if the wort is fully oxidized during mashing, the disulfides (bound thiols) are subsequently reduced during fermentation, thus converting them to active free thiol antioxidants in the beer.<ref name=lund/><ref name=lundm/><ref name=wu/> Of course, oxygen-limited packaging is also extremely important to prevent degradation of beer flavor. Beers with higher levels of free thiols taste better (i.e. less oxidized).<ref name=wumj/>

Reducing agents such as sulfite can re-activate oxidized thiols,<ref name=wu/><ref name=lundm/> although adding sulfite at packaging is not something we recommend.

Science FYI! A possible working mechanism for its ROS-scavenging ability is proposed: LTP thiol(s) is oxidized to the sulfenic acid by oxidants such as H<sub>2</sub>O<sub>2</sub>, which results in the destruction of a peroxide molecule in 1:1 stoichiometry. The free thiol can be recovered by two sequential reactions (reactions 2 and 3). The reaction 2 generates a disulfide (LTP-SSR) through reaction with a small molecule (HS-R) such as yeast thioredoxin. The reaction 3 uses sulfite or phenolic compounds to generate free thiol from the disulfide for the next round elimination of ROS.<ref name=wumj/>

Similar to sparkling wine, yeast-derived proteins can also positively affect the flavor stability of beer, even though massive yeast autolysis can generate off-flavors in beer.<ref name=picariello/>

===Foam===
Proteins in beer directly contribute to the formation of foam and its stability. See [[Foam]] for more information.

===Haze===
Proteins can play a significant role in the formation of beer haze. See [[Haze]] for more information.

==See also==
*[[Amino acids]]
*[[Glycoproteins]]
*[[Lipid transfer protein]]
*[[Protein Z]]
*[[Enzymes]]
*[[Foam]]
*[[Haze]]
*[[YAN testing]]


To review:
* [https://onlinelibrary.wiley.com/doi/full/10.1002/jib.630 Influence of high molecular weight polypeptides on the mouthfeel of commercial beer.]
*https://www.themodernbrewhouse.com/wp-content/uploads/2017/04/128-131.pdf

==References==