Editing Lipids

[[Category:off flavors]]
[[File:Linoleic-linolenic-fatty-acids.jpg|frame|Free fatty acids, the most common lipids in wort|alt=Linoleic and linolenic acid structures]]
Lipids are a complex class of macromolecules that are critical components of plant and animal cells. [[Barley]] is the major source of lipids in [[beer]],<ref name=bsp/><ref name=anniss>Anniss BJ, Reed RJR. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1985.tb04310.x Lipids in the brewery—a material balance.] ''J Inst Brew.'' 1985;91(2):82–87.</ref> and while the total lipid content varies by barley variety,<ref name=golston/><ref name=evans12/> [[malt]] generally contains about 2–4% lipids by weight.<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><ref name=bravie>Bravi E, Marconi O, Perretti G, Fantozzi P. [https://www.sciencedirect.com/science/article/abs/pii/S0308814612010138 Influence of barley variety and malting process on lipid content of malt.] ''Food Chem.'' 2012;135(3):1112–1117.</ref><ref name=mashing>Evans E. [[Library|''Mashing.'']]  American Society of Brewing Chemists and Master Brewers Association of the Americas; 2021.</ref> During [[malting]], these lipids serve as the initial source of energy for the new plant, supporting germination. Only a small portion of the lipids are consumed during malting, and the remainder are available for extraction during [[mashing]] and can be carried forward in the brewing process.<ref name=golston/><ref name=bravie/> The extracted lipids are of great interest to brewers because they can cause significant off-flavors (reduced flavor stability), as well as reducing [[foam]]. The [[flavor stability]] problems arise when certain lipids become oxidized, either by [[enzymes]] or whenever oxygen is introduced during wort production — at any point during [[milling]], [[mashing]], [[lautering]], [[boiling]], [[chilling]], and/or wort transfers. Therefore, limiting the ingress of oxygen, controlling the action of oxidative enzymes (lipoxygenases), and also limiting the amount of lipids going to the fermenter are all important parts of a quality brewing process.<ref name=golston/><ref name=kunze/><ref name=fix/> Effective [[low oxygen brewing|oxidation avoidance strategies]] can be implemented, and also lipids can be removed during many steps in the brewing process, reducing but not eliminating their impact.<ref name=golston/> However, lipids are essential components for [[yeast]] growth, so a small amount of lipids are beneficial to [[fermentation]] performance.<ref name=golston>Golston AM. [https://www.mbaa.com/publications/tq/tqPastIssues/2021/Pages/TQ-58-1-0322-01.aspx The impact of barley lipids on the brewing process and final beer quality: A mini-review.] ''Tech Q Master Brew Assoc Am.'' 2021;58(1):43–51.</ref>

Besides their effects on foam, flavor stability, and fermentation, lipids may have other influences as well. Endosperm lipids can bind with [[starch]] molecules, thereby increasing the [[gelatinization]] temperature, and also reducing the breakdown of that starch by blocking enzyme access.<ref name=gordon/><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><ref name=cozzolino>Cozzolino D, Degner S. [https://www.sciencedirect.com/science/article/abs/pii/S0963996916300047 An overview on the role of lipids and fatty acids in barley grain and their products during beer brewing.] ''Food Res Int.'' 2016;81:114–121.</ref> In other words, malt with higher lipid content is lower quality and produces wort with lower fermentability.<ref name=bravie/><ref>Cozzolino D, Roumeliotis S, Eglinton J. [https://onlinelibrary.wiley.com/doi/abs/10.1094/CCHEM-04-14-0071-R Relationships between fatty acid contents of barley grain, malt, and wort with malt quality measurements.] ''Cereal Chem.'' 2015;92(1):93–97.</ref> Lipid-starch complex formation can also interfere with [[iodine test]] results.<ref name=golston/> Lastly, lipid-starch-protein complexes can contribute to the formation of ''Oberteig'' in some cases (with uncontrolled oxygen levels in the mash), disrupting wort flow during the mash or lautering. See [[Protein#Oxidation|Protein oxidation]] for more information about Oberteig.

==Lipids in grain==
Biologically, lipids serve as cell membrane components, as energy storage, and as part of signaling pathways. In [[barley]], lipids are highly concentrated in the embryo, but the majority of lipids are found in the bran (aleurone, pericarp, and testa) and endosperm.<ref name=golston/> This is due to the embryo being a relatively small portion of the total kernel mass.

Lipids are broadly classified into two types: polar and neutral lipids.<ref name=golston/>
# Polar lipids are amphiphilic molecules, which have one end that is water-loving (“polar” or hydrophilic) and the other end is water-hating (“nonpolar” or hydrophobic). These are partially soluble in water. Polar lipids include phospholipids and glycolipids, and are major components of the amphiphilic lipid bilayer of yeast cell membranes.
# Neutral (nonpolar) lipids are less water soluble and are often referred to as fats or oils (hydrophobic). Often referred to as storage lipids, these are a necessary energy reserve within the barley kernel, which provides the embryo with an immediate energy source to support respiration as a new barley plant begins to grow. Within the barley kernel, neutral lipids are largely composed of triacylglycerides (TAGs). Sterols are another important type of nonpolar lipid present in wort that yeast utilize as structural and functional membrane components.

TAGs are composed of a glycerol molecule bound to three fatty acid (FA) molecules. FAs are hydrocarbon chains with a carboxylic acid on one end that can attach to glycerol. The hydrocarbon chains can vary in length and saturation. Saturated FAs have no double bonds in their hydrocarbon chain, while unsaturated FAs have a minimum of one double bond. When FAs are cleaved from the glycerol backbone, they are referred to as free FAs (FFAs). Hydrocarbon chain length and degree of unsaturation are two critical structural differences that differentiate each FA chain's chemical properties, such as water solubility and susceptibility to oxidation.<ref name=golston/>

==Impact of lipids==
Among the various types of lipids, free fatty acids predominate in wort and therefore are of the most interest to brewers.<ref name=fix/> In particular, oxidized (hydroxy) fatty acids are important because they are precursors to very potent flavor compounds related to beer staling.<ref name=kunze/><ref name=fix/>

===Flavor===
Hardly any polyunsaturated fatty acids (such as linoleic and linolenic acid) are present in the final beer because they are assimilated by [[yeast]] during fermentation. As such, they have no direct flavor impact.<ref name=bsp/> However, they are readily oxidized during the brewing process, resulting in the formation of lipid-derived products that carry through to the finished beer and can reduce the taste quality of beer enormously.<ref name=arts>Arts MJTJ, Grun C, De Jong RL, et al. [http://themodernbrewhouse.com/wp-content/uploads/2017/01/Oxidative-degradation-of-lipids-during-mashing..pdf Oxidative degradation of lipids during mashing.] ''J Agric Food Chem.'' 2007;55(17):7010–7014.</ref>
The compounds resulting from hydroxy fatty acid oxidation are known as aging [[carbonyl compounds|carbonyls]], and they damage [[flavor stability]] even in very small amounts.<ref name=kunze>Kunze W. Wort production. In: Hendel O, ed. [[Library|''Technology Brewing & Malting.'']] 6th ed. VLB Berlin; 2019:219–265.</ref><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><ref name=guidol>Guido LF, Boivin P, Benismail N, Gonçalves CR, Barros AA. [https://www.researchgate.net/profile/Luis-Guido-2/publication/226777056_An_early_development_of_the_nonenal_potential_in_the_malting_process/links/54e318780cf2de71a71df4ce/An-early-development-of-the-nonenal-potential-in-the-malting-process.pdf An early development of the nonenal potential in the malting process.] ''Eur Food Res Technol.'' 2005;220:200–206.</ref><ref name=kuhbeckreview/> The most researched lipid product is trans-2-nonenal (E2N), which is responsible for the papery or wet cardboard-like off-flavor and aroma in aged/oxidized beer.<ref name=golston/><ref name=kuroda>Kuroda H, Kojima H, Kaneda H, Takashio M. [https://www.jstage.jst.go.jp/article/bbb/69/9/69_9_1661/_pdf Characterization of 9-fatty acid hydroperoxide lyase-like activity in germinating barley seeds that transforms 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid into 2(E)-nonenal.] ''Biosci Biotechnol Biochem.'' 2005;69(9):1661–1668.</ref><ref name=caldas>Callemien D, Dasnoy S, Collin S. [https://pubs.acs.org/doi/abs/10.1021/jf051772n Identification of a stale-beer-like odorant in extracts of naturally aged beer.] ''J Agric Food Chem.'' 2006;54(4):1409–1413.</ref> Another example is trans,cis-2,6-nonadienal, which can give a cucumber off-flavor.<ref name=bsp/>

{| class="wikitable"
|+ style="text-align: center;" | Trans-2-nonenal (E2N)
|-
! Flavor
| Cardboard, papery, stale
|-
! Flavor threshold
| 0.03 ppb
|- 
! Boiling point
| 100–102°C
|}

It is important to understand that although the off-flavor from E2N '''appears''' during beer storage/aging, the compound is formed much earlier in the brewing process, primarily during mashing, lautering, and boiling.<ref>Kobayashi N, Kaneda H, Kano Y, Koshino S. [http://themodernbrewhouse.com/wp-content/uploads/2017/01/Kobayashi-Determination-of-Fatty-Acid-Hydroperoxides-Produced-During-the-Production-of-Wort.pdf Determination of fatty acid hydroperoxides produced during the production of wort.] ''J Inst Brew.'' 1993;99(2):143–146.</ref><ref name=bamlen/> After formation, E2N binds to other wort components, which temporarily masks the flavor so it cannot be tasted in wort or in fresh beer. The binding is reversible, so the E2N is released throughout the brewing process, particularly during beer aging (after fermentation completes).<ref name=golston/> This means that higher amounts of bound E2N in the wort lead to increased perception of staleness and cardboard flavor of beer after storage.<ref name=kuroda/> Lipids only cause this issue if we fail to manage our raw materials and processes appropriately.

Different levels of free fatty acids can influence the production of flavor compounds by yeast during fermentation.<ref name=bsp/><ref name=fix/> High lipid levels from excess [[trub]] suppress [[esters|ester]] production (particularly [[ethyl acetate]]).<ref name=kuhbeckreview/><ref name=gib125/> One possible reason for this is that elevated wort lipid content generally has a stimulating effect on yeast growth, creating an increased demand for acetyl-CoA to support fatty acid and sterol synthesis, which decreases the quantity available for ester formation.<ref name=golston/> The increased growth also causes an increase in the uptake of [[amino acids]] and therefore a greater production of [[higher alcohols]].<ref name=kuhbeckreview/> Another possible mechanism is that lipids repress the ''ATF'' genes, which encode alcohol acetyltransferases and have a major role in ester synthesis.<ref name=gib125/> A high or very low lipid concentration causes a higher peak concentration of vicinal diketones (e.g. [[diacetyl]]) during fermentation.<ref name=kuhbeckreview/> However, the peak is reached earlier and degradation is faster. Lastly, fermentation increases the levels of short-chain fatty acids, namely [[butryic acid|butyric]], [[isobutyric aicd|isobutyric]], [[isovaleric acid|isovaleric]], [[caprylic acid|caprylic]] and [[caproic acid]].<ref>Olšovská J, Vrzal T, Štěrba K, Slabý M, Kubizniaková P, Čejka P. [https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.9369 The chemical profiling of fatty acids during the brewing process.] ''J Sci Food Agric.'' 2019;99(4):1772–1779.</ref> However, these are generally (but not always) lower than their respective flavor thresholds.

Lipids in fermentation wort may also have a more direct effect by imparting caprylic (soapy) notes to the flavor/aroma profile, though this characteristic is associated with medium chain-length fatty acids and may in fact be ameliorated through the addition of longer chain fatty acids to the wort or by wort oxygenation. Shorter chain fatty acids may accumulate during fermentation due to the increased permeability of the yeast cell wall and contribute to stale or "yeasty" characters in beer.<ref name=gib125/>

===Fermentation===
Unsaturated fatty acids and sterols are necessary for the structure of the [[yeast]] cell membrane. The yeast can produce these fatty acids in sufficient amounts, but only when oxygen is present. This is the reason [[oxygenation]] is so important when pitching yeast. When oxygenation is inadequate, elevated amounts of lipids tend to enhance fermentation.<ref name=golston/><ref name=kuhbeckreview/> In this case, high levels of wort lipids improve fermentation performance, increase yeast viability, and provide the yeast greater resistance to high alcohol content.<ref name=bsp/><ref name=fix/><ref name=bravi/> Highly clarified worts with low dissolved oxygen levels exhibit decreased yeast activity and sluggish fermentations.<ref name=golston/>

Worts with excess trub containing high levels of lipids have also been shown to reduce [[sulfite]] production during fermentation (only applicable to yeast strains that product sulfite).<ref name=golston/> Sulfite is a desirable antioxidant that can help improve beer flavor stability during storage. High wort turbidity might also make yeast stay in suspension longer, potentially leading to hazy beer or other effects.<ref name=kuhbeckreview/>

=== Foam ===
While over 99.5% of barley lipids are lost throughout the brewing process, those that do survive can negatively impact [[foam]] stability, even at low levels.<ref name=fix/> In particular, both foam retention and lacing are reduced by longer chain fatty acids, while shorter chain fatty acids (C10 and shorter) have little effect.<ref name=gib125/> However, the exact relationship between wort cloudiness and foam stability is unclear.<ref name=kuhbeckreview/> Oxidized lipid products are especially harmful to foam stability, and increased oxidative enzyme activity may be part of the reason why a "protein rest" is so damaging to beer foam.<ref name=mashing/> Specialty [[malt]]s that are highly kilned can inhibit foaming when included in the grain bill, such as crystal 75L (Lovibond).<ref name=golston/> This appears to be a result of the elevated levels of triacylglycerides and lipid hydroperoxides in the wort due to the inclusion of these malts. However, there does not appear to be a simple correlation between the use of these specialty malts and foam inhibition.

==Lipid transformations==
Neutral lipids in [[barley]] consist of roughly 70–90% triacylglycerols (TAGs), 10–20% sterols and steryl glycosides, and less than 10% free fatty acids (FFAs).<ref name=golston/> The lipid profile of barley remains largely unchanged during [[malting]], although some degree of lipid oxidation may occur.<ref name=norja/><ref name=golston/><ref name=wackerbauer>Wackerbauer K, Meyna S, Marre S. [http://themodernbrewhouse.com/wp-content/uploads/2017/04/174-178.pdf Hydroxy fatty acids as indicators for ageing and the influence of oxygen in the brewhouse on the flavour stability of beer.] ''Monatsschrift Brauwiss.'' 2003;56(9/10):174–178.</ref><ref name=anniss/><ref name=garbe/><ref name=guidol/> The amount of oxidized lipids also increases the longer malt is stored.<ref name=wackerbauer/> However, [[mashing]] is when the real fun begins: the amount of FFAs rises drastically as the TAGs are degraded by lipase [[enzymes]]. Two of the most abundant FFAs are linoleic acid and linolenic acid,<ref name=bravi/> which are both sensitive to [[oxidation]] due to their double bonds.<ref name=bsp/><ref name=arts/><ref name=gordon>Gordon R, Power A, Chapman J, Chandra S, Cozzolino D. [https://www.mdpi.com/2311-5637/4/4/89/pdf A review on the source of lipids and their interactions during beer fermentation that affect beer quality.] ''Fermentation.'' 2018;4(4):89.</ref><ref name=cargon/> During brewing, these FFAs may become oxidized either enzymatically (by lipoxygenase enzymes) or non-enzymatically as a result of oxygen exposure.<ref name=stephenson/><ref name=kunze/><ref name=baedec/> The oxidized products give rise to a variety of compounds with negative effects on beer flavor.

[[File:Lipase-reaction.gif|frame|Enzymatic degradation by lipase|alt=Degradation of triacylglycerol to free fatty acids by lipase]]
Lipase is the enzyme responsible for the large increase in FFAs during mashing. It breaks apart TAGs into its components of glycerol and FFAs.<ref name=schwarzp>Schwarz P, Stanley P, Solberg S. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-60-0107 Activity of lipase during mashing.] ''J Am Soc Brew Chem.'' 2002;60(3):107–109.</ref><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> Besides barley, [[rice]] also contains lipase.<ref name=schwarzp/> Lipase is fairly stable at the range of temperatures used for mashing barley (up to around 158°F/70°C), so it is active throughout the duration of mashing, continually increasing the level of free fatty acids.<ref name=schwarzp/><ref name=golston/><ref name=cozzolino/><ref name=adb/><ref name=baedec>Baert JJ, De Clippeleer J, Hughes PS, De Cooman L, Aerts G. [https://pubs.acs.org/doi/abs/10.1021/jf303670z On the origin of free and bound staling aldehydes in beer.] ''J Agric Food Chem.'' 2012;60(46):11449–11472.</ref>

Lipoxygenases (LOX) occur in two forms, called LOX-1 and LOX-2.<ref name=bmp1>Davies N. Malts. In: Bamforth CW, ed. [[Library|''Brewing Materials and Processes: A Practical Approach to Beer Excellence.'']] Academic Press; 2016.</ref><ref name=adb/> These enzymes oxidize fatty acids during the brewing process, whether free or still bound to glycerol.<ref name=arts/><ref name=kunze/><ref name=golston/><ref name=garbe>Garbe LA, Barbosa de Almeida R, Nagel R, Wackerbauer K, Tressl R. [https://pubs.acs.org/doi/abs/10.1021/jf051993t Dual positional and stereospecificity of lipoxygenase isoenzymes from germinating barley (green malt):  Biotransformation of free and esterified linoleic acid.] ''J Agric Food Chem.'' 2006;54(3):946–955.</ref> Under its preferred conditions (below 60°C and relatively high mash pH), LOX-1 works very quickly and remains active until the temperature is increased.<ref name=arts/> In this case, even a relatively small amount of LOX can oxidize a large proportion of malt lipids during the mash.<ref name=norja>Kaukovirta-Norja A, Laakso S, Reinikainen P, Olkku J. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1993.tb01179.x Lipolytic and oxidative changes of barley lipids during malting and mashing.] ''J Inst Brew.'' 1993;99:395–403.</ref><ref name=bamenz>Bamforth CW. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1999.tb00025.x Enzymic and non‐enzymic oxidation in the brewhouse: A theoretical consideration.] ''J Inst Brew.'' 1999;105(4):237–242.</ref> Fortunately we have ways to minimize the activity of LOX during the mash (see below). LOX-2 is significantly less thermostable than LOX-1; thus, its contribution to FA oxidation during mashing is considered negligible.<ref name=golston/> Although they don't get as much attention as LOX, other enzymes in the wort are also involved with lipid oxidation, such as hydroperoxide lyase, which is partly responsible for converting oxidized lipids to E2N.<ref name=kuroda/><ref name=lewbam/><ref name=mashing/><ref name=cargon>Carvalho DO, Gonçalves LM, Guido LF. [https://ift.onlinelibrary.wiley.com/doi/abs/10.1111/1541-4337.12218 Overall antioxidant properties of malt and how they are influenced by the individual constituents of barley and the malting process.] ''Compr Rev Food Sci Food Saf.'' 2016;15(5):927–943.</ref><ref name=bamlen>Bamforth CW, Lentini A. The flavor instability of beer. In: Bamforth CW, ed. [[Library|''Beer: A Quality Perspective.'']] Academic Press; 2009:85–109.</ref>

FFAs are also oxidized by a non-enzymatic pathway.<ref name=adb/> Oxygen dissolved in wort quickly forms reactive oxygen species (ROS), reacting directly with fatty acids and other compounds (a process called autoxidation).<ref name=golston/><ref name=bamlen/> This autoxidation of lipids can occur very rapidly when oxygen levels are uncontrolled, fully oxidizing the lipids within the first 5 minutes of a mash.<ref name=arts/> Autoxidation yields similar products as the enzymatic pathway.<ref name=golston/> Lipid oxidation also occurs during wort [[boiling]] (via autoxidation).<ref name=gallardo/><ref name=baedec/> Furthermore, lipids present during boiling are also able to degrade amino acids to their corresponding [[Strecker aldehydes]] (staling compounds) by [[Maillard reaction]]s.<ref name=gallardo>Gallardo E, De Schutter DP, Zamora R, Derdelinckx G, Delvaux FR, Hidalgo FJ. [https://pubs.acs.org/doi/abs/10.1021/jf800094k Influence of lipids in the generation of phenylacetaldehyde in wort-related model systems.] ''J Agric Food Chem.'' 2008;56(9):3155–3159.</ref> Therefore, an increase in the amount of lipids in the boiling kettle produces increased levels of other staling compounds. Also, Maillard reaction products (e.g. formed during boiling) have been found to promote lipid oxidation,<ref name=gallardo/> further reason to prevent excessive lipids in the boiling kettle and avoid overly-intense boiling.

Oxidation of FFAs will result in the formation of hydroperoxides.<ref name=adb/> These hydroperoxides are reactive and degrade easily, forming products such as hydroxy fatty acids, phytoprostanes, and carbonyls.<ref name=arts/><ref name=adb/><ref name=sovrano>Sovrano S, Buiatti S, Anese M. [https://www.sciencedirect.com/science/article/abs/pii/S0308814605007399 Influence of malt browning degree on lipoxygenase activity.] ''Food Chem.'' 2006;99(4):711–717.</ref> Once formed, these oxidized lipid products can bind to other substances in the wort, creating a latent reserve of off-flavors (called "nonenal potential") that is protected from degradation (e.g. reduction by yeast) and removal (e.g. by evaporation) until being released during beer storage, causing off-flavors.<ref name=golston/> In particular, E2N is known to form reversible complexes with amino groups of proteins, peptides, or amino acids, which are all plentiful in wort.<ref name=golston/><ref name=calcol>Callemien D, Collin S. [https://www.tandfonline.com/doi/abs/10.1080/87559120903157954 Structure, organoleptic properties, quantification methods, and stability of phenolic compounds in beer—a review.] ''Food Rev Int.'' 2009;26(1), 1–84.</ref> Similarly, E2N can also reversibly bind with [[sulfite|SO<sub>2</sub>]] produced by the yeast during fermentation, temporarily masking its flavor.<ref name=golston/> The mechanism of E2N release from these complexes is not fully understood, although its rate of release is greatly influenced by beer storage temperature (higher storage temperature increases its release).<ref name=golston/><ref name=walhea>Walters MT, Heasman AP, Hughes PS. [https://www.researchgate.net/profile/Paul_Hughes8/publication/268296190_Comparison_of_-Catechin_and_Ferulic_Acid_as_Natural_Antioxidants_and_Their_Impact_on_Beer_Flavor_Stability_Part_2_Extended_Storage_Trials/links/552ce2640cf2e089a3acff3f.pdf Comparison of (+)–catechin and ferulic acid as natural antioxidants and their impact on beer flavor stability. Part 2: Extended storage trials.] ''J Am Soc Brew Chem.'' 1997;55(3):91–98.</ref><ref name=aroshe>Aron PM, Shellhammer TH. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.2010.tb00788.x A discussion of polyphenols in beer physical and flavor stability.] ''J Inst Brew.'' 2010;116(4):369–380.</ref><ref name=bamlen/> The antioxidant level of beer does not impact the release of E2N.<ref name=mcmmad>McMurrough I, Madigan D, Kelly RJ, Smyth MR. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-54-0141 The role of flavanoid polyphenols in beer stability.] ''J Am Soc Brew Chem.'' 1996;54(3):141–148.</ref> The oxidation of lipids during the brewing process also generates additional reactive oxygen species, inflicting further damage to other compounds in the wort (non-enzymatically).<ref name=arts/><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>

'''Summary of lipid transformations:'''
# Lipase enzymes break down triacylglycerols during the mash, releasing free fatty acids.
# The fatty acids become oxidized to some extent by either of the following mechanisms<ol style="list-style-type:lower-roman"><li>Enzymatically by lipoxygenase enzymes during mashing or earlier during malting, storage, or milling</li><li>Non-enzymatically by autoxidation at any point during wort production</li></ol>
# The oxidized fatty acids are further transformed by other enzymes and reactions and/or they bind to other wort components, masking the flavor and creating a latent reserve of staling compounds such as E2N.
# Most lipids are removed during mashing and boiling/chilling, along with the spent grain and trub respectively (discussed below).
# During fermentation, the yeast take up most of the remaining unoxidized free fatty acids.
# The latent E2N is released over time (faster at higher temperatures), causing a stale flavor.

==Avoiding negative effects==
Oxidation of lipids and FFAs is unavoidable, but steps can be taken throughout the brewing process to minimize the extent to which oxidation occurs. The strategy to avoiding the negative impact of lipids is three-fold:
# Prevent oxygen ingress into the wort (except when pitching yeast)
# Reduce the action of LOX
# Maximize lipid removal during the brewing process

The appearance of lipid-derived off flavors can also be delayed by storing the beer at a low temperature after fermentation completes.<ref name=walhea/> See [[Flavor stability]].

===Reducing oxygen===
Mashing is the most vulnerable step of beer production with regard to lipid oxidation,<ref name=arts/><ref name=adb/> and taking steps to avoid the presence of oxygen in the mash reduces the level of E2N and other off-flavor compounds.<ref name=golston/><ref name=wackerbauer/> The quality of beer is negatively influenced by [[oxidation]] at almost every stage of the [[brewing]] process, and lipid oxidation is just part of the picture — other beer components are also negatively affected. Therefore, appropriate recommendations to minimize oxidation are incorporated throughout the brewing process articles on this wiki. See [[Low oxygen brewing]] for an overview of best practices. Note that reducing oxygen exposure (along with the the use of [[antioxidants]]) only protects lipids against autoxidation; additional considerations are needed to reduce LOX activity.<ref name=arts/>

===Limiting LOX activity===
When its activity is properly controlled, lipoxygenase (LOX) is a relatively small part of the potential contributors to beer staling.<ref name=bmp1/><ref name=mashing/><ref name=guicur>Guido LF, Curto AF, Boivin P, Benismail N, Gonçalves CR, Barros AA. [https://pubs.acs.org/doi/abs/10.1021/jf0623079 Correlation of malt quality parameters and beer flavor stability:  multivariate analysis.] ''J Agric Food Chem.'' 2007;55(3):728–733.</ref><ref name=kanbam>Kanauchi M, Bamforth CW. [https://www.themodernbrewhouse.com/wp-content/uploads/2019/02/BrewingScience_bamforth_82-84.pdf A Challenge in the study of flavour instability.] ''BrewingScience - Monatsschrift Brauwiss.'' 2018;71(Sept/Oct):82–84.</ref> There are several ways that brewers can reduce  LOX activity in the mash:
* '''Low oxygen''' - Antioxidants and brewing methods that lower the oxygen ingress into wort reduce the action of LOX.<ref name=kunze/><ref name=adb/><ref name=derouck/><ref name=derouckg>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=golston/> This is redundant because we must keep the wort low oxygen in order to have any hope of avoiding massive oxidation damage to lipids and other compounds. See [[Low oxygen brewing]].
* '''Low mash pH''' - LOX has a relatively high optimal pH range, so lowering the mash pH reduces its activity by around 50% or more compared to higher pH.<ref name=bamlen/><ref name=kunze/><ref name=golston/><ref name=derouck/> Keeping mash pH under 5.8 is beneficial, and pH 5.4 or lower is preferred (measured at room temperature).<ref name=bsp/><ref name=derouck/><ref name=golston/><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> See [[Brewing pH]].
* '''High mash temperature''' - Mashing in at a temperature of at least 140°F (60°C) will reduce (but not eliminate) LOX activity since most of it is rapidly denatured.<ref name=poyri/><ref name=golston/><ref name=kunze/><ref name=wackerbauer/><ref name=mashing/> Mash-in temperatures of 144–145°F (62–63°C) are often recommended (avoid lower rests).<ref name=adb/><ref name=derouck/> See [[Mashing]].
* '''Short mash duration''' - Less time spent mashing, especially any time spent under 149°F (65°C) helps to limit LOX activity.<ref name=kunze/> This is one of the reasons why the high temperature and short duration of the ''Hoch-Kurz'' step mashing schedule is beneficial.
* '''Coarse crush''' - Fine milling causes more damage to the barley embryo and acrospires, which increases wort lipid and LOX levels.<ref name=golston/> Therefore, a more coarse crush is suggested to help reduce lipid oxidation.<ref name=adb/><ref name=kunze/><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> Best practices such as purging the oxygen from the grain and milling closer to mash-in can be helpful as well.<ref name=adb/><ref name=golston/> See [[Milling]].
* '''Null-LOX malt''' - Brewers may utilize a malt labeled as null-LOX or LOX-less, which has been bred to contain zero LOX enzymes toward the end of germination.<ref name=kuroda/><ref name=mashing/><ref name=bmp1/><ref name=derouck/><ref name=derouckg/> This is an effective approach because such malt has been shown to produce lower levels of oxidized lipid products, along with corresponding increases to foam and flavor stability.<ref name=golston/><ref>Hoki T, Saito W, Iimure T, et al. [https://www.sciencedirect.com/science/article/abs/pii/S0733521018303825 Breeding of lipoxygenase-1-less malting barley variety 'SouthernStar' and evaluation of malting and brewing quality.] ''J Cereal Sci.'' 2018;83:83–89.</ref><ref>Oozeki M, Sotome T, Haruyama N, et al. [https://www.jstage.jst.go.jp/article/jsbbs/advpub/0/advpub_16104/_article/-char/ja/ The two-row malting barley cultivar 'New Sachiho Golden' with null lipoxygenase-1 improves flavor stability in beer and was developed by marker-assisted selection.] ''Breed Sci.'' 2017:16104.</ref> However, the availability of null-LOX malt varieties is very limited, which may restrict its usage. See [[Malt]].
* '''Malt color''' - More-highly-kilned malt contains comparatively lower amounts of the LOX enzymes since the enzyme is destroyed to a greater extent by higher temperatures.<ref name=kunze/><ref name=norja/> In fact, LOX activity may decrease by 30–99.5% during the kilning process.<ref name=golston/> However, evidence suggests that higher kilning temperature may actually lead to higher levels of staling compounds in the wort (despite lower LOX activity).<ref name=guidol/><ref name=kuroda/><ref name=bmp1/> This is because the warmer kilning conditions promote lipid oxidization during kilning, creating a larger amount of staling compounds (nonenal potential) in the malt. On the other hand, it has been shown that modest additions of highly-kilned malt (i.e. 15% melanoidin, 10% caramel, or 1% black malt) decrease LOX activity in the mash. The increased [[melanoidins|melanoidin]] content of darker malt is believed to inhibit LOX, and the melanoidins also possess antioxidant capacity.<ref name=sovrano/>
*'''Storage time''' - LOX activity declines over time as malt is stored.<ref name=adb/><ref name=golston/> However, oxidation products increase during malt storage.<ref name=adb/> Use fresh ingredients!

===Reducing lipids in the wort===
Lipids are defined by their low solubility in water — in wort they tend to form solids that can be removed at certain points in the brewing process. Specifically, most of the lipids present in the mash are retained with the spent grain using proper [[lautering]] technique, and they can be removed along with the trub during the wort [[boiling]] process.<ref name=adb/><ref name=cozzolino/>

====Mashing & lautering====
Several factors can increase lipids extracted into the wort during mashing:
* Better modified malts<ref name=bsp/><ref name=lewbam>Lewis MJ, Bamforth CW. Chapter 12: Oxygen. In: Lewis MJ, Bamforth CW, eds. [[Library|''Essays in Brewing Science.'']] Springer; 2006:131–142.</ref>
* Finer milling<ref name=bsp/><ref name=bamlen/><ref name=lewbam/>
* Higher mashing & sparging temperature, especially above 149°F (65°C)<ref name=bsp/><ref name=golston/><ref name=evans12>Evans DE, Goldsmith M, Redd KS, Nischwitz R, Lentini A. [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2012-0103-01 Impact of mashing conditions on extract, its fermentability, and the levels of wort free amino nitrogen (FAN), β-glucan, and lipids.] ''J Am Soc Brew Chem.'' 2012;70(1):39–49.</ref><ref name=lewbam/>
* High percentages of malt (vs unmalted adjuncts)<ref name=bsp/>
* Decoction<ref name=bravi>Bravi E, Perretti G, Buzzini P, Della Sera R, Fantozzi P. [https://www.researchgate.net/profile/Elisabetta-Bravi/publication/233812024_Technological_stepyeast_biomass/links/0912f50bc95f43cc71000000/Technological-step-yeast-biomass.pdf Technological steps and yeast biomass as factors affecting the lipid content of beer during the brewing process.] ''J Agric Food Chem.'' 2009;57(14):6279–6284.</ref>

Adjusting these factors to decrease lipid extraction comes with certain trade-offs. For example, despite increasing lipids in the mash, higher mashing temperatures (i.e. step mashing) provide benefits to extraction and beer foam. Therefore it's not ideal to fully minimize lipid extraction during mashing because other aspects of beer quality may suffer. What we ''can'' do without sacrificing quality is to maximize the clarity of wort that goes into the brewing kettle by trapping solid lipid particles with the [[spent grains]] so they can be discarded.<ref name=kuhbeckreview/> During proper [[lautering]], the vast majority of lipids (up to 90%) are removed due to adsorption to the grain bed.<ref name=golston/> Also, precautions should be taken to reduce/minimize oxygen ingress during lautering and sparging, as lipid autoxidation can still occur.<ref name=golston/><ref name=kuhbeckreview/> By far the best way to accomplish these goals is to recirculate the mash using [[low oxygen brewing|low oxygen]] techniques, which traps coagulated lipid particles in the grain bed, producing clear wort. Manual [[vorlauf]] recirculation (draining the first runnings into a container and pouring it back into the mash) is a simple technique to minimize particles in the wort,<ref>Murphy L. [https://beerandbrewing.com/the-art-and-the-science-of-the-vorlauf-process/ The art and the science of the vorlauf process.] Craft Beer & Brewing website. 2016. Accessed January 27 2021.</ref> but this will obviously cause oxidation and therefore is not recommended. Squeezing the grain bed during lautering should be avoided because disturbance increases the amount of lipids (and other undesirable particles) in the wort.<ref name=golston/><ref name=bsp/><ref name=kuhbeckreview/> This is especially notable for brew in a bag (BIAB) lautering. Lastly, the no-sparge mashing method is beneficial since sparging increases the ratio of lipid extracted relative to sugar.<ref name=golston/><ref name=kuhbeckreview/> It is worth noting that lipases and LOX are denatured (deactivated) before reaching typical mash-out temperatures, so they will not have any further impact at this stage when step mashing.<ref name=golston/> On the other hand, single infusion brewers may still have free fatty acids being produced in the wort during lautering and sparging due to lipase activity.

Key points for lautering:
* Avoid oxygen ingress
* If possible, recirculate the mash
* Avoid disturbing the grain bed during lautering (e.g. do not squeeze)
* If possible, do not sparge

====Boiling & chilling====
The boil plays a significant part in the formation of latent lipid-derived staling compounds (nonenal potential) in the wort, which can carry over into the finished beer.<ref name=golston/><ref name=kuhbeckreview/> Lipid autoxidation during wort boiling has been shown to be responsible for up to 70% of nonenal potential in beers!<ref name=golston/> Again, brewers should avoid oxygen ingress by using modern methods such as gently filling the boil kettle from the bottom, minimizing splashing, avoiding leaky pumps, etc. (See [[Low oxygen brewing]])

FYI, practically none of the wort lipids are removed by evaporation.<ref name=golston/><ref name=anniss/>

During wort boiling and chilling, lipids, proteins, and other substances coagulate into solids call [[trub]]. In particular, denaturation of [[protein]]s renders them insoluble, as it exposes their hydrophobic regions, and lipids bind to this region because they are only partially soluble in water/wort. Consequently, the trub contains a large amount of fatty acids, as much as 50–70% lipids, and excessive trub in the wort makes it cloudy.<ref name=fix/><ref name=evans12/><ref name=gib125>Gibson BR. [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.2011.tb00472.x 125th anniversary review: improvement of higher gravity brewery fermentation via wort enrichment and supplementation.] ''J Inst Brew.'' 2011;117(3):268–284.</ref> As such, cloudy wort can contain up to 50 times the unsaturated fatty acid content of clear wort.<ref name=fix/><ref name=golston/> Therefore it's best to remove as much of the trub as reasonably possible.<ref name=fix/><ref name=golston/> Efficient removal of trub can lead to a 90% decrease of wort lipids.<ref name=golston/><ref name=anniss/><ref name=kuhbeckreview>Kühbeck F, Back W, Krottenthaler M. [https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/j.2050-0416.2006.tb00716.x Influence of lauter turbidity on wort composition, fermentation performance and beer quality – a review.] ''J Inst Brew.'' 2006;112(3):215–221.</ref> If excessive trub goes into the fermenter, lipid levels are likely to remain high into the finished beer.<ref name=anniss/> For home brewers, separation is typically accomplished via a whirlpool and/or allowing time to settle. During boiling or whirlpool/chilling, [[shear]] must be minimized to prevent disruption of the protein flocs leading to increased trub settling time.<ref name=golston/> In other words, it's helpful to minimize pumping. Likewise, any manner of disturbance to the trub pile should be minimized.

Key points for boiling & chilling:
* Avoid oxygen ingress
* Facilitate good trub formation
* Remove as much trub as possible by transferring only clear wort to the fermenter

====Fermentation====
Under optimal conditions (sufficient trub removal, pitching active yeast, etc), oxygenation of cold wort (8 ppm O<sub>2</sub>) has no significant impact on the level of lipid oxidation.<ref name=golston/> At this stage of the process, oxidation of wort compounds appears to play little to no part compared with the change of aroma and flavor from yeast metabolism. As mentioned above, the yeast take up fatty acids from the wort during fermentation. When the concentration of fatty acids is high, ''Saccharomyces'' takes them up by simple diffusion, while at low concentrations a facilitated transport system is used.<ref name=cozzolino/> Interestingly, several yeasts are known to secrete inducible lipases to degrade TAG to glycerol and fatty acids.<ref name=bravi/>

Hops are a source of lipids,<ref name=fix>Fix G. [[Library|''Principles of Brewing Science.'']] 2nd ed. Brewers Publications; 1999.</ref> although they supply very little lipid content to the wort compared to the amount from malt.<ref name=anniss/> However, it is possible for dry-hopping to contribute lipids to the final beer, some of which may cause off-flavors such as cheese-like aroma/flavor from [[isovaleric acid]] (a short-chain fatty acid) present in oxidized hops. Use fresh ingredients!

Key points:
*Pitch a good quantity of healthy and active yeast to facilitate rapid oxygen uptake
*Use non-oxidized hops for dry hopping

== See also ==
* [[Mashing]]
* [[Lautering]]
* [[Low oxygen brewing]]
* [[Oxidation]]
* [[Trub]]
* [[Foam]]

To review:
* Kobayashi, N.; Kaneda, H.; Kuroda, H.; Kobayashi, M.; Kurihara, T.; Watari, J.; Shinotsuka, K. Simultaneous determination of mono-, di- and trihydroxyoctadecenoic acids in beer and wort. J. Ins. Brew. 2000, 106, 107−110.

==References==