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==Proteins in the boil==
==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 LTP1 and Protein Z, due to their 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/>
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 LTP1 and Protein Z, due to their 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/>


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==Proteins during fermentation==
==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.
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.


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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.
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 (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>


==Proteins in beer==
==Proteins in beer==
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Three major protein components are present in beer:<ref name=jin/><ref name=steiner/><ref name=wu/>
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/>
# 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.<ref name=jin/><ref name=steiner/><ref name=han/>
# 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/>
# 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/>


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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.
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> 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 is believed to be mainly from LTP1 because it is rich in cysteine and is a major component of beer protein.<ref name=lund/><ref name=lundm/> 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/> 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/> As discussed above, thiols can serve as antioxidants during mashing. Not that the free thiol concentration diminishes as they are exposed to oxygen, removing the antioxidative ability of LTP1 and the other 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 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.
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> 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.
 
Reducing agents such as sulfite can re-activate oxidized thiols,<ref name=wu/><ref name=lundm> although adding sulfite at packaging is generally not something we recommend. See [[Packaging]].
 




Typically fresh beer contains many reduced protein thiols (PrSH), meaning that these proteins, in our vernacular, are in a reduced (redox) state. However they are lost as beer ages. Peroxide then appears on cue once these thiols are oxidized and no longer visible using specific staining techniques. The reduced redox state can be restored by sulfite, which is a reducing agent.<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>


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/>


LTP survives the brewhouse, retaining redox thiols that appear to be redox active in the packaged beer. These are also reactive and protective in tests designed to measure their effectiveness against inhibition of yeast growth in the presence of reactive oxygen species.<ref name=wu/>


Yeast thioredoxin (TRX) is another thiol-rich candidate ripe for consideration as a functional element of anti-ROS cascades. At one time we anticipated that TRX, which is secreted by yeast during fermentation, could be linked to beer stability. That was in fact the start of our move into beer proteomics. Yeast cells protect themselves against oxidative stress with interlinking processes that can transfer electrons within cells to where they are needed to quench reactive oxygen species. More contentiously, they can transfer reducing equivalents to outside the cell, where presumably they may carry out different or perhaps the same defence strategies. The trouble with this view, at least on face value, is that, outside the cell, the rest of the machinery for recycling oxidized TRX is just not present. Yet it might be possible — if yeast happens to secrete small thiols like cysteine as some mammalian cells do — this might be how LTP is reduced during fermentation after probably being oxidized in the wort and in the kettle. Taking together the data of beer proteomics and thiol proteins, we propose that there could be a thiol-based cycle operating in beer that involves oxidized thiols and reversible reduction after peroxide destruction using sulfite or some reductant molecules.<ref name=wu/>


Lastly, what of the relevance to beer makers? Well, barley breeders might have something to say about this and even now it seems that there are plans for cultivars that have higher levels of LTPs as well as thioredoxin. One way or another, by plant breeding and/or by the use of yeast genetically modified organisms, it may be possible to identify and amplify the levels of desirable proteins in beer. However, if we count in lifetimes, we doubt it. There may be other options, however. Hioe et al. (50) reported that hop cones, with an inordinate extra load of leaf material, in some cases showed improvements in beer stability and in the destruction of peroxide that forms in the brewhouse operations. Green hopped beer also showed remarkable stability, judged by the sustained appearance of protein thiols, as well as from sensory evaluation over years.<ref name=wu/>


LTP1 has a prominent role in maintaining redox balance of beer.<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> To further confirm LTP1's free radical scavenging activity in a physiological context, it was tested against six reactive oxygen species using a Saccharomyces cerevisiae-based assay. The antioxidant activity of LTP1 protected the yeast against the toxic effects of all six oxidants (Figure 5b). LTP1 was most effective against menadione, increasing yeast growth by 11-fold, and it counteracted all the six oxidants and increased yeast growth approximately 4 or 5-fold against H2O2, linoleic acid hydroperoxide (LAH), peroxynitrite and diamide. These findings demonstrated barley LTP1 has free radical scavenging and antioxidant capacity. In terms of brewing, LTP1’s activity against hydrogen peroxide and LAH is significant. H2O2 and LAH are thought to major ROS involved in flavor deterioration process. Elimination of these ROS abrogates the cause of oxidative process. Linoleic acid is found to be the most abundant lipid derived from malt and its oxidation by hydrogen peroxide or hydroxyl radical can lead to formation of LAH which can in turn trigger oxidative reactions, resulting in generation of precursors of the stale-tasting aldehydes.
<ref name=wumj/> To further confirm LTP1's free radical scavenging activity in a physiological context, it was tested against six reactive oxygen species using a Saccharomyces cerevisiae-based assay. The antioxidant activity of LTP1 protected the yeast against the toxic effects of all six oxidants (Figure 5b). LTP1 was most effective against menadione, increasing yeast growth by 11-fold, and it counteracted all the six oxidants and increased yeast growth approximately 4 or 5-fold against H2O2, linoleic acid hydroperoxide (LAH), peroxynitrite and diamide. These findings demonstrated barley LTP1 has free radical scavenging and antioxidant capacity. In terms of brewing, LTP1’s activity against hydrogen peroxide and LAH is significant. H2O2 and LAH are thought to major ROS involved in flavor deterioration process. Elimination of these ROS abrogates the cause of oxidative process. Linoleic acid is found to be the most abundant lipid derived from malt and its oxidation by hydrogen peroxide or hydroxyl radical can lead to formation of LAH which can in turn trigger oxidative reactions, resulting in generation of precursors of the stale-tasting aldehydes.


This high content of thiol cysteines in the protein is the basis for its radical scavenging and antioxidant activities. However, native barley LTP1 would not have antioxidant activity because all its thiol groups are occupied in the formation of disulfide bonds. The labeling of LTP1 thiols in beer demonstrated that the disulfide bonds in the native LTP1 were disrupted and linearised, most likely due to denaturing steps of malting, wort boiling and brewing. These free thiols were maintained during brewing and in packaged beer by a variety of factors. One of them could be the glycation of glycine and lysine residues with sugars such as glucose and xylose via the Maillard reaction [21]. The foam stabilising property of LTP1 has also been attributed to its glycosylation [19]. A possible working mechanism for its ROS-scavenging ability is proposed: LTP thiol(s) is oxidised to the sulfenic acid by oxidants such as H2O2, 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/> Beers with higher levels of free thiols taste better.
This high content of thiol cysteines in the protein is the basis for its radical scavenging and antioxidant activities. However, native barley LTP1 would not have antioxidant activity because all its thiol groups are occupied in the formation of disulfide bonds. The labeling of LTP1 thiols in beer demonstrated that the disulfide bonds in the native LTP1 were disrupted and linearised, most likely due to denaturing steps of malting, wort boiling and brewing. These free thiols were maintained during brewing and in packaged beer by a variety of factors. One of them could be the glycation of glycine and lysine residues with sugars such as glucose and xylose via the Maillard reaction [21]. The foam stabilising property of LTP1 has also been attributed to its glycosylation [19]. A possible working mechanism for its ROS-scavenging ability is proposed: LTP thiol(s) is oxidised to the sulfenic acid by oxidants such as H2O2, 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/> Beers with higher levels of free thiols taste better.
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