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Isomerization of Alpha Acids
===Acids===
'''Alpha acids''' (also called "humulones" and abbreviated as "α-acids") in hops mostly consist of humulone, cohumulone, and adhumulone. Trace amounts of other forms of humulones are also present but are difficult to quantify and currently have limited research: posthumulone, perhumulone, adprehumulone, and acetohumulone <ref name="Hao_2020">[https://www.tandfonline.com/doi/full/10.1080/03610470.2020.1712641 Junguang Hao, R.A. Speers, Heliang Fan, Yang Deng & Ziru Dai (2020) A Review of Cyclic and Oxidative Bitter Derivatives of Alpha, Iso-Alpha and Beta-Hop Acids, Journal of the American Society of Brewing Chemists, 78:2, 89-102, DOI: 10.1080/03610470.2020.1712641.]</ref><ref name="Leker_2022">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2022.2079944 Jeremy Leker & John Paul Maye (2022) Discovery of Acetohumulone and Acetolupulone a New Hop Alpha Acid and Beta Acid, Journal of the American Society of Brewing Chemists, DOI: 10.1080/03610470.2022.2079944 ]</ref>. The ratio of these individual acids to each other can vary based on hop variety much like total iso-α-acid percent, though generally the primary acids are humulone and cohumulone. Cohumulone has been identified by some researchers as a source of a more harsh bitterness, although similar research contradicts this statement <ref>[http://www.scielo.br/scielo.php?pid=S0100-40422000000100019&script=sci_arttext&tlng=es Fundamentals of beer and hop chemistry. Denis De Keukeleire. 1999.]</ref>. While Being hydrophobic, alpha acids are mostly insoluble in wort at typical brewing pH (alpha acids become much more soluble as the pH rises towards 5.9 to 7, which is not typical for wort production <ref name="Bastgen_2019">[https://www.tandfonline.com/doi/full/10.1080/03610470.2019.1587734 Influencing Factors on Hop Isomerization Beyond the Conventional Range. Nele Bastgen, Tobias Becher & Jean Titze. 2019. DOI: https://doi.org/10.1080/03610470.2019.1587734.]</ref>). During boiling, the alpha acids are isomerized into iso-alpha acids (also called isohumulones) which are formed during boiling that are soluble. Isomerization leads to roughly a 70%/30% split between diastereomeric isomers called ''cis'' and ''trans'' iso-α-acids respectively, with ''cis'' iso-α-acids being more stable over time and more bitter<ref name="Schönberger and Kostelecky, 2012"> [http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.2011.tb00471.x/abstract Schönberger and Kostelecky, 2012]</ref>. Alpha acids themselves do not taste bitter, but isomerized alpha acids (iso-α-acids/isohumulones) contribute to the bitterness of beer and have antimicrobial properties. Isocohumulone is often cited as being more harshly bitter than the other iso-α-acids, but studies of taste perception of individual iso-α-acids have not agreed with this. However, iso-cohumolone is slightly more soluble than the other acids and therefore a hop with a higher cohumulone composition may result in a beer with higher iso-α-acid for hops of equal iso-α-acid percent and use in brewing but different iso-α-acid breakdown<ref name="Schönberger and Kostelecky, 2012"/>. Alpha acids are susceptible to oxidation and the alpha acid content of a hop will decrease with storage.
There is evidence to show that during wort boiling iso-humulone and perhaps also iso-cohumulone bind with the head forming proteins, Lipid Transfer Protein (LTP) and Protein Z, to help form foam-positive structures in beer. These iso-alpha acids bind less so with LTP than they do with Protein Z. The resulting bound structures have been described as "vesicles", which are protein "bubbles" (but with no gas in them) with thick surface layers <ref>[https://www.sciencedirect.com/science/article/pii/S0268005X19325391 Vesicular structures formed from barley wort proteins and iso-humulone. Yi Lu, Peter Osmark, Björn Bergenståhl, Lars Nilsson. 2020.]</ref>. See also [https://www.youtube.com/watch?v=5F8vmuTV5Mg Escarpment Labs presentation on the science of beer foam].
'''Beta Acids''' (lupulones) are similar in structure to alpha acids and have the analogous individual beta acids (lupulone, colupulone, adlupulone, prelupulone, postlupulone, adprelupukone, and postlupulone acetolupulone <ref name="Dušek_2014">[http://pubs.acs.org/doi/abs/10.1021/jf501852r Qualitative Determination of β‑Acids and Their Transformation Products in Beer and Hop Using HR/AM-LC-MS/MS. Martin Dušek, Jana Olšovská, Karel Krofta, Marie Jurková, and Alexandr Mikyška. 2014.]</ref><ref name="Hao_2020" /><ref name="Leker_2022" />) to individual alpha acids. In their original form, beta acids do not contribute to the flavor of beer because they are not soluble in beer unless the pH of the boiling wort is significantly raised to around 7 pH (which is not typical in brewing conditions) and the original gravity is relatively low (2-8°P) <ref name="Bastgen_2019" />. They are also not able to isomerize during wort boiling. Beta acids do not become soluble in wort or beer unless they are chemically modified by a process such as oxidation <ref name="Algazzali_2014" />, nor are they soluble in beer when dry hopping <ref name="Maye_EBC2017">John Paul Maye. EBC 2017 Presentation. 2017.]</ref>. Oxidized beta acids are soluble and can contribute to bitterness in beer. Oxidized beta acids are discussed more under [[Hops#Acids_2|aged hops]].
====Isomerization of Alpha Acids====
The isomerization of alpha acids into iso-alpha acids is mostly dependent on alpha acid content of the hops, time (to a certain extent), temperature, original gravity, hop rate (hop weight), and IBU saturation. Other variables also affect isomerization to a lesser extent such as pH and calcium concentration <ref>[https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1964.tb06356.x CHANGES IN HOP ACIDS CONCENTRATIONS ON HEATING IN AQUEOUS SOLUTIONS AND UNHOPPED WORTS. H. O. Askew. 1964.]</ref><ref name="Malowicki_2005">[http://pubs.acs.org/doi/abs/10.1021/jf0481296 Isomerization and Degradation Kinetics of Hop (Humulus lupulus) Acids in a Model Wort-Boiling System. Mark G. Malowicki and Thomas H. Shellhammer. 2005.]</ref><ref name="justice_2018">[https://www.mbaa.com/publications/tq/tqPastIssues/2018/Pages/TQ-55-3-1205-01.aspx Tracking IBU Through the Brewing Process: The Quest for Consistency. Aaron Justus. Director of R&D and Specialty Brewing, Ballast Point Brewing. MBAA TQ 2018; vol. 55, no.3. https://doi.org/10.1094/TQ-55-3-1205-01.]</ref>. The higher the gravity of wort above 1.050 SG, the more proteins coagulate and drop iso-alpha acids out of solution (lower gravity worts are not affected by this). During fermentation, yeast cells can absorb iso-alpha acids, which results in further loss of iso-alpha acids in the finished beer <ref name="Bastgen_2019" />. Lower flocculating yeast strains tend to reduce the IBU in finished beer more than high flocculating yeast <ref name="justice_2018" />. Significant isomerization of alpha acids can occur in water without sugar at all (temperatures around boiling are still required), which is relevant in the production of [http://www.garshol.priv.no/blog/331.html "hop tea" in traditional farmhouse brewing] where hops are steeped in hot water for some time, and this is said to extract bitterness from the hops <ref name="Malowicki_2005" /><ref>[http://www.ijbbb.org/papers/161-E005.pdf Kinetic Modeling of Hop Acids during Wort Boiling. Yarong Huang, Johannes Tippmann, and Thomas Becker. 2013.]</ref><ref>[http://www.garshol.priv.no/blog/331.html Lars Marius Garshol. "Raw ale". Larsblog. 05/06/2015. Retrieved 12/17/2018.]</ref>. Aside from boiling hops in wort, [https://www.frontiersin.org/articles/10.3389/fnut.2022.843808/full Hydrodynamic Cavitation] at a 90°C temperature is another method that results in the isomerization of alpha acids into iso-alpha acids; although an additional 10 minutes of boiling at 100°C was needed to remove DMS and achieve hot break. This method reportedly had a 33% savings in energy costs in [https://www.sciencedirect.com/science/article/abs/pii/S0960308524000166 one study].
Malowicki and Shellhammer determined a calculation that predicts the isomerization rates of alpha acids into iso-alpha acids at different temperatures. Beginning at the boiling temperature of 100°C/212°F, which could be considered a rate of 100%, at 96°C/205°F the rate is 72%, and at 90°C/194°F the rate is 43%. This rate continues to drop significantly as the temperature of the wort decreases. At 82°C/180°F isomerization occurs at a rate of 17%. At a temperature of 50°C/122°F, the isomerization rate is at 1%, and finally 0% at 45°C/113°F(note that [https://www.tandfonline.com/doi/full/10.1080/03610470.2021.1878684 Kishimoto et al. (2021)] reported that isomerization begins somewhere between 60-70°C <ref name="Kishimoto_2021">[https://www.tandfonline.com/doi/full/10.1080/03610470.2021.1878684 Toru Kishimoto, Satoko Teramoto, Akiko Fujita & Osamu Yamada (2021) Evaluation of Components Contributing to the International Bitterness Unit of Wort and Beer, Journal of the American Society of Brewing Chemists, DOI: 10.1080/03610470.2021.1878684.]</ref>). This fact has several impacts on brewing processes. For example, when brewing at higher altitudes where the boiling point of wort is less than 100°C/212°F, the isomerization of alpha acids into iso-alpha acids will be reduced to whatever the rate is at that lower temperature. "Hop stands" or "whirlpool additions" where hops are left in contact with hot wort that is less than boiling temperature will continue to isomerize alpha acids <ref name="Malowicki_2005" /><ref>[https://www.mbaa.com/publications/tq/tqPastIssues/2017/Pages/TQ-54-3-0806-01.aspx A Look at Isomerization Reduction Due to Altitude. John Palmer. MBAA TQ 2017 http://dx.doi.org/10.1094/TQ-54-3-0806-01.]</ref>.
The hopping rate (weight) and IBU saturation have a drastic effect on IBU's. Aaron Justice reported a higher utilization when using Polaris hops (17.6% alpha acids) versus Tettnang hops (1.9% alpha acids). Two beers were brewed using enough of each of the hop varieties to target a calculated 40 IBU. However, the beer brewed with Polaris hops had 42.6 IBU and the beer brewed with Tettnang hops had only 28 IBU. It was hypothesized that the higher surface area of more hop matter reduces IBU's by binding to iso-alpha acids and possibly other compounds that register on the standardized IBU test. Justice also reported that more IBU pickup from whirlpooling was possible in beers without hops added in the boil, indicating that IBU saturation can limit IBU's, with a maximum IBU being around 100 <ref name="justice_2018" />.
The pH of the wort can also appears to have a small no significant effect on the isomerization of alpha acids to iso-alpha acids, although this variable is less significant than other variables such as alpha acid content, time (to a certain extent), temperature, and original gravity. Aaron Justice reported a trending slight rise in the conversion of alpha acids to iso-alpha acids when the boil pH was raised from 5.05 (~40% of aa's converted to iso-aa's) to 5.35 (~50% of aa's converted to iso-aa's). However, an Eppendorf BioSpectrometer using ASBC Wort-23 and ASBC Beer-23A was used to measure IBU'S, not isomerized alpha acids specifically (chromatography must be used to measure iso-alpha acids without measuring other bittering compounds) <ref name="justice_2018" />. Bastgen et al. (2019) found that at a boil pH of 5.6, the percentage of iso-alpha acids increased by 32% by extending the boil from 60 minutes to 120 minutes. However, there was no increase at all in iso-alpha acids when the boil pH was 7, but a pH of 7 is not typical in the brewing process <ref name="Bastgen_2019" />. Time is thought to play a large role in isomerization, however, Justice reported that the majority of the IBU from iso-alpha acids in 60 minute additions and in whirlpool additions occurs within the first 10 minutes, with only a 12-30% increase after another 50 minutes of boiling/whirlpooling (higher gravity beers had more isomerization during the final 50 minutes while lower gravity beers had less isomerization during the final 50 minutes of boiling) <ref name="justice_2018" />. Blogger and "garage scientist", John Paul Hosom, who developed the so-called "[https://alchemyoverlord.wordpress.com/2021/11/10/ibus-and-the-smph-model/ SMPH Model for estimating IBU's], hypothesized that non-iso-alpha acid bittering compounds that impact spectrophotometer IBU measurements such as oxidized alpha acids are affected by wort pH, rather than the isomerization rate of alpha acids into iso-alpha acids <ref>[https://alchemyoverlord.wordpress.com/2021/11/10/ibus-and-the-smph-model/ John Paul Hosom. "The Effect of pH on Utilization and IBUs". Alchemyoverlord blog. Retrieved 01/29/2023.]</ref>.
See also:
* [http://brulosophy.com/other-projects/hop-chronicles/ Brulosophy's "The Hop Chronicles", an attempt to characterize hop flavor and aroma.]
* [https://www.hopsteiner.com/blog/blending-hops-to-match-target-hop-profile/ Hopsteiner presentation on specific blends of hops that match the flavor profile of another hop.]
 
===International Bitterness Unit===
Beer bitterness is often described in terms of International Bitterness Units (IBU), or more accurately, Bitterness Units (BU). The European Brewery Convention has adopted the [https://europeanbreweryconvention.eu/new-international-method-on-bitter-compounds-in-dry-hopped-beers/ “E.B.C. Bitterness Units,”] determined in a similar way and recently updated for dry hopped beers to account for humulinones, as a uniform method that best expresses the true bitter flavor value of beer <ref name="asbc_ibu">[https://www.asbcnet.org/Methods/BeerMethods/Pages/default.aspx ASBC Methods of Analysis website. Retrieved 02/11/2022.]</ref>. These measurements seek to measure the amount of iso-alpha acids, which contribute the majority of bitterness to beer. There are, however, other compounds that contribute to bitterness, such as oxidized alpha and beta acids (see [[Hops#Chemistry_and_Characteristics|Aged Hops]] below). These methods include using [https://en.wikipedia.org/wiki/Spectrophotometry spectrophotometry], [https://en.wikipedia.org/wiki/High-performance_liquid_chromatography High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV)], and [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry liquid chromatography–mass spectroscopy (LC–MS)]. The ASBC describes these methods in depth on [https://www.asbcnet.org/Methods/BeerMethods/Pages/default.aspx their website] (see method 23; requires membership to read). While many brewers argue that the IBU measurement is not that helpful for communicating bitterness to consumers, it is generally agreed upon that IBU measurements are very useful to brewers who are seeking consistency in their products <ref>[https://beerandbrewing.com/dictionary/eej03p6ZUI/ The Oxford Companion to Beer definition of International Bitterness Units (IBUs). Retrieved 02/12/2022.]</ref>.
 
Compounds other than iso-alpha acids present several challenges to traditional methods of measuring BU's. Many hop compounds other than iso-alpha acids that have varying levels of perceived bitterness are detected at the same wavelength as iso-alpha acids using spectrophotometry. In addition, oxidized alpha acids are known to contribute to bitterness. As a result, updated methods of using High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) and liquid chromatography–mass spectroscopy (LC–MS) are generally recommended for measuring bitterness units in dry hopped beers (although models that account for bitterness contribution from compounds other than iso-alpha acids have not yet been completed). In addition to these challenges, iso-alpha acids and other hop compounds that contribute to bitterness degrade over time, thus the perceived bitterness of beer tends to become weaker as beer ages <ref>[https://www.chromatographyonline.com/view/liquid-chromatography-mass-spectrometry-analysis-of-hop-derived-humulone-and-isohumulone-constituents-in-beer-the-bitter-truth-of-hops-utilization-during-brewing Liquid Chromatography–Mass Spectrometry Analysis of Hop-Derived Humulone and Isohumulone Constituents in Beer: The Bitter Truth of Hops Utilization During Brewing. Bruce C. Hamper, Nicholas Viriyasiri, Aaron Boland, Lorna Espinosa, Hunter J. Campbell, Kurt Driesner, Michael McKeever. January 1, 2022. LCGC Europe, January 2022, Volume 35, Issue 01. Pages: 32–37.]</ref><ref name="asbc_ibu"/>.
 
Measuring BU's directly requires costly laboratory equipment, and so researchers and enthusiasts have made some progress in creating models that attempt to estimate bitterness units. These models are generally based off of boil time, alpha acid percent of the hops used, weight of the hops used, post-boil volume of wort, and gravity of the wort. These include the [http://www.realbeer.com/hops/research.html Tinseth] model, the Garetz model, and the [http://realbeer.com/hops/FAQ.html#units Rager] model. These models are often used in brewing software, such as [https://beersmith.com/blog/2021/09/23/hop-utilization-models-for-beer-brewing-compared/ BeerSmith™] and [https://www.brewersfriend.com/ibu-calculator/ Brewer's Friend]. [https://alchemyoverlord.wordpress.com/2021/11/10/ibus-and-the-smph-model/ Another model called the 'SMPH' model] has been proposed by John Paul Hosom as potential updated model that addresses their limitations in regard to newer hopping techniques such as whirlpool hopping and dry hopping, as well as accounting for IBU's from non-iso-alpha acid compounds Other limitations include differences in brewhouse size and efficiencies. For example, the Tinseth model was developed on a homebrew system using whole leaf hops (see the Experimental Brewing podcast interview with Glenn Tinseth link below).
 
See also:
* [http://scottjanish.com/dry-hopping-effect-bitterness-ibu-testing/ "Dry Hopping Effect on Bitterness and IBU Testing" by Scott Janish.]
* [https://www.experimentalbrew.com/podcast/episode-32-ibu-lie Experimental Brewing interview with Glenn Tinseth (52 minutes in).]
* [https://www.craftbeer.com/craft-beer-muses/beer-ibus-fact-fiction-misconceptions "Last Call for IBUs: Fact, Fiction and Their Impact on Your Beer," by Chris McClellan.]
* [http://thebrulab.libsyn.com/episode-076-a-modern-method-for-predicting-ibu-w-john-paul-hosom The Brü Lab Podcast Episode 076 | A Modern Method For Predicting IBU w/ John Paul Hosom]
==Antimicrobial Properties==
Hops are known to have antimicrobial properties against Gram-positive bacteria. This includes bacteria that can be present in beer both as spoilage organisms and as intentionally added in sour and mixed fermentation beer such as ''[[Lactobacillus]]'' and ''[[Pediococcus]]''. Certain other Gram-negative bacteria found in beer , such as ''Acetobacteraciae'' are Gram-negative , and are not susceptible to the antimicrobial properties of hops <ref name="Hough_1957">[https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1957.tb06267.x J. S. Hough, B.Sc, Ph.D., G. A. Howard, M.Sc., Ph.D., and C. A. Slater, Ph.D. 1957.]</ref><ref name="Macrae_1964">[https://onlinelibrary.wiley.com/doi/abs/10.1002/j.2050-0416.1964.tb02001.x SIGNIFICANCE OF THE USE OF HOPS IN REGARD TO THE BIOLOGICAL STABILITY OF BEER: I. REVIEW AND PRELIMINARY STUDIES. R. M. Macrae. 1964.]</ref>. Certain Gram-positive bacteria strains that have adapted to the brewing environment , such as some strains of ''Lactobacillus brevis'' and ''L. paracasei'' <ref>[https://proceedings.science/slacan-2023/papers/prospects-for-the-development-of-a-new-hopped-and-functional-sour-beer-survival?lang=en Marcos Edgar Herkenhoff; Susana Marta Isay Saad. PROSPECTS FOR THE DEVELOPMENT OF A NEW HOPPED AND FUNCTIONAL SOUR BEER: SURVIVAL OF PROBIOTIC STRAINS OF LACTICASEIBACILLUS PARACASEI SUBSP. PARACASEI IN HIGH HOPPED BEERS (HUMULUS LUPULUS L.).. In: CADERNO DE RESUMOS DO 15° SLACAN - SIMPóSIO LATINO AMERICANO DE CIêNCIA DE ALIMENTOS E NUTRIçãO, 2023, Campinas. Anais eletrônicos... Campinas, Galoá, 2023. Disponível em: <https://proceedings.science/slacan-2023/trabalhos/prospects-for-the-development-of-a-new-hopped-and-functional-sour-beer-survival?lang=en> Acesso em: 23 nov. 2023.]</ref>, are known to be more resistant to the antimicrobial effects of hops. The antimicrobial effect is characterized as inhibiting the growth and lactic acid production of lactic acid bacteria, however, this does not always also include cell death as ''Lactobacillus'' that has been inhibited by hops can later be revived <ref name="Macrae_1964" />. Multiple mechanisms The effectiveness of hops to inhibit Gram-positive bacteria is also dependent on pH; at a lower pH, hops have a greater effect on inhibiting bacteria <ref name="Almaguer_2015" />. Hop extracts have also been proposed demonstrated to be antimicrobial <ref>[https://www.sciencedirect.com/science/article/abs/pii/S0963996923003770 Yan Li, Sevim Dalabasmaz, Sabrina Gensberger-Reigl, Marie-Louise Heymich, Karel Krofta, Monika Pischetsrieder. Identification of colupulone and lupulone as the main contributors to explain why hops are antimicrobially activethe antibacterial activity of hop extracts using activity-guided fractionation and metabolome analysis. Food Research International. 2023.]</ref>.
Multiple mechanisms have been proposed to explain why hops are antimicrobially active. One mechanism of the antimicrobial activity of hops is due to the role of iso-alpha alpha acids and possibly similar hop acids (such beta acids and oxidized hop acids) as ionophores, or compounds which can transport ions across cell membranes. While their antimicrobial properties are strong, alpha and beta acids in beer and wort and their effects on brewing are generally disregarded because they do not solubilize <ref name="Fernandez and Simpson, 1993"> [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.1993.tb02782.x/full Fernandez and Simpson (1993)] </ref><ref name="Sakamoto and Konings, 2003"> [http://www.sciencedirect.com/science/article/pii/S0168160503001533 Sakamoto and Konings (2003)]</ref>. The protonated iso-α-acid (the form of the acid with an associated H+ ion, an H+ ion is a proton) is the antimicrobially active form. This means that for a beer with a given iso-α-acid concentration, the antimicrobial effects will be stronger at lower pH values because a greater percentage of the acid will be protonated. Protonated iso-α-acids act against bacteria by crossing into the cell and dissociating (releasing H+ ions from the iso-α-acid and decreasing the pH within the cell <ref name="zhao_1027">[https://www.frontiersin.org/articles/10.3389/fmicb.2017.00239/full#B28 Heterogeneity between and within Strains of Lactobacillus brevis Exposed to Beer Compounds. Yu Zhao, Susanne Knøchel and Henrik Siegumfeldt. 2017. DOI: https://doi.org/10.3389/fmicb.2017.00239.]</ref>), therefore disrupting the cellular proton gradient which is necessary for cells to function, before binding an equal charge in metal ions and crossing back out of the cell. Cells with resistance to hop bitter acids are better able to eject disassociated iso-α-acids from the cell and therefore preserve their proton gradients. The mechanism to expel iso-α-acids appears to be specific toward this type of compound rather than by a more general antimicrobial resistance mechanism such as multi-drug resistant bacteria possess <ref name="Sakamoto and Konings, 2003"/>. The anti-microbial power of iso-α-acids is pH dependent. At a higher pH (5.6) iso-α-acids begin to lose their anti-microbial properties, but at a typical beer pH (4.3) iso-α-acids inhibited a sample of 6 strains of ''L. brevis'' that exhibited a range of general hop tolerance in one study <ref name="zhao_1027" />. Hop resistant bacteria cultured in the absence of hop acids can lose their resistance if grown in an environment without antibacterial hop compounds<ref name="Fernandez and Simpson, 1993"/> and some hop resistant microbes need to be acclimated to hop acids by growth in sub-limiting levels of antibacterial acids before they are able to resist higher levels <ref name="Sakamoto and Konings, 2003"/>.
Another antimicrobial mechanism resulting from oxidative stress has been attributed to both iso-α-acids and humulinic acids <ref name="Schurr et al, 2015"> [http://www.sciencedirect.com/science/article/pii/S0740002014002470 Benjamin C. Schurr et al, Hannes Hahne, Bernhard Kuster, Jürgen Behr, Rudi F. Vogel.Molecular mechanisms behind the antimicrobial activity of hop iso-α-acids in Lactobacillus brevis. Food Microbiology, Volume 46, (2015), Pages 553-563, ISSN 0740-002., https://doi.org/10.1016/j.fm.2014.09.017.] </ref>. Humulinic acids are either not bitter tasting or much less bitter than iso-α-acids but are similar in structure to and are formed from the degradation of iso-α-acids as well as during the aging of hops <ref>[https://www.sciencedirect.com/science/article/abs/pii/S0040402001981992 The absolute configuration of the isohumulones and the humulinic acids. D.De Keukeleire, M.Verzele. 1971. https://doi.org/10.1016/S0040-4020(01)98199-2.]</ref>. Humulinic acids consist of the "cis-" and" trans-" forms of humulinic acid, cohumulinic acid and adhumulinic acid <ref>[https://www.sciencedirect.com/science/article/pii/B9781855734906500088 "8 - The chemistry of hop constituents," Editor(s): Dennis E. Briggs, Chris A. Boulton, Peter A. Brookes, Roger Stevens, In Woodhead Publishing Series in Food Science, Technology and Nutrition, Brewing. Woodhead Publishing, 2004. Pages 255-305. ISBN 9781855734906. https://doi.org/10.1533/9781855739062.255.]</ref>. This oxidative stress-driven antimicrobial activity is due to the potential for oxidation-reduction (redox) reactions within bacterial cells between Mn2+ ions and these specific hop acids. The redox potential is due to different conditions inside (higher pH, higher Mn2+) and outside (lower pH, lower Mn2+) of the bacterial cell <ref name="Behr and Vogel, 2010"> [http://aem.asm.org/content/76/1/142.short Behr and Vogel, (2010)] </ref><ref name="Schurr et al, 2015"/>. Iso-α-acids or humulinic acids passing into the cell, form complexes with Mn2+ and transfer electrons out of the cell <ref name="Behr and Vogel, 2010"/>. By targeted molecular modifications [http://www.sciencedirect.com/science/article/pii/S0740002014002470 Schurr et al. (2015)] determined that the Mn oxidative stress-driven antimicrobial effect of iso-α-acids was more important than the antimicrobial effect of the ionophore proton transfer discussed above in the overall antimicrobial activity of hops. Thus, the antimicrobial effects of humulinic acids have been found to be even stronger than iso-alpha acids, suggesting that aged hops retain at least some antimicrobial properties at least partially from humulinic acids <ref name="Schurr et al, 2015"/>. One study found that adding Mn2+ to a lager beer minimized this effect and slightly increased the cell count of 6 strains of ''L. brevis'' that had a range of hop tolerances <ref name="zhao_1027" />.
The oxidized forms of hop acids have been shown to have a limited inhibitory effect on Gram-positive bacteria. This might explain the anecdotal experiences of some brewers that have tried using aged hops that were high alpha varieties and produced beer that wasn't sour. Stevens et al. (1961) reported that a strain of ''Lactobacillus'' that was cultured from infected beer was inhibited by alpha acids at 40 ppm, beta acids at 10 ppm, iso-alpha acids at 160 ppm, and oxidized beta acids (cohulupone) at 200 ppm. So, while the oxidized beta acids had the least inhibitory power, a high concentration was still inhibitory <ref name="Stevens_1961">[https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1961.tb01830.x R. Stevens, Ph.D., F.R.I.C., and D. Wright, Ph.D. 1961.]</ref>. Oxidized alpha acids (humulinones) have only been tested for antibacterial properties at a concentration of 50 ppm or less. At 50 ppm, oxidized alpha acids were not able to inhibit two strains of ''Lactobacillus'' that were isolated from infected beer, as reported by Hough et al. (1957) <ref name="Hough_1957" />. See [[Hops#Acids_2|oxidized hop acids]] for more information on oxidized hop acids.
==Hop Derived Compounds In Beer and Biotransformations==
[[File:Svedlund 2022.jpg|thumb|400px|An overview of the biotransformation reactions occurring in certain yeast with the required genetic/enzymatic capability. Abbreviations: 3MH 3-mercaptohexanol, 3MHA 3-mercaptohexyl acetate, Cys cysteine, GSH glutathione, TPA terpene alcohol. Credit: Henrik Svedlund.
 
Source: [https://link.springer.com/article/10.1007/s00253-022-12068-w Svedlund, N., Evering, S., Gibson, B. et al. Fruits of their labour: biotransformation reactions of yeasts during brewery fermentation. Appl Microbiol Biotechnol 106, 4929–4944 (2022). https://doi.org/10.1007/s00253-022-12068-w]]]
 
The flavor and aroma compounds found in leaf/pellet hops are different than the hop-derived flavor and aroma compounds found in finished beer (other than in the case of dry hopping). The brewing process (particularly boiling), and fermentation greatly affect the composition of flavor and aroma compounds that are found in beer. For example, boiling wort and hops isomerizes non-bitter alpha acids into bitter iso-alpha acids. During the boiling of the wort, many compounds found in hops are evaporated, such as many of the various sulfur compounds found in hops. The terpene hydrocarbons which make up most of the hop oil content in hops (myrecene, humulene, and caryophyllene) are completely removed by fermentation. It is believed that these terpene hydrocarbons stick to the yeast cells and fall out of solution during fermentation <ref name="Praet_2012">[http://www.sciencedirect.com/science/article/pii/S1373716311001636 Biotransformations of hop-derived aroma compounds by Saccharomyces cerevisiae upon fermentation. Tatiana Praet, Filip Van Opstaele, Barbara Jaskula-Goiris, Guido Aerts, Luc De Cooman. 2012.]</ref>.
A "biotransformation" is any change in a chemical's structure that is initiated by a living organism <ref>[https://en.m.wikipedia.org/wiki/Biotransformation "Biotransformation". Wikipedia. Retrieved 05/10/2019.]</ref>. It has been hypothesized that biotransformations of some kind are taking place in beer during fermentation and explain changes to hop compounds during fermentation and beer storage. Some carbonyl compounds found in hops (citral, geranial, nerol, [https://en.wikipedia.org/wiki/Citronellal citronellal], and methyl ketones) can be used as a food source by yeast during fermentation. ''Cyclic ethers'' such as linalool oxides, karahana ether, hop ether, and rose oxide (aroma of roses <ref>[http://www.thegoodscentscompany.com/data/rw1035651.html "(Z)-rose oxide ". Good Scents Company. Retrieved 12/29/2016.]</ref>), increase after fermentation and have been identified as secondary metabolites produced by yeast during metabolism from hop derived precursors. ''Esters'' found in hops can be converted into ethyl esters by yeast during fermentation; for example, geranyl esters found in Cascade hops can be hydrolyzed into geraniol (flowery). The terpenoid [https://en.wikipedia.org/wiki/Citronellol citronellol] (citrus and floral <ref>[https://eic.rsc.org/magnificent-molecules/citronellol/2000020.article "There are no flies on Emma Stoye". Emma Stoye. Education in Chemistry website. 06/01/2016. Retrieved 01/10/2017.]</ref>) can be esterified by yeast fermentation into citronellyl acetate (fresh, rosy, fruity odor reminiscent of geranium oil <ref>[https://shop.perfumersapprentice.com/p-6034-citronellyl-acetate.aspx "Citronellyl acetate". Perfumers Apprentice website. Retrieved 01/10/2017.]</ref>). Yeast strains differ in their ability to convert these compounds. For example, one study found that lager yeast was able to form acetate esters of geraniol and citronellol, but ale yeast was not <ref name="Praet_2012" />.  In addition to biochemical changes to hop compounds, yeast derived flavor compounds that are independent of hops such as esters and phenols can affect the overall sensory characteristics of hop compounds in beer. Therefore, different strains of yeast can greatly impact the overall organoleptic experience of hop flavors and aromas in finished beers <ref>[https://www.mdpi.com/2304-8158/12/5/1064 Kumar, A.; Warburton, A.; Silcock, P.; Bremer, P.J.; Eyres, G.T. Yeast Strain Influences the Hop-Derived Sensory Properties and Volatile Composition of Beer. Foods 2023, 12, 1064. https://doi.org/10.3390/foods12051064.]</ref>.  ===Terpenes===
Terpenes and terpenoids (monoterpene alcohols) can also be transformed by fermentation. Studies have found that geraniol and nerol can transform into linalool by a strain of ''S. cerevisiae'', as well as nerol and linalool into alpha-terpineol, which can then be further transformed to terpin. Geraniol can also be converted into citronellol, and the content of geraniol and citronellol can be increased in finished beer by increasing the initial content of geraniol, which is found in higher quantities in some varieties of hops (Citra, for example). Linalool, nerol, and alpha-terpineol gradually decrease during fermentation and aging (perhaps being transformed into [https://en.wikipedia.org/wiki/Ether ethers], which is a class of organic compound that contains an oxygen atom connected to two alkyl or aryl groups), while nerol and citronellol gradually increase. Geraniol also decreases during fermentation, but not as drastically as linalool. It has been hypothesized that the bioconversion of geraniol into citronellol could be by means of glycosidic activity (although evidence for glycosidic activity in ''S. cerevisiae'' in regards to hop derived compounds is very weak; see [[Hops#Glycosides|Hop Glycosides]]). Post-fermentation dry hopping preserves linalool and alpha-terpineol, and limits citronellol to trace levels <ref name="Praet_2012" />.
[https://onlinelibrary.wiley.com/doi/abs/10.1002/j.2050-0416.2010.tb00428.x Takoi et al. (2012)] used Citra hops with a high content of geraniol added late in the boil, and reported a steep decline on geraniol during the first three days of fermentation with a lager yeast strain. Linalool had a gradual decline but ended up at higher levels than geraniol in the finished beer. Citronellol had a sharp increase during the first three days of fermentation and then remained at a stable level until the end of fermentation. However, after storing the beer at 15°C (59°F) for 1 week, the amount of citronellol more than doubled. This indicated that active fermentation may not be required for the transformation of geraniol into citronellol (the yeast was filtered before packaging the finished beer, after a storage time of 6-8 days at 13–15°C and then at 0°C for 2–3 weeks). The enzyme NADPH dehydrogenase 2, encoded by the OYE<sub>2</sub> gene in some yeast strains, was proposed as the mechanism for this transformation. Interestingly, Takoi et al. (2012) also showed that coriander seeds, which also have high levels of linalool and geraniol, have a nearly exact same effect on beer, with a beer made with 0.5 g/L of coriander seed resulting in 20 ppb of citronellol and a beer made with 0.75 g/L of coriander seed resulting in 30 ppb of citronellol. The Citra beer had a citrus and "green" aroma, while the coriander beers had a very floral aroma with a slight citrus impression. They also conducted a sensory experiment with different levels of geraniol and citronellol added to linalool to see if small amounts of these would affect the flavor of a large dosage of linalool, and the results confirmed that small increases of geraniol and citronellol increased flowery and fruity flavors even in the presence of high dosages of linalool <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1002/j.2050-0416.2010.tb00428.x The Contribution of Geraniol Metabolism to the Citrus Flavour of Beer: Synergy of Geraniol and β‐Citronellol Under Coexistence with Excess Linalool. Kiyoshi Takoi, Yutaka Itoga, Koichiro Koie, Takayuki Kosugi, Masayuki Shimase, Yuta Katayama, Yasuyuki Nakayama, Junji Watari. 2012. DOI: https://doi.org/10.1002/j.2050-0416.2010.tb00428.x.]</ref>. The data for the Citra beer is shown below:
[[File:Biotransformation Takoi 2012.png|[https://onlinelibrary.wiley.com/doi/abs/10.1002/j.2050-0416.2010.tb00428.x Takoi et al. 2012]]]
Other yeast species can also convert monoterpenes. For example, a strain of ''Kluyveromyces lactis'' was found to reduce geraniol to citronellol. This strain and a strain of ''Torulaspora delbrueckii'' produced linalool from both geraniol and nerol, and could also form geraniol from nerol <ref>[https://www.ncbi.nlm.nih.gov/pubmed/10790686 Biotransformation of monoterpene alcohols by Saccharomyces cerevisiae, Torulaspora delbrueckii and Kluyveromyces lactis. King A1, Richard Dickinson J. 2000.]</ref>. Many species of ''Debaryomyces'', ''Kluyveromyces'', and ''Pichia'' were found to transform geraniol into linalool, and nerol into linalool and alpha-terpineol <ref>[https://www.ncbi.nlm.nih.gov/pubmed/18357555 Biotransformation of acyclic monoterpenoids by Debaryomyces sp., Kluyveromyces sp., and Pichia sp. strains of environmental origin. Ponzoni C, Gasparetti C, Goretti M, Turchetti B, Pagnoni UM, Cramarossa MR, Forti L, Buzzini P. 2008.]</ref>. Colomer et al. (2020) measured the monoterpenes in two experiments before and after inoculating with different strains of ''Brettanomyces'' that had varying degrees of beta-glucosidase activity. They found that the strains with the least beta-glucosidase activity had the least impact on biotransformation, but the increase in beta-citronellol was higher than what has been reported in biotransformation studies with ''Saccharomyces'' <ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.610 Biotransformation of hop derived compounds by Brettanomyces yeast strains. Marc Serra Colomer, Birgitte Funch, Natalia Solodovnikova, Timothy John Hobley, Jochen Förster. 2020. DOI: https://doi.org/10.1002/jib.610.]</ref>. See [[Brettanomyces#Hop_Biotransformation|''Brettanomyces'' hop biotransformation]] for more information.
 
Sulfur-based compounds known as ''thiols'' have also been shown to be produced by yeast fermentation from hop derived precursors (suspected to be S-glutathione). So far, science has found that these include the volatile thiols 3-sulfanyl-4-methylpentan-1-ol (3S4MP; grapefruit) and 3-sulfanyl-4-methylpentyl acetate (3S4MPA; passionfruit, grapefruit). These thiols were found in beers dry hopped separately with Amarillo, Hallertau Blanc, and Mosaic hop varieties. The amounts of these two thiols were higher than expected based on the content of these thiols in the hops alone <ref name="Cibaka_2016" />. See also this [https://www.facebook.com/groups/MilkTheFunk/permalink/1373899592638251/ MTF thread speculating on how ''Brettanomyces'' might produce thiols].
In general, different yeast strains have a large impact on how hops are perceived in the final beer, including both perceived bitterness and flavors. For example, POF+ (phenolic positive) strains of ''[[Saccharomyces|Saccharomyces cerevisiae]]'' tends to mask the hop-derived aromas in dry hopped beers <ref name="Sharp_Presentation" />. A beer hopped with the Tradition hop variety produced fruit flavors when fermented with Abbaye ale yeast, and woody/spicy flavors when fermented with US-05. When the beer was brewed with Citra hops, with US-05 the beer had sweet fruits/citrus flavors and more bitterness, but when fermented with the Abbaye ale strain the beer had a more one dimensional sweet fruit/floral flavor and less bitterness <ref>"Influence of yeast strain on hop aroma development in dry hopped beers." Christina Schönberger, Elisabeth Wiesen, Benedikt Matsche, Barth Innovations Yves Gosselin, Stephan Meulemans, Fermentis. Presentation slides at 35th Congress EBC.</ref>.
See also:
:<youtube height="200" width="300">GCQ22HSDDUQ</youtube> <youtube height="200" width="300">r09eb46k97I</youtube>
* [https://www.crowdcast.io/c/itcl8mhhsdsa Escarpment Labs presentation by Richard Preiss on biotransformation.]
* [https://www.masterbrewerspodcast.com/282 MBAA Podcast #282: New Belgium's tests with mid-fermentation dry hopping.]
====Glycosides====Hops contain In addition to terpenes and monoterpene alcohols being found in hops in a free form, they are also present in the form of glycosides, which are flavor compounds that are bound to a sugar molecule. Plants use glycosides for a range of metabolic purposes. The amounts and types of glycosides in hops varies by hop variety, and can be affected by plant fertilization and harvest timing <ref name="Svedlund_2022">[https://link.springer.com/article/10.1007/s00253-022-12068-w Svedlund, N., Evering, S., Gibson, B. et al. Fruits of their labour: biotransformation reactions of yeasts during brewery fermentation. Appl Microbiol Biotechnol 106, 4929–4944 (2022). https://doi.org/10.1007/s00253-022-120.]</ref>. In their bound form, glycosides are flavorless. Studies on hop compounds elude to the possibility of compounds being produced by the glycosidic activity of ''S. cerevisiae'', however direct evidence of glucosidic activity in ''S. cerevisiae'' is lacking. Daenen (2008) reviewed the glycosidic activity of many strains of ''S. cerevisiae'', and found that only a few strains expressed any real glucosidic activity and none that exhibited exo-beta-glucosidase which would be required to break glycosidic bonds in the beer/wort. Daenen did find that enzymatic activity from some strains of ''Brettanomyces'' can efficiently release these bound compounds and release their flavor and aromatic potential <ref name="Praet_2012" />. Beta-glucosidase enzyme can also be added to beer to enhance the breakdown of glycosides and intensify hop-derived flavors and aromas. For example, one study showed an increase in citrus, orange, grapefruit, and tropical pineapple in a Cascade dry hopped beer that had beta-glucosidase enzymes added to it <ref>"Optimizing hop aroma in beer dry hopped with Cascade utilizing glycosidic enzymes (presentation slides)." Kaylyn Kirkpatrick from New Belgium Brewing Co. Young Scientist Symposium, Chico, CA 2016.</ref>. There is also some evidence to support that there is higher glucosidase activity in seeded hops, which are generally not used in the brewing industry <ref>"Seeded and "Unseeded Hops - a Quality Comparison (presentation slides)." Martin Zarnkow. EBC 2015.</ref>. Hops also contain polyhenols that are bound in glycosidic form that could contribute a small amount of bitterness <ref>[https://www.tandfonline.com/doi/abs/10.1080/03610470.2021.2024112?journalCode=ujbc20 Martin Biendl, Stefanie Ritter & Christina Schmidt (2022) Monitoring of Glycosidically Bound Polyphenols in Hops and Hop Products Using LC-MS/MS Technique, Journal of the American Society of Brewing Chemists, DOI: 10.1080/03610470.2021.2024112.]</ref> (see also [https://traffic.libsyn.com/secure/forcedn/thebrulab/056_Applying_the_Science_-_High_Hop_Loads_w_Jordan_Folks.mp3 Bru Lab Podcast Episode 055; Hop Bitterness And Polyphenols w/ Dr. Martin Biendl]).
The effects of beta-glucosidase on hops may be limited even in ideal conditions using pure beta-glucosidase that is highly efficient in beer where beta-glucosidase activity by yeast is limited. Sharp et al. (2017) determined that hops contain such a small amount of glycosides that their release doesn't contribute much to hop flavor and aroma. While previous studies focused on hop extracts, they studied glycosides in whole leaf hops for the first time and found non-significant levels of hop-derived monoterpenes from glycosides even when using pure beta-glucosidase extracted from almonds. For example, linalool was increased by 16.5 μg/L when using the highest hopping rate, but this amount has little impact on the overall aroma and flavor of the beer. The terpenes citronellol, terpineol, nerol, and geraniol were also not increased in significant amounts, however, the fatty alcohol 1-octanol (waxy, green, citrus, orange, aldehydic, fruity <ref>[http://www.thegoodscentscompany.com/data/rw1021071.html Octanol. The Good Scents Company. Retrieved 03/31/2017.]</ref>) was increased significantly <ref name="Sharp_2017">[http://onlinelibrary.wiley.com/doi/10.1002/jib.418/abstract The effect of hopping regime, cultivar and β-glucosidase activity on monoterpene alcohol concentrations in wort and beer. Daniel C. Sharp, Jan Steensels, Thomas H. Shellhammer. 2017. DOI: 10.1021/jf2042517.]</ref>. The alcohol octanol can be esterified into octyl acetate, which is a classically "citrusy" aroma, so perhaps certain yeasts can create this ester during mid-fermentation hopping <ref>[https://pubs.acs.org/doi/abs/10.1021/jf2042517 Eric G. Dennis, Robert A. Keyzers, Curtis M. Kalua, Suzanne M. Maffei, Emily L. Nicholson, and Paul K. Boss. 2012.]</ref>.
See [[Glycosides]] for more information on glycosides.
 
===Thiols===
Sulfur-based compounds bound to a hydrogen atom known as ''thiols'' have also been shown to be produced by yeast fermentation from the hop derived precursors cysteine or glutathione via yeast β-lyase activity. This β-lyase activity is expressed by strains of ''Saccharomyces cerevisiae'' that have the ''IRC7'' gene. This activity takes place within the yeast cells which uptake the bound thiol precursors and then expel the free thiols. Most industrial brewing and wine strains of ''S. cerevisiae'' do not have a functional version of this gene due to a lack of selective pressure. Even in strains with a functional version of the ''IRC7'' gene, the expression of the gene is repressed in nitrogen rich substrates such as wort <ref>[https://www.masterbrewerspodcast.com/227 Dr. Laura Burns and Lance Shaner. MBAA Podcast Episode 227 Interview. 10/18/2021. Retrieved 02/10/2023.]</ref>(~4 mins in). The cysteine and glutathione precursors are also found in malt, hops, certain varieties of wine grapes (Sauvignon blanc, Gewürztraminer, Semillon, Chardonnay and Riesling grapes), and rice, with levels varying greatly depending on varieties of the plants. In addition to some strains of ''Saccharomyces cerevisiae'' that express the genes to produce thiols, strains of the yeasts ''Metschnikowia pulcherrima'', ''Torulaspora delbrueckii'', ''Lachancea thermotolerans'', ''Candida zemplinina'', and the bacteria ''Lactobacillus plantarum'' have also been found to produce thiols <ref name="Svedlund_2022" />. So far, science has found that these include the volatile thiols:
* Grape and hop based thiols:
** 3-mercaptohexan-1-ol (3MH; also referred to as 3-sulfanylhexan-1-ol, 3SH; ''tropical fruit'')
** 4-mercapto-4-methylpentan-2-one (4MMP; also referred to as 4-methyl-4-sulfanylpentan-2-one, 4MSP; ''blackcurrant'')
* Hop based thiols (Nelson Sauvin hops):
** 3-sulfanyl-4-methylpentan-1-ol (3S4MP; ''grapefruit'')
** 3-mercaptopentanol (3MP; also referred to as 3-sulfanylpentan-1-ol, 3SP)
Acetylation (acetate) variations:
* 3-sulfanyl-4-methylpentyl acetate (3S4MPA; ''passionfruit'', ''grapefruit'')
* 3-mercaptohexyl acetate (3MHA; ''passionfruit'')
Typically, in beer and wine, the amount of free thiols that are formed from these precursors is less than 1%, perhaps due to poor activity of β-lyase activity in acidic media and inhibition by polyphenols. However, these thiols were found in beers dry hopped separately with Amarillo, Hallertau Blanc, and Mosaic hop varieties. The amounts of these two thiols were higher than expected based on the content of these thiols in the hops alone <ref name="Cibaka_2016" />. Dry hopping temperature plays a role, with with 18-24°C being optimum for 3Mh and 3MP, and 28°C being optimum for 3S4MP. Mash hopping can potentially increase thiols, while using copper in the brewing or winemaking process can reduce them <ref name="Svedlund_2022" />. In order to get around the nitrogen caused suppression of the IRC7 gene in brewing yeast, [[Omega Yeast Labs]] has bioengineered a yeast strain called [https://omegayeast.com/news/cosmic-punch-new-thiol-boosting-strain Cosmic Punch™ (British V OYL-011)] to produce significant amounts of thiols from hops and malted grains. [https://berkeleyyeast.com/available-yeast-strains/ Berkeley Yeast] also offers bioengineered yeast strains that produce thiols. See also this [https://www.facebook.com/groups/MilkTheFunk/permalink/1373899592638251/ MTF thread speculating on how ''Brettanomyces'' might produce thiols].
 
See also:
* [https://topcrop.co/collections/thiols "Thiols" articles on Top Crop from Omega Labs.]
* [http://scottjanish.com/the-locksmith-utilizing-bioengineered-yeast-and-high-bound-thiol-precersour-hops-and-phantasm-powder-to-thiol-drive-beer/ "The Locksmith: Utilizing Bioengineered Yeast and High Bound Thiol Precersour Hops and Phantasm Powder to Thiol Drive Beer," by Scott Janish.]
* [http://scottjanish.com/thiol-driver/ "Thiol-Driver," by Scott Janish.]
* [https://www.charlesfaram.co.uk/news/technical-article-introduction-to-thiols-in-hop-oils/ Charles Faram Blog; Introduction to Thiols in Hop Oils.]
* [https://www.masterbrewerspodcast.com/227 IRC7 + CRISPR = Cosmic Punch - MBAA podcast with Dr. Laura Burns and Lance Shaner from Omega Yeast.]
* [https://www.falsebottomedgirls.com/podcast/episode/212472ad/thiols-with-laura-burns-of-omega-yeast False Bottom Girls podcast interview with Dr. Laura Burns from Omega Yeast.]
* [https://www.masterbrewerspodcast.com/268 MBAA Podcast Episode 268 Free Thiol Release with Cécile Chenot.]
* [https://www.themadfermentationist.com/2023/05/are-thiols-scam-thiolized-yeast.html Practical advice from Michael Tonsmeire on The Mad Fermentationist blog.]
* [https://beerandbrewing.com/podcast-episode-306-ron-beatson-brand-ambassador-nz-hops/ Craft Beer & Brewing Magazine interview with New Zealand hop breeder Ron Beatson.]
===Lightstruck===
In [[lambic]] brewing, the term '''aged hops''' refers to hops (usually Noble varieties such as Tettnang, Saaz, Target, and Hallertau) which have been aged for 3-5 years in non-refrigerated conditions, and in burlap sacks or some other oxygen permeable bag <ref>[http://www.horscategoriebrewing.com/2016/04/hops-in-spontaneous-fermentation.html Dave Janssen. "Hops in spontaneous fermentation". Hors Catégorie Brewing blog. 04/28/2016. Retrieved 04/09/2018.]</ref><ref>[http://jesterkingbrewery.com/home-for-our-aged-hops "Home for Our Aged Hops". Jester King's blog. Retrieved 11/18/2016.]</ref>. It should be noted that the term "aged hops" can also refer to any sort of hop aging (especially in scientific literature), including short-term hop aging (1-6 months, for example) at refrigerated or non-refrigerated temperatures, and in oxygen-rich or vacuum sealed packaging. Much of the information below references hops that have been aged in warm conditions for shorter time periods than what hops are aged for in lambic brewing. The additional aging of hops that are used in lambic brewing or similar beers might have different effects than what has been studied in hops that are aged for shorter periods of time.
For techniques and usage amounts of aged hops, see [[Hops#Aged_Hops_in_LambicAged_Hops_in_Lambic_and_Other_Spontaneous_Fermentation_Beer|Aged Hops in Lambic]].
===Aging Hops===
Typically, only low alpha acid hops are used (high alpha acid hops may lead to more hop character and higher inhibition of lactic acid bacteria than desired possibly due to oxidized acids). The hops are typically bound in burlap sacks/paper bags or something similar that allows for exposure to oxygen, and then they are left to age in preferably low humidity conditions at room temperatures to warm temperatures (warmer temperatures will encourage faster aging). Changes in the environment such as temperature shifts are not a concern; for example [https://web.archive.org/web/20180204131402/http://jesterkingbrewery.com/home-for-our-aged-hops Jester King Brewery in Austin Texas ages hops in a horse barn]. The hops should are traditionally aged for 2+ years. Monitor for mold growth during this time, and discard any hops that show visible signs of mold growth. Some brewers prefer to age the hops until the cheesy character ([[Isovaleric Acid]]) is gone, while other brewers do not mind the presence of this cheesy character (for example, some [[Lambic|lambic]] beers display isovaleric acid character even after packaging). While hop leaves are generally preferred over pelletized hops, if aging pellets, it has been advised to break the pellets up so that the entirety of the hop material is exposed to the air.
Freshly harvested hops (also called "wet hops") should not be aged. Freshly harvested hops should be dried first, as is normal for hop processing, before aging in order to prevent [[Mold|mold]] growth (see this [https://www.canr.msu.edu/news/drying_hops_on_a_small_scale article from Michigan State University] on measuring moisture levels and [https://www.homebrewersassociation.org/how-to-brew/how-to-harvest-prepare-and-store-homegrown-hops/ this AHA article on drying hops] for home growersand this [https://www.masterbrewerspodcast.com/117 MBAA podcast interview with Val Peacock on industrial hop drying]).
See also:
===Chemistry and Characteristics===
====Acids====During agingand if exposed to oxygen, both alpha and beta acids compounds in hops oxidize and degrade with warmer temperatures and more oxygen exposure having a greater impactinto different compounds known collectively as "oxidative polar compounds" or OPC's. Hao et al. The generally accepted theory is that oxygen interacts directly (2020) reported 39 different OPC's in aged hops, with hop 15 of them derived from alpha acids. This event is called "autooxidation". An alternative theory to this is that oxygen indirectly oxidizes , 15 derived from isomerized alpha acids by first oxidizing the hop oils , and turning them into pro9 from beta-oxidants, which then oxidize the hop acids which are mixed in with the oils within the lupulin glands <ref name="Algazzali_2014Hao_2020" />. The Many of these oxidation of hop acids corresponds with an increase derived compounds are considered "hard resins" (meaning they are soluable in the [http://methods.asbcnet.org/summaries/hops-12.aspx Hop Storage Index (HSIalcohol)], which is a practical way of measuring the overall freshness of hopsand contribute bitterness and antimicrobial properties to beer. As the oxidation Examples of hop oils rises"soft resins", the measured HSI number on a lot of hops increases <ref name="Lam et al.which are not soluable in alcohol, 1986"/>are alpha and beta acids <ref name="Maye_2016Almaguer_2015">[httphttps://wwwonlinelibrary.hopsteinerwiley.com/wp-contentdoi/uploadsfull/201610.1002/07/TQ-53-1-0227-01jib.160 Almaguer, C.pdf Humulinone Formation in Hops and Hop Pellets and Its Implications for Dry Hopped Beers, Schönberger, C. John Paul Maye, Robert SmithGastl, and Jeremy LekerM. 2016, Arendt, E.]</ref>K. These oxidized compounds lead to a higher amount of non-alpha-acid bitterness compounds in aged hops and have Becker, T. (2014), Humulus lupulus – a remarkable effect on the bitterness of the beerstory that begs to be told. The bitterness from oxidized hop compounds has been described as more earthyA review, harsh, and astringent than the sharperJ. Inst. Brew., cleaner bitterness from iso-alpha acids <ref name="Mikyška_2012">[http120://onlinelibrary289– 314.wiley.com/doi/DOI: 10.1002/jib.40/abstract Assessment of changes in hop resins and polyphenols during long-term storage. Alexandr Mikyška and Karel Krofta. 2012160.]</ref>.
Aging hops while exposed to oxygen develops a cheesy aroma due to [[Isovaleric Acid|isovaleric acid]], isobutyric acid, and 2-methylbutyric acid. These acids are produced by the oxidative cleavage ====Summary of acyl side chains of the hop resins <ref nameOxidized Hop Acids==="Briggs_2004" />. These cheesy oxidation compounds can be esterified to form wine-like and fruity tasting compounds (see [[Hops#Esters|Esters]] below and [[Aging_and_Storage#Acids_and_Esters|Aging and Storage]]) <ref name="Shellhammer, Vollmer and Sharp, CBC 2015"/>.
Storage conditions {| class="wikitable sortable" style="width:75%;"|-| colspan="5" align="center" style="padding: 2em;" | '''Alpha Acid Derived Compounds <ref name="Hao_2020" />'''|- ! Oxidized Compound ! Precursor ! Beer Soluble (Y/N/?) ! Bitterness Level ! Notes|-| Humulinones (cohumulinone, n-humulinone, and adhumulinone) || Alpha acids (cohumulone, humulone, and variety play a large role adhumulone) || Y || 66% as bitter as iso-alpha-acids || In hops, alpha-acids are degraded to humulinones in the presence of air, with higher temperatures leading to more rapid conversion. |-| tricyclooxyisohumulones A and B, deisopropyltricycloisohumulone, and tricycloperoxyisohumulone A || Alpha acids || ? || ? || Along with humulinones and 4'-hydroxy-allohumulinones, they are considered aging indicators of of hop oxidation.|-| Abeo-isohumulone || Humulinone || ? || Not reported; might contribute to bitterness of beers with high amounts of aged hops. || Derived from the oxidation of humulinone in how acid content hops. Found in naturally aged Saaz and Nugget hops changes over timea long period of storage. |-| 4'-hydroxy-allohumulinones || Humulinones || ? || Not reported; might contribute to bitterness of beers with high amounts of aged hops. || Believed to be derived from the oxidation of humulinone in hops. Beta acids They are generally more resistant to oxidation stable than alpha acids. A study by Mikyška humulinone over time and Krofta (2012) found that after 12 months are thus thought to be useful indicators of the degree of storage at 20°C oxidation in open air, pellet hops lost 64.|-| 4'-88% of their alpha acid content Hydroxyallo-cis-humulinones and 51cis-oxyhumulinic acids || 4'-hydroxy-allohumulinones || Y || Contributes to bitterness quality || These are formed during boiling where 4'-83% hydroxy-allohumulinones are isomerized into 4'-Hydroxyallo-cis-humulinones for the first 60 minutes of the beta acid contentboiling, with and then the beta 4'-Hydroxyallo-cis-humulinones are slowly degraded into cis-oxyhumulinic acids dropping off more significantly after 6 months (alpha acid content declined steadily throughout 60 minutes of boiling. After 120 minutes of boiling, 40-50% of the aging period)4'-hydroxy-allohumulinones is transformed into 4'-Hydroxyallo-cis-humulinones and cis-oxyhumulinic acids. These amounts varied with different Czech hop varieties (Saazare thought to effect the bitterness quality of beer, and as hard resins they could help improve head retention <ref>[https://www.researchgate.net/publication/286063120_Contributions_of_hop_hard_resins_to_beer_quality Almaguer, Cynthia & Gastl, SládekMartina & Arendt, PremiantElke & Becker, Th. (2012). Contributions of hop hard resins to beer quality. BrewingScience. 65. 118-129.]</ref>.|-| Scorpiohumulinols A/B and Agnusdicyclohumulinols A/B || 4'-hydroxy-allohumulinones || ? || ? || Potentially makes up the majority (70%), and beta acids degraded slower than alpha acids as seen below of compounds derived from the degradation of 4'-Hydroxyallo-cis-humulinones in beer stored at 40°C over 12 weeks.|-| colspan="5" align="center" align="center" style="padding: 2em;" | '''Beta Acid Derived Compounds <ref name="Mikyška_2012Hao_2020" /> '''|-! Oxidized Compound !! Precursor !! Beer Soluble (Y/N/?) !! Bitterness Level !! Notes|-| Hulupones (cohulupone, hulupone) || Beta acids (colupulone, lupulone) || Y || 84% as bitter as iso-alpha-acids (short-lasting alpha-acid-like bitterness); colupulone has a flavor threshold of 7.9 umol/L || Degradation rate of beta acids into hulupones increases with temperature. Has a relatively low absorption in wort (4.8–10.4%). May also degrade into other unknown compounds other than hulupinic acids.|-| Hulupinic Acids || Hulupones || Y || Contributes marginal bitterness to beer; flavor threshold of 69 umol/L || Has a relatively low degradation in wort.|-| Tricyclocolupone epimers A/B || Beta acids (percentages listed below are how much percent was lostcolupulone) || Y || Contributes long-lasting lingering and harsh bitterness; flavor threshold of 38-54 umol/L || Found to survive the brewing process up to 50% in a Pilsner style beer.|-| Dehydrotricyclocolupones epimers A/B || Beta acids (colupulone):|| Y || Contributes long-lasting lingering and harsh bitterness; flavor threshold of 40 umol/L || Found to not survive the brewing process in a Pilsner style beer, so it might not contribute to beer bitterness.|-{| class="wikitable sortable"Hydroperoxy-tricyclocolupone epimers A/B || Beta acids (colupulone) || Y || Contributes long-lasting lingering and harsh bitterness; flavor threshold of 20 umol/L || Found to not survive the brewing process in a Pilsner style beer, so it might not contribute to beer bitterness.
|-
! Storage !! Oil !! Saaz (pellet) <ref name="Mikyška_2012" | Hydroxy-tricyclocolupone epimers A/> !! Sládek B || Beta acids (pelletcolupulone) <ref name="Mikyška_2012" /> !! Premiant (pellet) <ref name="Mikyška_2012" /> !! Agnus (pellet) <ref name="Mikyška_2012" /> !! Saaz (leaf) <ref name="krofta_2013">[http:|| Y || Contributes long-lasting lingering and harsh bitterness; flavor threshold of 14-17 umol//www.actahort.org/books/1010/1010_26.htm Stability of Hop Beta Acids and Their Decomposition Products during Natural Ageing. K. Krofta, S. Vrabcová, A. Mikyska, M. Jurková, T. Cajka L || Found to not survive the brewing process in a Pilsner style beer, J. Hajslováso it might not contribute to beer bitterness. 2013.]</ref> !! Vital (leaf) <ref name="krofta_2013" /> !! Pure Beta Acid <ref name="krofta_2013" />
|-
| colspan="9" Nortricyclocolupone | '''Open air at 20°C for 12 months'''| Beta acids (colupulone) || Y || Contributes bitterness; flavor threshold of 90 umol/L || Found to survive the brewing process.
|-
| Epoxycohulupone and Epoxyhulupon || Alpha Beta acids (colupulone, lupulone) || -80% ? || -88.3% || -64.3% ? || -78Newly discovered; not much is known about them.2% || ||
|-
| || Beta acids || -60.5% || -83% || -53.7% || -51% || -50% || -77.colspan="5% |" align="center" align="center" style="padding: 2em;" | '''Iso-99%Alpha Acid Derived Compounds <ref name="Hao_2020" />'''
|-
| colspan="9" | '''Vacuum sealed at 20°C for 12 months'''! Oxidized Compound !! Precursor !! Beer Soluble (Y/N/?) !! Bitterness Level !! Notes
|-
| ''cis''-alloisohumulone and ''trans''-alloisohumulone || Alpha Iso-alpha acids (''cis''-isohumulone and ''trans''-isohumulone) || -20.6% Y || ? || ''cis''-alloisohumulone was found in trace levels in a Pilsner style beer at all different aging points, but ''trans''-24alloisohumulone remained undetected.9% |- | ''trans''-22.2% humulinic acid and ''cis''-humulinic acid || Iso-alpha acids (''cis''-isohumulone and ''trans''-21.7% isohumulone) || Y || No bitterness ||Minor levels in wort, but stable through the brewing process. Higher levels were detected in beers aged in oxygen permeable PET bottles versus glass bottles. Humulinic acid content increases over the storage of beer over many years as iso-alpha acids decrease. It has been suggested that the decomposition of iso-alpha acids into humulinic acids over time plays a role in the decrease in perceived bitterness of aged beer.
|-
| Hydroperoxy-''cis''-alloisohumulones and Hydroperoxy-''trans''-alloisohumulones || Beta Iso-alpha acids (''cis''-isohumulone and ''trans''-isohumulone) || Y ||? | | Iso-alpha acids degrade into these in beer that is exposed to oxygen after just 1-2hours. ''Cis''-isohumulone degrade into hydroperoxy-''cis''-alloisohumulones, while ''trans''-isohumulone degrade into Hydroperoxy-''trans''-alloisohumulones and tricyclohumol.7They are also formed during the wort boiling process, but are reduced by 50% || during the fermentation process. Hydroperoxy-''trans''-alloisocohumulone increases as beer is stored in bottles over many years (1, 4, and 10 years were measured in one study), while hydroperoxy-''cis''-alloisocohumulone is less stable and decreases.7% || In PET bottles, both types of hydroperoxy-2alloisohumulones decreased to zero after 4 years of storage at room temperature.1% || While the degredation of iso-alpha acids into hydroperoxy-alloisohumulones occurs very quickly when in the presence of oxygen, the degradation of hydroperoxy-1alloisohumulones occurs much more slowly during storage at room temperature.2% || || ||
|-
| colspan="9" Hydroxyl-''cis''-alloisohumulones and Hydroxyl-''trans''-alloisohumulones || Hydroperoxy-''cis''-alloisohumulones and Hydroperoxy-''trans''-alloisohumulones | | Y || ? || Hydroperoxy-alloisohumulones are further degraded into their respective hydroxyl-alloisohumulones (''cis''and 'Vacuum sealed 'trans'') via metal ions or UV light. Similar to hydroperoxy-alloisohumulones, hydroxyl-''trans''-alloisohumulones slowly increase during storage of bottles at 2°C for 12 monthsroom temperature over many years (1, 4, and 10 years), while hydroxyl-''cis''-alloisohumulones slowly decrease. In PET bottles, both types degrade to nearly 0 (from 0.39 umol/L to 0.08 umol/L) after 4 years at room temperature. When beer is intentionally oxidized, the hydroperoxy-alloisohumulones degrade very quickly over 24 hours into their hydroxyl-alloisohumulones derivatives.
|-
| Tricyclohumulactol || Alpha acids || Hydroperoxy-1.1% alloisohumulones || -5.5% Y || -0.3% ? || -1Only detected in a model solution; not detected in fresh or aged beer possibly due to low concentrations and/or coelution with isobaric molecules, exposing a technical difficulty of detecting these compounds in beer.4% || || ||
|-
| Scorpiohumols || Hydroxyl-''trans''-alloisohumulone || Y || ? || Same as Tricyclohumulactol.|-| Tricyclocohumol, Tricyclocohumene, Isotricyclocohumene, Tetracyclocohumol, and Eptitetracyclocohumol || ''Trans''-isohumulone (iso-alpha acid) || Y || Bitterness thresholds in beer as umol/L: 30, 5, 10, 70, 70 (''Trans''-isohumulone is 20). Characterized as "lingering harsh bitterness". || The most abundant oxidation derived hop compounds in aged beer. All are derived from ''trans''-isohumulone, which is the iso-alpha acid that is less stable than ''cis''-isohumulone. ''Trans''-isohumulone degrades into these faster at warmer temperatures and lower pH (no degradation at a pH of 6, slower degradation at a pH of 3, and very fast degradation at a pH of 1). Even differences as small as 0.1 pH (4.2, 4.3, and 4.4 pH values were tested) can make a significant difference in the degradation rate of ''trans''-isohumulone into tricyclocohumol, with lower pH values leading to faster degradation. After storing beer in brown bottles at 28°C for 8 months, these degradation products were detected at high amounts. Tricyclohumol remains relatively stable, even in the presence of oxygen.|- |} ====Acids====During aging, both alpha and beta acids oxidize and degrade with warmer temperatures and more oxygen exposure having a greater impact. The generally accepted theory is that oxygen interacts directly with hop acids. This event is called "autooxidation". An alternative theory to this is that oxygen indirectly oxidizes acids by first oxidizing the hop oils and turning them into pro-oxidants, which then oxidize the hop acids which are mixed in with the oils within the lupulin glands <ref name="Algazzali_2014" />. The oxidation of hop acids corresponds with an increase in the [http://methods.asbcnet.org/summaries/hops-12.aspx Hop Storage Index (HSI)], which is a practical way of measuring the overall freshness of hops. As the oxidation of hop oils rises, the measured HSI number on a lot of hops increases <ref name="Lam et al., 1986"/><ref name="Maye_2016">[http://www.hopsteiner.com/wp-content/uploads/2016/07/TQ-53-1-0227-01.pdf Humulinone Formation in Hops and Hop Pellets and Its Implications for Dry Hopped Beers. John Paul Maye, Robert Smith, and Jeremy Leker. 2016.]</ref>. These oxidized compounds lead to a higher amount of non-alpha-acid bitterness compounds in aged hops and have a remarkable effect on the bitterness of the beer. The bitterness from oxidized hop compounds has been described as more earthy, harsh, and astringent than the sharper, cleaner bitterness from iso-alpha acids <ref name="Mikyška_2012">[http://onlinelibrary.wiley.com/doi/10.1002/jib.40/abstract Assessment of changes in hop resins and polyphenols during long-term storage. Alexandr Mikyška and Karel Krofta. 2012.]</ref>. Aging hops while exposed to oxygen develops a cheesy aroma due to [[Isovaleric Acid|isovaleric acid]], isobutyric acid, and 2-methylbutyric acid. These acids are produced by the oxidative cleavage of acyl side chains of the hop resins <ref name="Briggs_2004" />. These cheesy oxidation compounds can be esterified to form wine-like and fruity tasting compounds (see [[Hops#Esters|Esters]] below and [[Aging_and_Storage#Acids_and_Esters|Aging and Storage]]) <ref name="Shellhammer, Vollmer and Sharp, CBC 2015"/>. Storage conditions and variety play a large role in how acid content in hops changes over time. Beta acids are generally more resistant to oxidation than alpha acids. A study by Mikyška and Krofta (2012) found that after 12 months of storage at 20°C in open air, pellet hops lost 64-88% of their alpha acid content and 51-83% of the beta acid content, with the beta acids dropping off more significantly after 6 months (alpha acid content declined steadily throughout the aging period). These amounts varied with different Czech hop varieties (Saaz, Sládek, Premiant, and Agnus), and beta acids degraded slower than alpha acids as seen below <ref name="Mikyška_2012" /> (percentages listed below are how much percent was lost): {| class="wikitable sortable"|-! Storage !! Oil !! Hallertau Magnum (Pellet, 14% AA) <ref name="Kishimoto_2021" /> !! Saaz (Pellet) <ref name="Mikyška_2012" /> !! Sládek (Pellet) <ref name="Mikyška_2012" /> !! Premiant (Pellet) <ref name="Mikyška_2012" /> !! Agnus (Pellet) <ref name="Mikyška_2012" /> !! Saaz (Leaf) <ref name="krofta_2013">[http://www.actahort.org/books/1010/1010_26.htm Stability of Hop Beta Acids and Their Decomposition Products during Natural Ageing. K. Krofta, S. Vrabcová, A. Mikyska, M. Jurková, T. Cajka , J. Hajslová. 2013.]</ref> !! Vital (Leaf) <ref name="krofta_2013" /> !! Pure Beta Acid <ref name="krofta_2013" /> |-| colspan="10" | '''Open air at 20°C for 12 months'''|- align="right"| || Alpha acids || || -80% || -88.3% || -64.3% || -78.2% || || |- align="right"| || Beta acids || || -60.5% || -83% || -53.7% || -51% || -50% || -77.5% || -99%|- | colspan="10" | '''Open air at 40°C for 3 months'''|- align="right"| || Alpha acids || -100% || || || || || || |||- | colspan="10" | '''Vacuum sealed at 20°C for 12 months'''|- align="right"| || Alpha acids || || -20.6% || -24.9% || -22.2% || -21.7% || || |||- align="right"| || Beta acids || || -2.7% || -1.7% || -2.1% || -1.2% || || |||-| colspan="10" | '''Vacuum sealed at 2°C for 12 months'''|- align="right"| || Alpha acids || || -1.1% || -5.5% || -0.3% || -1.4% || || |||- align="right"| || Beta acids || || -1.7% || -2.3% || -0.4% || -0.5% || || ||
|-
|}
{| class="wikitable sortable"
|-
! Storage !! Oil !! Sybilla (leafLeaf) <ref name="Skomra_2020" /> !! Puławski (leafLeaf) <ref name="Skomra_2020" /> !! Magnat (leafLeaf) <ref name="Skomra_2020" /> !! Magnum (leafLeaf) <ref name="Skomra_2020" />
|-
| colspan="9" | '''Open air at 5°C for 12 months''' (Harvest dates I, II, II, and only for Magnat/Magnum IV)
|}
'''Oxidized alpha acids''' (humulinones) are similar in taste perception to iso-α-acids, but have been described as less bitter (an average of about 66% as bitter on a 1 to 1 basis). The quality of the bitterness from oxidized alpha acids has been described in one study as "smoother and less lingering" than iso-alpha acids; this was attributed to humulinones being more polar than iso-alpha acids and therefore do not stick or linger on the tongue as long as iso-alpha acids <ref name="Shellhammer, Vollmer and Sharp, CBC 2015"/><ref name="Maye_2016" />. While the taste threshold of iso-alpha acids is 5-6 mg/L in light lager, the threshold for humulinones has been measured to be 8 mg/L in light lager (note that this is an average; tasters vary widely in how much bitterness they perceived from different bitter compounds) <ref name="Algazzali_2014" />. Humulinone content increases in hops after being pelletized (whole leaf hops have less humulinones). In fresh pellet hops that have a relatively low humulinone content, the humulinones contribute little to the bitterness of the beer when boiled, however when dry hopped they readily dissolve into the beer and have a significant impact on the beer's bitterness. With heavy dry hopping, the humulinones also decrease iso-alpha acid content of beer with more than about 25 IBU's, but not in beer with less than about 20 IBU. The decrease in iso-alpha acids and perceived bitterness/IBU is partially made up for the bitterness of the humulinones themselves (humulinones are picked up in IBU measurements with a [http://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry spectrophotometer] and as such it has been suggested that IBU's be [http://masterbrewerspodcast.com/004-dry-hopping-its-effects-on-bitterness-and-the-ibu-test-0 measured more accurately with HPLC]). In beers with less than 20 IBU, high dry hopping rates greatly increase the bitterness/IBU due to the bitter humulinones<ref name="Maye_2016" />. Humulinones also increase foam creation and stability by binding with the foam positive protein, Protein Z, via hydrogen bonding <ref>[https://www.sciencedirect.com/science/article/abs/pii/S0308814623020678 Chen Xu, Xuanqi Zhang, Mingyang Sun, Hanhan Liu, Chenyan Lv. Interactions between humulinone derived from aged hops and protein Z enhance the foamability and foam stability. Food Chemistry, Volume 434, 2024, 137449, ISSN 0308-8146, https://doi.org/10.1016/j.foodchem.2023.137449.</ref>.  The rate of humulinone formation is limiting, meaning . Maye et al. (2016) showed that humulinone formation occurs occurred rapidly during hop pelletization, and the concentration peaks during this time (these researchers found that further exposure to air did not increase humulinone content). Scientists believe They speculated that this is because when whole leaf hops are baled, only 20% of lupulin glands are broken, whereas when they are pelletized 100% of the lupulin glands are broken. In contrast, Taniguchi et al. (2013) found that humulinone formed slowly over 40 weeks in pellets that were stored at 20°C, but at 40°C and 60°C storage temperature they formed quickly and then diminished to very low levels at 10 weeks and 2 weeks respective to the higher storage temperatures. This demonstrated that warm storage increases the rate of humulinone formation. In addition, the oxidized hop compound 4′-hydroxy-allohumulinone increased trailing behind the humulinone formation and at a similar concentration at 20°C over 40 weeks of storage, and were much more stable than humulinones when stored at 40-60°C for 40 weeks <ref>[https://pubs.acs.org/doi/10.1021/jf3047187 Identification and Quantification of the Oxidation Products Derived from α-Acids and β-Acids During Storage of Hops (Humulus lupulus L.). Yoshimasa Taniguchi, Yasuko Matsukura, Hiromi Ozaki, Koichi Nishimura, and Kazutoshi Shindo. Journal of Agricultural and Food Chemistry 2013 61 (12), 3121-3130. DOI: 10.1021/jf3047187.]</ref>. The exact mechanism by which alpha acids are converted to humulinones is not known . The amount of humulinone correlates strongly with the [http://methods.asbcnet.org/summaries/hops-12.aspx Hop Storage Index (HSI)] <ref name="Maye_2016" />. Humulinone content in long-aged hops (1+ years) has not been studied.
'''Oxidized beta acids''' produce some compounds that also contribute to the perception of bitterness, specifically hulupones. Unlike humulinones which form relatively quickly from the oxidation of alpha acids, hulupones form at a much slower rate <ref name="Dušek_2014" />. Also unlike humulinones, they survive boiling and fermentation. While some sensory analysis of beers containing oxidized beta acids describes the resulting bitterness as "harsh and clinging", another analysis by Krafta et al (2013) described the bitterness of oxidized beta acids in beer when added in their pure form at the beginning of the boil as "pleasant and not lingering". The more degradation of beta acids into oxidized beta acids that occurs in hops, the more bitter beers brewed with these hops will be <ref name="krofta_2013" />. Two other compounds other than hulupones have been identified as being produced by the oxidation of beta acids, epoxycohulupone and epoxyhulupone. Their effect on beer flavor is not yet known; however, it is believed that hulupones have a greater impact on beer flavor and bitterness than these compounds <ref name="Dušek_2014" />.
Other compounds have been associated with the oxidation of beta acids and are extracted during wort boiling. These are described as giving a long-lasting, lingering bitterness on the palate. They include hydroxytricyclo-lupulone, dehydrotricyklolupulone, and hydroperoxytricyklolupulone <ref>[http://www.sciencedirect.com/science/article/pii/S0308814609001770 Structure determination and sensory evaluation of novel bitter compounds formed from β-acids of hop (Humulus lupulus L.) upon wort boiling. Gesa Haseleu, Daniel Intelmann, Thomas Hofmann. 2009.]</ref>.
 
====IBU====
The overall effect of oxidized compounds in aged hops has been shown by Val Peacock, a former scientist at Anheuser-Busch. Peacock stored hops at four different temperatures for 18 months. His data showed that although the alpha acid content in the hops and the iso-alpha acid content in the beers brewed with them decreased the older the hops were stored, the measured IBU of the different beers was about the same. This is because the oxidized acids in hops show up in the same spectrum as iso-alpha acids when using the ASBC method of measuring IBUs with a spectrophotometer <ref name="Aron">[https://www.homebrewersassociation.org/how-to-brew/resources/conference-seminars Dr. Patricia Aron. "Bitterness and the IBU: What’s It All About?" HomebrewCon 2017 Presentation. ~34 mins in. Retrieved 09/05/2017.]</ref>. This data is shown below. Caleb Buck's experiment [[Hops#Aged_Hops_in_Lambic|seen below]] supports this. Although it has not been shown that oxidized alpha and/or beta acids can inhibit lactic acid bacteria, if they do, then this might help explain reports <ref>[https://homebrewingfun.blogspot.com/2019/11/one-gallon-spontaneous-fermentation.html Adam Kielich. "One Gallon Spontaneous Fermentation Beer Batch 5 Recipe and Brewday". Brain Sparging on Brewing. 11/16/2019.]</ref> of using aged hops that originally had a high alpha acid content retaining a strong inhibitory effect towards lactic acid bacteria.
|}
[https://www.tandfonline.com/doi/full/10.1080/03610470.2021.1878684 Kishimoto et al. (2021)] reported similar results when making beers with forcibly aged hops. They aged Magnum hop pellets (14% AA) at 40°C and exposed to air for a number of different days: 3, 5, 7, 10, 14, 21, 30, and 90 days. The hops aged for 14 days had 9.8% AA, 21 days had 4.4% AA, 30 days had 1.2% AA, and 90 days had 0% AA. The IBU (measured with a spectrophotometer using Method Beer-23A from the American Society of Brewing Chemists) for the different beers made with the aged hops was more or less the same despite how much alpha acids were left in the aged hops. However, beers brewed with the different aged hops reflected a rapid decrease in iso-alpha acids in the beers made with the aged hops. Perceived bitterness also decreased for the beers made with the aged hops, but not as much as the decrease in ppm of iso-alpha acids, again demonstrating the oxidized hop compounds carry some bitterness, but less bitterness than iso-alpha acids <ref name="Kishimoto_2021" />. See alsoFigure 5 from Kishimoto et al. (2021): [[File:Kishimoto 2021 Fig5.jpg|600px|[https://www.tandfonline.com/doi/abs/10.1080/03610470.2021.1878684 Toru Kishimoto, Satoko Teramoto, Akiko Fujita & Osamu Yamada (2021) Evaluation of Components Contributing to the International Bitterness Unit of Wort and Beer, Journal of the American Society of Brewing Chemists, DOI:10.1080/03610470.2021.1878684.] Uploaded with permission from Toru Kishimoto.]]* It is therefore advised that brewers ask hop providers that offer aged hops what the original alpha acid percentage was when the hops were fresh, as well as the variety. This could be an important factor when determining how bitter the aged hops will taste, and potentially also how much they will negatively impact lactic acid bacteria growth. See also [[Hops#Aged_Hops_in_Lambic_and_Other_Spontaneous_Fermentation_Beer|"Aged Hops in Lambic" below]] and [https://www.facebook.com/groups/MilkTheFunk/permalink/2503097546385111 this MTF thread on IBU's from aged hops].]
====Oils====
Hop oils also generally degrade over time, however, their degradation rates are more complex. [http://pubs.acs.org/doi/abs/10.1021/jf00070a043 Lam et al. (1986)] found that aging both cascade and North American grown Hallertauer Mittelfrueh resulted in an increase in grapefruit-like character, although the compound that caused this was not identified. In the case of Cascade the intensity of this flavor correlated with the age of the hops <ref name="Lam et al., 1986"> [http://pubs.acs.org/doi/abs/10.1021/jf00070a043 Aging of Hops and Their Contribution to Beer Flavor. Lam et al. 1986.] </ref>. In the Hallertauer hops, aging resulted in an increase in a spicy/herbal character <ref name="Lam et al., 1986"/>, which is in agreement with reports of oxidized sesquiterpenes (specifically humulenol II, humulene diepoxides, caryophyllene, and to a lesser extent humulene monoepoxides and alpha-humulene) contributing a spicy/herbal flavor to beer <ref name="Goiris et al., 2002">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.2002.tb00129.x/abstract Goiris et al., 2002]</ref><ref name="Mikyška_2012" />. Many of the oils followed in the Lam et al. (1986) study which increased during a short accelerated aging period (2 weeks at 90°F) then decreased during extended aging (60 additional days at 90°F). The cascade hops lost more of the fruity/citrusy hop oils (myrecene, linalool, and geranial) than Hallertauer, suggesting that different strains of hops can withstand aging better than others. The concentration of hop oils are affected by the brewing process and fermentation (see the table) <ref name="Lam et al., 1986"/>. Another study found that beta-ionone (classified as a ketone, and characterized as "floral" and "woody" <ref>[http://www.thegoodscentscompany.com/data/rw1006632.html Beta-ionone. Good Scents Company. Retrieved 11/22/2016.]</ref>) increased in beers brewed with hops that were aged for 30 days at 40°C versus beers brewed with aged hops <ref name="kishimoto_2007" />.
A recent 2017 study at the Shellhammer lab looked at how trained panelists and consumers perceived a lager beer dry hopped with slightly oxidized Hallertau Mittelfrüh hops (exposed to oxygen once, then stored at 38°C for two weeks) versus highly oxidized (daily exposure to oxygen and stored at 38°C for two weeks). They found that the trained panelists detected more characteristics that are associated with noble hops; e.g. more woody, earthy, and herbal characteristics in the lager beers dry hopped with oxidized hops. They also found the oxidized hopped beers to be more bitter (probably due to oxidized alpha and beta acids). Consumers were not statistically able to tell the difference. The study determined that oxidized hops might serve to provide nuanced increases in noble hop character <ref>[http://www.asbcnet.org/publications/journal/vol/2017/Pages/ASBCJ-2017-1287-01.aspx Aroma Properties of Lager Beer Dry-Hopped with Oxidized Hops. Daniel M. Vollmer, Victor Algazzali, and Thomas H. Shellhammer. 2017.]</ref>. In a similar study by Hengyuan et al. (2023), aging Saaz hops for a short amount of time (aged at 30 ℃ in a ventilated environment for 5 days) reduced the grassy and resinous character and improved the spicy and woody character of single hopped beers (hopped at three points in the boil and dry hopped). Consumers also preferred this beer over the other samples, including fresh Saaz. However, over-aging Saaz hops resulted in less preference with consumers (30 ℃ for 10 days, 40 ℃ for 5 days, or 40 ℃ for 10 days). This effect was not demonstrated by Simcoe hops, however, indicating that short aging is only beneficial for some hop varieties. Under the same aging condition with Simcoe hops, consumers preferred the fresh Simcoe, and any degree of aged Simcoe was less preferred. The beers with aged Simcoe hops were described as having less fruity and floral character and more dry, thin bitterness <ref>[https://www.bio-conferences.org/articles/bioconf/abs/2023/04/bioconf_icbb2023_01016/bioconf_icbb2023_01016.html Aging of Hops and Their Effects on India Pale Ale Flavor. Hengyuan Xu, Shaokang Sun, Xiaochen Wang, Haojun Zhang and Cong Nie. BIO Web Conf., 59 (2023) 01016. DOI: https://doi.org/10.1051/bioconf/20235901016.]</ref>.
* [http://scottjanish.com/increasing-bitterness-dry-hopping/ "Increasing Bitterness By Dry Hopping", article by Scott Janish on oxidized alpha acids.]
===Aged Hop Suppliers===
* [https://www.mainiacalyeastyakimachief.com/commercial/hop-wire/introducing-the-ych-aged-hops/ Mainiacal Yeast -program YCH offers 1-2 oz bags for homebrewers and 44 lb bags of aged hops (small lots ; contact for homebrewers)more information.]
* <s>[http://www.hopsdirect.com/choice-debittered-aged-hops-leaf/ Hops Direct "Choice Debittered/Aged Hops" (Leaf - Cascade).]</s>
* <s>[https://hopsdirect.com/products/choice-debittered-pellets Hops Direct "Choice Debittered/Aged Hops" (Pellet - Columbus).]</s>* [https://www.freshops.com/shop/hop/aroma-hopproduct/lambic-hops/ Freshhops "Lambic Hops" (Leaf - Willamette)".]* [httphttps://www.yakimavalleyhops.com/Lambic2oz_pproducts/hopslambichops3.htm lambic-hop-pellets Yakima Valley Hops "Lambic / Aged Hops" (Pellet).]
* [http://www.farmhousebrewingsupply.com/lambic-hop-blend-4-oz-2015/ Farmhouse Brewing Supply "Lambic Hop Blend" (Pellet - Blend of varieties that are aged for ~5 years and then pelletized <ref>Private correspondence with Dustin Carver by Farmhouse Brewing Supply. 03/22/2016.</ref>).]
* [httphttps://www.themaltmiller.co.uk/index.php?_a=viewProd&productId=592 The Malt Miller (UK).]* [http:/product/www.brewstore.co.uk/specially-aged-fuggles-hops-100-grams Brew Store UK / The Malt Miller (Leaf - Fuggles).]* [http://www.brewstore.co.uk/specially-aged-hallertau-hops-100-grams Brew Store UK (Leaf - Hallertau).]
* [https://northwesthopfarms.com/products/lambic-aged-hops Northwest Hop Farms (BC, Canada).]
* [https://www.yakimachiefbsgcraftbrewing.com/ YCH lambic-hop-blend BSG sometimes offers 44 lb bags of aged pellet hopsfor commercial brewers; contact for more informationdetails.]* See [https://bsgcraftbrewingwww.facebook.com/groups/MilkTheFunk/permalink/4326634927364688 this MTF thread] on experiences with these hops BSG sometimes offers aged pellet hops for commercial brewers; contact for details.]* [https://www.ebay.com/itm/Lambic-Hops-pellet-or-whole-cone/172249202875?hash=item281ada40bb:m:mX2CSrKvWb0-3WIIuBh0aLA&var=471070249004 Ted from Hop Heaven on eBay sells 8 oz and 1 lb bags of aged pellet and leaf hops.] See this [https://www.facebook.com/groups/MilkTheFunk/?comment_tracking=%7B%22tn%22%3A%22R%22%7D reviews on permalink/4338527352842112/ MTFthread].* [https://shop.humle.se/humle/lambichumle/ Humlegårdens (Sweden); several varieties on reviews of aged whole leaf hops, 100 gram quantitiesthis product. ]
* [https://shop.humle.se/humle/lambichumle/ Humlegårdens (Sweden); several varieties of aged whole leaf hops, 100 gram quantities.]
===Dry Hopping===
[https://beerandbrewing.com/dictionary/96jvGQTSdT/ Dry hopping] is the technique of adding hops to beer that has finished fermenting or is in the process of fermenting. Historically, dry hopping is associated with English pale ales of the 1800's, and it was re-discovered by Fritz Maytag of Anchor Brewing Company in 1975 when the company brewed the first American IPA, Liberty Ale <ref>[https://beerandbrewing.com/dictionary/96jvGQTSdT/ "The Oxford Companion to Beer definition of dry hopping," Garrett Oliver. Craft beer & Brewing Magazine. Retrieved 11/16/2023.]</ref><ref>[https://northamericanbrewers.org/liberty-ale/ "Liberty Ale". North American Brewers Association. April 28, 2022. Retrieved 11/16/2023.]</ref><ref>[https://www.youtube.com/watch?v=4ijHO5TFG3I Scott Ungermann, Brewmaster of Anchor Brewing Company. The Doug Piper Gourmet Brewing Podcast. July 14th, 2023.]</ref>(20 mins in). Brewers have had positive and interesting results dry-hopping sour and funky beer. Often fresh American or New Zealand varieties that complement fruit flavors are chosen, however, other varieties have been used as well, including English and German hops. Just as in dry hopping normal beers, dry hopping sour/funky should be done after the beer has matured. Dry hopping for around 1-3 days before [[packaging]] the beer is adequate for extraction, depending on whether or not the beer is recirculated or agitated (agitation of the beer while on contact with the dry hops attains full extraction in 24 hours) <ref>[http://ir.library.oregonstate.edu/xmlui/bitstream/handle/1957/34093/Wolfe_thesis.pdf?sequence=1 A Study of Factors Affecting the Extraction of Flavor When Dry Hopping Beer (master thesis). Peter Harold Wolfe. 2012.]</ref>. Hopping rates generally range from 0.5-1 ounces per 1 gallon of beer (1-2 pounds per bbl or 3.7-7.5 grams per liter) to achieve hop-forward flavors, although lesser rates can be used to achieve a more subtle character (see the threads below) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1693639013997639/?comment_id=1693660390662168&comment_tracking=%7B%22tn%22%3A%22R4%22%7D Nate Walter and Dan Pixley. Milk The Funk Facebook group. 05/21/2017.]</ref>.
Dry hopping can contribute to bitterness in beer through oxidized alpha acids and oxidized beta acids. Oxidized alpha acids can also reduce iso-alpha acids in beers that begin with more than 25 IBU from iso-alpha acids, potentially reducing percieved bitterness after dry hopping (see [[Hops#Acids_2|Oxidized Alpha Acids]] above). Some alpha acids will also dissolve into the beer, which are estimated as being 10% as bitter as iso-alpha acids. Dry hopping also has a linear impact on the pH of beer regardless of the starting IBU or pH: the pH rises by 0.14 per pound of hop pellets per barrel of beer in a beer that started with a pH of 4.2 (~0.5 ounces per gallon or 3.7 grams per liter) <ref name="Maye_2016" /><ref name="Shellhammer, Vollmer and Sharp, CBC 2015"/>. This rise in pH might be less in more acidic beers that are dry hopped since pH is a logarithmic scale. Dry hopping can also reduce head retention in beers, although this is variety dependent (one study found that dry hopping with Eureka and Apollo hops increased head retention, while dry hopping with Bravo, Centennial, and Cascade decreased head retention). Extended dry hopping times (after 3 days) can also reduce head retention <ref name="Maye_EBC2017" />.
See also:
* [http://thebrulab.libsyn.com/episode-079-impact-dry-hopping-has-on-bitterness-ibu-and-ph-w-dr-john-paul-maye Bru Lab podcast Episode 079 - Impact Dry Hopping Has On Bitterness, IBU, and pH w/ Dr. John Paul Maye.]
* [https://www.therarebarrel.com/index.cfm?method=blog.blogDrilldown&blogEntryID=8141D6FA-EE78-5BCE-E08E-704DB6EEA279&originalMarketingURL=blog/Dry-hopped--Changing-process-leads-to-bottling The Rare Barrel reports on an anecdote that dry hopping in a less sour beer extracts better hop aroma, and ''Brettanomyces'' preserves the character.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/2358742877487246/ MTF thread by Dave Janssen on experiences with doing long-term dry hopping with noble hops in sour beers.] See also [http://www.horscategoriebrewing.com/2018/11/saison-from-1911-finishing-hops-and.html Dave's research on a 1911 saison that was dry hopped long term].
====The Freshening Power of the Hop (Hop Creep)====
Also known as "dry hop creep", it was first discovered in 1893 by Brown and Morris that dry hopping increases the ABV of beers and dries them out. Dry hop creep, along with the secondary fermentation of ''Brettanomyces'' in long aged British ales of the late 19th and early 20th centuries, were once characterized as the potential mechanisms by which long aged ales could retain a high level of carbonation in casks <ref>[https://archive.org/details/principlespracti00syke "The principles and practice of brewing" Sykes, Walter John. London, C. Griffin and Company, limited, 1907. Pgs 384-388.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/4709953772366133 Gareth Young. Milk The Funk Facebook group thread about English brewers historically relying on Brettanomyces and dry hop creep for carbonation in long aged ales. 06/17/2021.]</ref>. It was proposed that the likely cause is the release of glycolytic enzymes that break down starches into sugars which viable yeast can then ferment. Brewers normally aim to control the final alcohol percentage in a beer through brewhouse operations rather than postfermentation dilutions with lower/higher alcohol beers or water. This approach to brewing is called "brewing to final gravity." Due to the need to have a predictable ABV for government regulatory reasons, unexpected fermentation is, therefore, a concern for many breweries <ref name="Kirkendall_2018">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2018.1469081?journalCode=ujbc20 The Freshening Power of Centennial Hops. Jacob A. Kirkendall, Carter A. Mitchell & Lucas R. Chadwick. 2018. DOI: https://doi.org/10.1080/03610470.2018.1469081.]</ref>. Hop creep can also result in additional attenuation and higher carbonation after packaging, as well as diacetyl production.
Historically, there have been two studies published on the phenomenon of hops releasing glycolytic enzymes that break down starches during dry hopping: [http://barclayperkins.blogspot.com/2018/03/why-dry-hop.html Brown and Morris (1893)] and [https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1941.tb06070.x Janicki et al. (1941)]. More recently, several researchers and brewers have revisited this phenomenon. Brown and Morris (1893) discovered that hops could break down maltodextrin, but failed to identify the enzymes from the hop plant material and hypothesized (probably incorrectly) that tannins were inhibiting the enzymes. Janicki et al. (1941) came to similar conclusions regarding the enzymes and tannin inhibitors, and they also concluded that the enzyme activity was independent of hop variety, geography, age, storage conditions, pH values between 4.1 and 4.8, and that one or more additional unknown factors were at play <ref name="Kirkendall_2018" />.
Kirkendall et al. (2018) found that hop varieties also have a varying ability to ferment dextrins. They reported the following ABV increases when dry hopped in a pale ale at one pound per barrel: Centennial hops (+0.27%), Citra (+0.12%), Simcoe (+0.33%), Cascade (+0.49%) and Amarillo (+0.49%). Prolonged contact with Centennial hops (42 days) increased the ABV even more so and resulted in a nearly 1% ABV increase. Rousing the hops into suspension hastened the increase in ABV compared to samples that were left still. From their results, it appears as though contact with hops during dry hopping continues the breakdown of starches and dextrins into fermentable sugars. They also concluded that dry hopping at a temperature that is too cold for the yeast strain in the beer to ferment resulted in no change in ABV. They compared the enzymatic activity of Centennial hops that were stored at -20°C versus room temperature storage and found that there was no significant difference, indicating that the unidentified enzymes are relatively stable <ref name="Kirkendall_2018" />.
Kirkpatrick and Shellhammer (2018) found that the enzymes responsible for the conversion of dextrins into sugars include amyloglucosidase (removes glucose from non-reducing ends of α-1,4 and branching α-1,6 linkages, with a preference for α-1,4 linkages and longer chain oligosaccharides), α-amylase (hydrolyzes randomly along glucopolysaccharides to produce maltose, maltotriose, maltopentaose, and maltohexaose products from amylose as well as maltose, glucose, and branched dextrins from amylopectin), β-amylase (saccharifiying enzyme, cleaving maltose in small amounts from nonreducing ends of glucopolysaccharides, and to a minor extent, maltotriose), and limit dextrinase (debranches limit dextrins at α-1,6 linkages, producing linear α-1,4 chains which can further be degraded by the combined action of amylases). They were able to successfully extract them from Cascade pellet hops using commercially available assays (enzyme specific para-nitrophenyl blocked oligosaccharide substrates). The amount of α and β-amylase found in Cascade hops was well below that of malted barley, but within the range reported in other plant leaves. These enzymes are denatured by high temperatures, and as such would be denatured when boiling hops. They reported a similar increase in ABV of 1.3% after 40 days when dry hopping a beer with Cascade hops (and a decrease of 1.9°P) at a rate of 10 g/L. They also found that the hops contained glucose and a small amount of fructose, which accounted for a sugar increase of 0.02−0.03 °P per gram of hops. More studies on whether or not the amount of dry hopping has a large effect needs to be done, and whether or not warmer temperatures speed up the enzymatic breakdown of dextrins, and the authors hypothesized that the rate of dextrin break down could be slowed by dry hopping at lower temperatures <ref name="Kirkpatrick_2018">[https://pubs.acs.org/doi/pdf/10.1021/acs.jafc.8b03563 Evidence of Dextrin Hydrolyzing Enzymes in Cascade Hops (Humulus lupulus). Kaylyn R. Kirkpatrick and Thomas H. Shellhammer. 2018. DOI: DOI: 10.1021/acs.jafc.8b03563.]</ref>. Rubottom and Shellhamer (2023) later demonstrated that drying hops at 150°F reduced the amount of enzymes in dried pellitized hops versus drying them at 130°F <ref>[https://www.tandfonline.com/doi/abs/10.1080/03610470.2023.2194838 Evaluating the Impact of High and Low Kilning Temperatures on Popular American Aroma Hops. Lindsey N. Rubottom, Thomas H. Shellhammer. Received 07 Feb 2023, Accepted 21 Mar 2023, Published online: 28 Apr 2023. https://doi.org/10.1080/03610470.2023.2194838.]</ref>.
The Kirkpatrick and Shellhammer (2018) also reported that the exposure time of the beer to the dry hop material played a significant role in the breakdown of dextrins. Most of the breakdown of dextrins occurs within 5 days (+0.7% ABV), but continued slowly up until at least 40 days (+1.3%). They also tested removing the hops via centrifuge and storing the beer at 10°C or 20°C. Their results suggested that the effect of the enzymatic breakdown of dextrins by hops appears to only be active when in contact with the hops and that once the beer is removed then this breakdown of dextrins stops. The authors suggest that to avoid as much breakdown of starches and over-attenuation from dry hops as possible, brewers can limit the amount of time sits on the hops and reduce the temperature, however, it is also important to consider how this might impact the product's flavor and careful measures should be taken to balance the over-attenuation problem and overall beer quality <ref name="Kirkpatrick_2018" />. After removing the beer from the hops, a second diacetyl rest has been suggested as a way to clean up any diacetyl or off-flavors that the yeast produces from the additional fermentation during dry hopping <ref>[https://www.rockstarbrewer.com/how-dry-hop-creep-causes-diacetyl-in-beer-and-how-brewers-can-minimise-the-risk/ STEVE 'HENDO' HENDERSON. How “Dry Hop Creep” Causes Diacetyl In Beer and How Brewers Can Minimise The Risk. Rockstar Brewer Academy website. 09/03/2018. Retrieved 10/05/2018.]</ref>. Other recommended solutions to avoiding hop creep is pasteurizing, filtering, or cold crashing out the yeast before dry hopping, storing the beer cold so that the yeast remains inactive, reducing dry hopping amounts, and dry hopping before fermentation is finished <ref>[http://beersmith.com/blog/2019/03/31/dry-hop-creep-over-carbonation-and-diacetyl-in-beer/ Brad Smith. BeerSmith blog. 03/13/2019. Retrieved 07/23/2019.]</ref>. [https://www.mbaa.com/publications/tq/tqPastIssues/2021/Pages/TQ-58-3-0705-01.aspx Teraoka et al. (2021)] argued that enzymes present in dry hops are not derived from the hop plants themselves, but microbes living on the hops and that the reported variability of starch degrading enzymes present in hops is due to the varying environmental conditions created by drying hops that encourage or discourage microbial growth. It has been reported that proteobacteria are the dominant type of bacteria that grow on hops, with the majority of them being Pseudomonas and Sphingomonas, with Pseudomonas having being previously reported to produce alpha-amylase. However, the authors recommend that further research is needed to identify which microbe species might be producing these enzymes on hops. They found that hops treated with the anti-microbial chemical sodium azide resulted in hops with much lower starch degrading enzymes. They also reported that alpha-amylase decreased in dried leaf and pellet hops over 3 months of anaerobic storage (storage temperature did play a role in the degradation rate of the enzymes). Alpha-amylase stayed relative consistent in fresh hops during storage. Beta-amylase remained stable over the three month storage time regardless of hop type. The authors also acknowledge another possible variable being that hop seed material might contain starch; however, seeded hops are generally not used in the brewing industry <ref>[https://www.mbaa.com/publications/tq/tqPastIssues/2021/Pages/TQ-58-3-0705-01.aspx "Do Starch-Degrading Enzymes in Hop Samples Originate in Microorganisms?" Ryohei Teraoka (1), Makoto Kanauchi (1), and Charles W. Bamforth (2). 1. Department of Food Management, Miyagi University, Hatatate Taihaku-ku Sendai Miyagi, 982-0215, Japan. 2. Sierra Nevada Brewing Company, Chico, CA 95928, U.S.A.]</ref>. See also [https://www.masterbrewerspodcast.com/229 MBAA Podcast episode 229 "Is Hop Creep Caused by Microorganisms?" with Dr. Charlie Bamforth]. [https://www.mdpi.com/2311-5637/7/2/66/html Bruner et al (2021)] investigated whether or not yeast strain selection has an impact on hop creep with the goal of finding if any strains would reduce the potential for dry-hop creep. They examined 30 different ''Saccharomyces'' yeasts from different yeast labs, and measured the [https://beerandbrewing.com/dictionary/gaKDNn0yxE/ real degrees of fermentation (RDF)] that occurred after dry-hopping post fermentation. With the exception of two strains, all of the yeasts re-fermented the beer after dry hopping, indicating that yeast strain selection won't reduce the chances for dry hop creep. There were two exceptions to this. The first was a diastatic strain of ''Saccharomyces cerevisiae'' (SafAle™ BE-134), which was able to break down starches in the beer prior to dry hopping due to the [[Saccharomyces#Diastatic_strains_of_Saccharomyces_cerevisiae|diastatic properties]] of this yeast strain. The other exception was a strain of ''Saccharomyces mikatae'' that is used as a co-fermenter in wine fermentations and is a poor attenuater of wort. The study also found no correlation between flocculation and attenuation from dry-hop creep, but suggested that further research be done to investigate the belief that higher flocculating strains could reduce the negative effects of dry hop creep <ref>[https://www.mdpi.com/2311-5637/7/2/66/html Bruner J, Marcus A, Fox G. Dry-Hop Creep Potential of Various Saccharomyces Yeast Species and Strains. Fermentation. 2021; 7(2):66. https://doi.org/10.3390/fermentation7020066.]</ref>.
See also:
* [https://www.thebrewingnetwork.com/hop-and-brew-school-ep8-hop-creep/ Methods for avoiding hop creep and diacetyl production from hop creep explained by Nick Zeigler ("Hop and Brew School" podcast).]
* [http://masterbrewerspodcast.com/098-dry-hop-creep MBAA podcast episode 98 on dry hop creep.]
* [https://brulosophy.com/podcasts/the-bru-lab/ Episode 008 | The Freshening Power Of Hops with Jake Kirkendall on the The Brü Lab podcast.]
* [https://www.onedropbrewingco.com.au/cleanfusion One Drop Brewing Co's "Clean Fusion" method of using a centrifuge to remove yeast before dry hopping in order to avoid hop creep.]
===Aged Hops in [[Lambic]] and Other [[Spontaneous Fermentation|Spontaneous Fermentation Beer]]===
[[File:Cantillon aging hops.jpg|thumbnail|right|Brasserie-Brouwerij Cantillon aging their hops; image provided by Dave Janssen.]]
Modern lambic traditionally uses aged hops at a moderate rate to help limit and select for microbes and regulate acid production. Modern Lambic brewers cite rates in the range of roughly 450 grams of hops per Hl hectoliter of finished beer (0.6 ounces per gallon) <ref name="Jean Van Roy on Basic Brewing Radio"> [http://hwcdn.libsyn.com/p/e/a/2/ea26e00136fe1638/bbr05-30-13cantillon.mp3?c_id=5723890&expiration=1443888327&hwt=8dd886677defabdd73669cdc262ef446 Jean van Roy on Basic Brewing Radio] </ref> (~43 min in) (see also the notes pertaining hopping rates on the [[Cantillon]] page), with some brewers possibly going above this range. The age of hops used depends on the producer and their preferences/stock. Cantillon uses hops that are roughly 3 years old<ref>D. Janssen personal communication with Jean Van Roy, 9-Nov-2013</ref>, while 3 Fonteinen reports using hops that are over 10 years old<ref name="Drie Fonteinen on Belgian Smaak"> [http://www.belgiansmaak.com/armand-debelder-michael-blancquaert-drie-fonteinen/ Drie Fonteinen on Belgian Smaak] </ref> (~48 minutes in). Jester King reported using 0.66 - 0.75 pounds of whole leaf aged hops per BBL (0.34-0.39 ounces per gallon) in their spontaneously fermented ales <ref>Averie Swanson. "Sour Power! A Pro Brewer Spontaneous Fermentation Roundtable". HomebrewCon seminar. 2018.</ref> (~31:00 mins in). Lambic brewers either add their hops while still collecting wort, sometime before the wort comes to a boil<ref>[https://www.facebook.com/groups/Lambic.Info/permalink/1831338433787524/ Video of Cantillon wort reaching a boil from Bill on Lambic.info]</ref> (also known as "first wort hopping"), or shortly after boil is reached<ref name="Drie Fonteinen on Belgian Smaak"/> (~48 min in). The hops are then boiled with the wort for essentially the full length of the boil <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1593059604055581/?comment_id=1593928187302056&reply_comment_id=1593938693967672&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Dave Janssen on MTF. 02/24/2017.]</ref><ref>[http://www.lambic.info/Brewing_Lambic#Hopping "Brewing Lambic", section "Hopping". Lambic.info website. Retrieved 02/24/2017.]</ref>. The resulting lambic beers are often surprisingly bitter, especially when young. Historically, there is [http://www.horscategoriebrewing.com/2016/04/hops-in-spontaneous-fermentation.html some evidence] that lambic brewers used a combination of aged hops and fresh dried hops. Not all aged hops are the same; different varieties/sources result in different levels of residual alpha/beta acids (probably not zero), oxidized acids, IBU's, perceived bitterness, and inhibition of lactic acid bacteria. Varieties with high acids and hop oils probably have more residual acids and oils, and aging times/conditions may not be ideal enough to completely age high alpha/beta/oil hop varieties. Therefore, it is impossible to give a blanket statement on how much aged hops to use given a specific lot of aged hops. Andrew Holzhauer from Funk Factory Geuzeria suggests tasting aged hops for bitterness and adjusting the amount of hops depending on how bitter they taste <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2723907944304069/?comment_id=2723956477632549&reply_comment_id=2724104977617699&comment_tracking=%7B%22tn%22%3A%22R%22%7D Andrew Holzhauer. Milk The Funk Facebook group on how much aged hops to use. 06/13/2019.]</ref>, while James Howat from Black Project suggests making a small batch and having the wort/beer analyzed for IBU's and adjusting accordingly.
For example, homebrewer Caleb Buck performed an experiment comparing two different hopping rates for spontaneously fermented beer at home using whole leaf aged hops that were independently tested to have 0.5% alpha acids and 0.2% beta acids and were obtained from Hops Direct in Junuary 2016 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2302073339820867/?comment_id=2302314029796798&reply_comment_id=2303737466321121 Caleb Buck. Milk the Funk Facebook group thread on Caleb's aged hop experiment. 10/01/2018.]</ref>. The two rates tested were 0.3 ounces of aged hops per gallon and 0.6 ounces per gallon, both added at the beginning of the boil <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3970005893027595/?comment_id=3970763046285213 Caleb Buck. Milk The Funk Facebook group. Addendum to when hops were added to the boil for his associated hop experiment. 10/14/2020.] </ref>. Samples of the two worts were sent to Sweetwater Science Labs to perform IBU analysis using the ASBC standard IBU test. Interestingly, the results were 72 IBU and 127 IBU respectively. The unexpectedly high IBU might be due to the variety of aged hop, as well as oxidized hop acids showing up in the standard IBU test (see Peacock's data [[Hops#Acids_2|here that showed that aged and fresh dried hops produce a similar IBU]]). After about 7 months, one of the 0.3oz/gal batches got down to a pH of 3.6, a second batch at 0.3oz/gal got to a pH of 4, while the 0.6oz/gal batches remained within a pH of 4.2 - 4.3. From this experiment, Caleb will attempt using only 0.15 oz/gal of aged hops which should be closer to 30 IBU and so that more acidity can be achieved. James Howat from Black Project Spontaneous Ales suggests making sample wort with the hops that will be used for a larger batch and sending that sample off for IBU testing in order to more easily achieve the desired IBU's. More detail can be found on [http://www.archaicpursuit.com/2018/08/2017-coolship-experiment-hopping-rate.html?m=1 Caleb Buck's collected data on cooling rates, acidity from hopping rates, and other collected data over a multi-year, multi-batch experiment] and [http://hwcdn.libsyn.com/p/b/d/2/bd2703ec2214a5c7/bbr09-27-18souribus.mp3 Caleb's interview on this experiment on BasicBrewing Radio].
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1923361737692031/ MTF discussion on using aged hops in other styles of beers, including historical references and tips on producing lambic-like character from aged hops and commercial cultures.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/2534320159929516/ MTF discussion on leaving hops beer fermented with ''Brettanomyces'' long term; inspired by historical English brewing methods.]
 
===Spent Hops===
There has been some research and experimentation on using spent hops (mostly spent dry hops) in a second beer.
* [https://www.facebook.com/groups/MilkTheFunk/permalink/3280694625292062/ MTF thread containing summaries of science and historical use of re-using spent hops.]
 
===Hop Extract Products===
* [https://www.stitcher.com/show/craft-beer-brewing-magazine-podcast/episode/episode-202-brandon-capps-of-new-image-brews-better-ipas-through-chemistry-86471398 Craft Beer & Brewing Magazine Podcast Episode 202: Brandon Capps of New Image Brews Better IPAs Through Chemistry.]
==See Also==
===External Resources===
* [https://www.tandfonline.com/doi/suppl/10.1080/03610470.2023.2232267/suppl_file/ujbc_a_2232267_sm7384.pdf "Free Exhaustive Literature Review on Hops (Humulus lupulus L.)," Keven Bélanger Harbour; Journal of the American Society of Brewing Chemists.]
* [https://www.barthhaas.com/resources/hop-harvest-guide#!download BarthHaas Hop Harvest Guide with flavor wheels.]
* [https://appellationbeer.com/blog/hop-queries/ "Hop Queries"; Stan Hieronymus's hops newsletter.]
* [https://www.youtube.com/channel/UCYi3RcKUPk9hCOxaN4TCoHQ/videos Hop Growers of America video presentations (YouTube).]
* [https://ext.vt.edu/agriculture/commercial-horticulture/hops.html Virginia Tech Virginia Cooperative Extension "Hops" webpage: growing and agriculture resources.]
* [https://lisameoli.wordpress.com/2015/11/04/the-hop-plant-dissected/ "The Hop Plant Dissected" by Lisa Meoli, 2015.]
* [http://www.thebrewingnetwork.com/hop-and-brew-school-ep11-hops-and-sour-beer/ Hop and Brew School podcast interview with Vinnie Cilurzo from Russian River Brewing, Jay Goodwin from The Rare Barrel and Charlie Johnson from the Ronin Fermentation Project on using fresh and aged hops in sour beer.]
* [https://pubs.acs.org/doi/10.1021/acs.jafc.7b04055 Overview of scientific evidence of health benefits of polyphenols.]
* [https://cryopopblend.com/wp-content/uploads/2021/10/Survivable-Compounds-Handbook.pdf Survivable Compounds Handbook.]
* [https://www.youtube.com/watch?v=52nl1Fh6qnQ The hops that built craft beer – a documentary | The Craft Beer Channel.]
==References==

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