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Hops
,→Isomerization of Alpha 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>. 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, alpha acids are isomerized into iso-alpha acids (also called isohumulones) 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 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>.
==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]]''. Gram-negative bacteria found in beer, such as ''Acetobacteraciae'', 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" />. 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 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 the 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"/>.
===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 the 1960's 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" />.