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Hops
,→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, with colupulone and lupulone being suggested as the strongest inhibitors <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><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, Volume 169, 2023. 112832. ISSN 0963-9969. https://doi.org/10.1016/j.foodres.2023.112832.]</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 <ref>[https://kuleuven.limo.libis.be/discovery/search?query=any,contains,LIRIAS4199525&tab=LIRIAS&search_scope=lirias_profile&vid=32KUL_KUL:Lirias&offset=0 Goedseels, M., & Michiels, C. (2024). Mechanisms of resistance against hop beta acids in Listeria monocytogenes.]</ref> 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, 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" />.