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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]]''. Certain other 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 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 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), 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'' in one study <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>. 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 Schurr et al., (2015)] </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>. 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"/>.
===Bacterial Resistance to Hop Compounds===
Due to the multiple mechanisms for hop antimicrobial activity, multiple resistance mechanisms are necessary for a Gram-positive bacterial cell to successfully be hop-tolerant<ref name="Behr and Vogel, 2010"> [http://aem.asm.org/content/76/1/142.short Behr and Vogel, (2010)] </ref>. Hop resistance of bacteria will vary by species as well as within a species with individual strains. The environment in which strains are cultured and maintained may also influence their hop tolerance. It is possible for a small subpopulation of individual cells that have a higher tolerance to hops to eventually dominate the overall population <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>. The hop tolerance of lactic acid bacteria strains decreases when they are cultured in hop-free environments and strains cultured in media with increasing concentrations of hop compounds show an increase in hop tolerance<ref name="Sakamoto and Konings, 2003"/>. The stability of hop resistance, or the rate at which it is lost when bacteria are cultured in unhopped wort, varies by strain. It can take up to 1 year for maximum loss of hop resistance, suggesting that in some strains have a relatively stable hop resistance<ref name="Sakamoto and Konings, 2003"/>. Because of this intra-species variability and dependence on how the strains were cultured, it is difficult to give specific advice about the hop-tolerance of a wide range of strains offered from different sources. As a general rule, some common lactic acid bacteria species used in sour beer and found as beer spoilage organisms like ''Lactobacillus brevis'', ''Lactobacillus lindneri'' and ''Pediococcus delbrueckii'' have some resistance to hops<ref name="Sakamoto and Konings, 2003"/>. Brewers seeking to make acidic beers with higher doses of hops may wish to seek out one of these species. Some hop-tolerant species benefit from pre-culturing in media with below-limiting concentrations of compounds before being used in more highly hopped wort or beer<ref name="Simpson and Fernandez, 1992"> [http://onlinelibrary.wiley.com/doi/10.1111/j.1472-765X.1992.tb00636.x/abstract Simpson and Fernandez, 1992]</ref>.
See also [[Pediococcus#Hop_Resistance|''Pediococcus'' hop resistance]] and [[Lactobacillus#Hop_Tolerance|''Lactobacillus'' hop tolerance]].