Mechanisms of Hop Inhibition Include the Transmembrane Redox Reaction

Abstract

In this work, a novel mechanistic model of hop inhibition beyond the proton ionophore action toward (beer spoiling) bacteria was developed. Investigations were performed with model systems using cyclic voltammetry for the determination of redox processes/conditions in connection with growth challenges with hop-sensitive and -resistant Lactobacillus brevis strains in the presence of oxidants. Cyclic voltammetry identified a transmembrane redox reaction of hop compounds at low pH (common in beer) and in the presence of manganese (present in millimolar levels in lactic acid bacteria). The antibacterial action of hop compounds could be extended from the described proton ionophore activity, lowering the intracellular pH, to pronounced redox reactivity, causing cellular oxidative damage. Accordingly, a correlation between the resistance of L. brevis strains to a sole oxidant to their resistance to hop could not be expected and was not detected. However, in connection with our recent study concerning hop ionophore properties and the resistance of hop-sensitive and -tolerant L. brevis strains toward proton ionophores (J. Behr and R. F. Vogel, J. Agric. Food Chem. 57:6074-6081, 2009), we suggest that both ionophore and oxidant resistance are required for survival under hop stress conditions and confirmed this correlation according to the novel mechanistic model. In consequence, the expression of several published hop resistance mechanisms involved in manganese binding/transport and intracellular redox balance, as well as that of proteins involved in oxidative stress under "highly reducing" conditions (cf. anaerobic cultivation and "antioxidative" hop compounds in the growth medium), is now comprehensible. Accordingly, hop resistance as a multifactorial dynamic property at least implies distinct resistance levels against two different mechanisms of hop inhibition, namely, proton ionophore-induced and oxidative stress-induced mechanisms. Beyond this specific model of hop inhibition, these investigations provide general insight on the role of electrophysiology and ion homeostasis in bacterial stress responses to membrane-active drugs.The inflorescences of the hop plant Humulus lupulus are traditionally used for beer brewing, due to the pleasant bitterness of hop compounds in beer and their additional inhibitory effect on bacteria. These antibiotic and bacteriostatic properties of hops comprise several inhibitory mechanisms. The described effects of hop compounds on bacteria are permeability changes in the bacterial cell wall (30), leakage of the cytoplasmic membrane and a subsequent inhibition of respiration, and protein, DNA, and RNA synthesis (42), as well as changes in leucine uptake and proton ionophore activity (33). In a recent study (5), we characterized the latter ionophore properties of hop compounds in a cell-free model system via bilayer lipid membrane (BLM) techniques in connection with growth challenges of hop-sensitive and -resistant Lactobacillus brevis strains. The antibacterial action of iso-␣-acids as prot...

The Microbiology of Malting and Brewing

“…This impairs enzymatic activity and nutrient transport, halting growth and ultimately killing the cell (220,221). In addition, iso-alpha-acids participate in transmembrane redox reactions in association with manganese, causing oxidative stress to the bacterial cell (222), which explains the manganese-dependent enhancement of transmembrane potential observed previously (219,221) (Fig. 4).…”

“…Hop stress in L. brevis also induces expression of a broad range of proteins involved in redox homeostasis, DNA repair, and protein repair, facilitating a shift toward energy balance and metabolic regulation to cope with low-pH conditions and oxidative stress (225). The multiple modes of inhibition exerted by hop challenge and the complex response elicited in resistant bacteria indicate that hop-resistant LAB are specialized for growth in beer through resistance to oxidative and acid stress (219,222). Early work demonstrated that iso-alpha-acids have no impact on the growth of Gram-negative bacteria (226), but no further work has been done to determine the mechanism for resistance.…”

Reduction and restoration of culturability of beer-stressed and low-temperature-stressed Lactobacillus acetotolerans strain 2011-8

“…Thus, the lag between stimulus and response yields a timeframe conducive to antimicrobial activity. Recently, the antibacterial action of hop compounds was suggested to involve potent redox reactivity, resulting in cellular oxidative damage (Behr and Vogel, 2010). Accordingly, in the absence of active phenotypic adaptation, the VPNC cells of beer-spoilage L. acetotolerans are probably sensitive and not equipped to detoxify superoxide and a proportion or all of these cells remain non-culturable.…”

125th Anniversary Review: Microbiological Instability of Beer Caused by Spoilage Bacteria

“…One explanation for this phenomenon is that the cells respond to Mn 2+ limitation by upregulating these enzymes, thus compensating for the reduced levels of intracellular manganese and the lost activities of Mn 2+ -dependent enzymes 18 . Very recently, the antibacterial mechanisms of hop compounds were suggested to involve proton ionophoric actions and redox-reactive uncoupler activities occurring in parallel 19,162 . Accordingly, it is plausible that beer spoilage LAB have to cope with oxidative stress induced by hop compounds, in addition to PMF depletion.…”

“…Accordingly, it is plausible that beer spoilage LAB have to cope with oxidative stress induced by hop compounds, in addition to PMF depletion. Thus, the observed upregulation of Mn 2+ -dependent enzymes responsible for redox homeostasis is most likely part of a defensive response to the oxidative stress caused by hop bitter acids 19,162 . On the other hand, a morphological shift into smaller rods was observed in beer-adapted L. brevis and L. lindneri cells (Fig.…”