Characterization of a Highly Hop-ResistantLactobacillus brevisStrain Lacking Hop Transport

Abstract

Resistance to hops is a prerequisite for lactic acid bacteria to spoil beer. In this study we analyzed mechanisms of hop resistance of Lactobacillus brevis at the metabolism, membrane physiology, and cell wall composition levels. The beer-spoiling organism L. brevis TMW 1.465 was adapted to high concentrations of hop compounds and compared to a nonadapted strain. Upon adaptation to hops the metabolism changed to minimize ethanol stress. Fructose was used predominantly as a carbon source by the nonadapted strain but served as an electron acceptor upon adaptation to hops, with concomitant formation of acetate instead of ethanol. Furthermore, hop adaptation resulted in higher levels of lipoteichoic acids (LTA) incorporated into the cell wall and altered composition and fluidity of the cytoplasmic membrane. The putative transport protein HitA and enzymes of the arginine deiminase pathway were overexpressed upon hop adaptation. HorA was not expressed, and the transport of hop compounds from the membrane to the extracellular space did not account for increased resistance to hops upon adaptation. Accordingly, hop resistance is a multifactorial dynamic property, which can develop during adaptation. During hop adaptation, arginine catabolism contributes to energy and generation of the proton motive force until a small fraction of the population has established structural improvements. This acquired hop resistance is energy independent and involves an altered cell wall composition. LTA shields the organism from accompanying stresses and provides a reservoir of divalent cations, which are otherwise scarce as a result of their complexation by hop acids. Some of the mechanisms involved in hop resistance overlap with mechanisms of pH resistance and ethanol tolerance and as a result enable beer spoilage by L. brevis.Resistance to hops is a prerequisite for the ability of lactic acid bacteria to grow in beer and thus cause beer spoilage. Hop compounds, mainly iso-␣-acids, were described as ionophores which dissipate the pH gradient across the cytoplasmic membrane and reduce the proton motive force (PMF). Consequently, the low intracellular pH (pH in ) interferes with essential enzyme reactions and PMF-dependent nutrient uptake is hampered, resulting in the death of cells of hop-sensitive strains (34,37,49).Several mechanisms involved in the hop resistance of lactobacilli have recently been characterized (13, 34-36, 38, 24-44, 47, 48). The proteins contributing to hop resistance include multidrug resistance (MDR) transporters that excrete the hop compounds into the outer medium (35, 45) and proton export systems that maintain the intracellular pH. HitA is a putative divalent cation transporter present predominantly in beerspoiling lactobacilli (13). An alteration of the teichoic acids in the cell wall (50) and a changed lipid composition of the cytoplasmic membranes (34) might additionally contribute to the hop resistance. However, the role of these hop resistance mechanisms in beer-spoiling lactobacilli is not fully understood...

Sake and Beer Spoilage Lactic Acid Bacteria - A Review

“…Nonetheless these membrane-bound defense mechanisms are inevitably energy-consuming, and malate, citrate, pyruvate and arginine were identified as energy sources for ATP production in beer, where most nutrients are depleted by brewing yeast 108 . As for energy-independent defense systems, lipoteichoic acids located in the cell wall were reported to provide a reservoir of divalent cations, which are otherwise scarce as a result of complexation with hop bitter acids 11,12,134 . The ratio of 16:0 fatty acid content to 16:1 fatty acid content, which is considered important in proton ionophore resistance, was also reported to increase substantially upon hop adaptation of beer spoilage LAB 11 .…”

“…As for energy-independent defense systems, lipoteichoic acids located in the cell wall were reported to provide a reservoir of divalent cations, which are otherwise scarce as a result of complexation with hop bitter acids 11,12,134 . The ratio of 16:0 fatty acid content to 16:1 fatty acid content, which is considered important in proton ionophore resistance, was also reported to increase substantially upon hop adaptation of beer spoilage LAB 11 . The morphological studies further showed that beer spoilage LAB occur in beer as shorter rods (Fig.…”

“…In fact, L. brevis strains, isolated from sources other than beer and related environments, were reported to generally lack beer spoilage ability 68,70 . Beer spoilage L. brevis strains are typically isolated from beer brewing environments, and are suggested to possess numerous layers of hop-resistance mechanisms that appear to have been acquired stepwise during their long history of beer adaptation processes 11,12,102 . Interestingly, the sequencing analysis of gyrB indicated that beer spoilage L. brevis strains form a distinct subgroup within this species 70 .…”

“…Therefore hop resistance ability has been known as a distinguishing character for beer spoilage strains of LAB. The hop resistance mechanisms are regarded as multifactorial dynamic properties and appear to consist of various active and passive defense systems 11,106 . The active defense mechanisms involve efflux pumps, such as HorA and HorC, which have been proposed to transport hop bitter acids out of cells 46,90 .…”

Effects of Lysozyme on the Microbiological Stability and Organoleptic Properties of Unpasteurized Beer

“…They exert bacteriostatic effects on most Gram-positive bacteria as they act as proton ionophores and dissipate the transmembrane pH gradient. Several LAB species, such as Lactobacillus spp., have been found to have acquired beer-spoilage ability due to hop resistance genes (horA and horC), and consideration must be given to the fact that altering the hop composition of beer can have a profound impact on the organoleptic and physico-chemical properties of the final product 6,21,22,33,35 .…”