The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated ΔpH. The bile acid concentrations that dissipated ΔpH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (ΔΨ). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated ΔpH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4× MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8× MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the ΔpH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2× MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane ΔpH and ΔΨ dissipation), potassium ion, and other cellular components and eventually cell death.
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