Active ingredients of Piper betle extracts and their antibacterial effects on Levilactobacillus brevis

Levilactobacillus brevis, a common beer spoilage bacterium, causes acidification during brewing. In this study, we aimed to investigate the antibacterial activities of Piper betle extracts against L. brevis and identify their active constituents. A crude ethanol extract was obtained from P. betle leaves and further fractionated into chloroform, ethyl acetate (PBAE), and aqueous extracts via liquid-liquid partitioning. Among all extracts, PBAE exerted the strongest antibacterial effects, forming an inhibition zone (diameter = 57.5 ± 0.71 mm) larger than that formed by the P. betle ethanol extract (diameter = 45.5 ± 0.71 mm) and the gentamicin control (diameter = 21.0 ± 0.1 mm), which served as a reference benchmark. PBAE also caused morphological deformation and increased the membrane permeability of L. brevis, resulting in intracellular DNA and protein leakage and elevated culture medium conductivity. Two major antibacterial compounds, 4-allylcatechol (minimum inhibitory concentration = 50 μg/mL) and 5-allylbenzene-1,2,3-triol (minimum inhibitory concentration = 200 μg/mL), were identified in PBAE via high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Overall, our results suggest PBAE as a natural antibacterial agent against beer spoilage bacteria.