Synergistic antimicrobial activity of essential oils in combination with phage endolysin against Salmonella Typhimurium in cooked ground beef

Abstract

This study was designed to evaluate the antimicrobial activity of selected essential oils (AITC, carvacrol, eugenol, and thymol) in combination with Salmonella phage-encoding endolysin (LysPB32) against Salmonella Typhimurium KCCM 40253 in vitro and in cooked ground beef. The minimum inhibitory concentrations (MICs) of AITC, carvacrol, eugenol, and thymol against S. Typhimurium were determined in the absence and presence of LysPB32 using broth microdilution assay. In vitro antimicrobial susceptibility of S. Typhimurium to the essential oils in combination with LysPB32 was evaluated at 37 °C for 24 h using time-kill curve assay. The N-phenyl-1-napthylamine (NPN) uptake and relative bacterial fitness were measured to evaluate the outer membrane permeability and reproductive success. The morphological alterations of these treated S. Typhimurium cells were observed through a holotomographic 3D microscopy. The MICs of AITC, carvacrol, eugenol, and thymol against S. Typhimurium were significantly decreased from 6.0 to 2.9 mg/mL, 3.0 to 0.8 mg/mL, 2.8 to 0.7 mg/mL, and 1.4 to 0.7 mg/mL, respectively, when treated with LysPB32 (100 μg/mL). The combinations of carvacrol, eugenol, and thymol combined with LysPB32 effectively reduced the numbers of S. Typhimurium to below the detection limit (20 CFU/mL) after 12 h incubation at 37 °C, caused by membrane disruption. The relative bacterial fitness was significantly decreased in S. Typhimurium treated with carvacrol in the absence and presence of LysPB32. The combinations of carvacrol and eugenol with LysPB32 most effectively inhibited the growth of S. Typhimurium in cooked ground beef after 24 h of storage at 37 °C (>2 log reduction) and 7 days of refrigerated storage (>2 log reduction). Therefore, this study could provide the possibility of using the combinations of essential oils and phage endolysin as synergistic antimicrobials and also pave the way for designing effective food preservatives to enhance food safety and quality.