Evaluation of a liquorice extract as a novel antimicrobial

The foodborne disease listeriosis is rare but has a high mortality rate therefore it is a major concern for the food industry. It is caused by the Gram-positive bacterium Listeria monocytogenes which can survive mild food preservation conditions, (e.g., high salt and low pH), and its psychrotrophic nature means that it can grow at refrigeration temperatures. Hence listeriosis is associated with the consumption of a broad range of ready-to-eat (RTE) foods, including sliced deli meats. Liquorice extract is common a food flavouring that is known to have antimicrobial activity and this project was designed to investigate the antimicrobial activity of a waste product produced during the production of liquorice flavourings for the food industry. The antimicrobial efficacy of the liquorice extract was used to challenge different types of bacteria to determine whether this food by-product had antimicrobial activity. A range of both Gram-positive and Gram-negative bacteria were tested and it was found that the extract was only effective against Gram-positive bacteria suggesting that the extract had a similar mode of action in each case with a concentration of 50 μg ml-1 resulting in total growth inhibition, therefore this was defined as the MIC. The use of bacterial strains containing plasmids expressing bioluminescence revealed that at sub-MIC concentrations (12.5 μg ml-1) the metabolic state of the cells was reduced, indicated that membrane integrity was affected. The mechanism of action of the liquorice extract was further investigated using a fluorescent LIVE/DEAD® stain. Exposure to the MIC for this extract (50 μg ml-1) resulted in total growth inhibition and the presence of non-viable cells was detected using fluorescence microscopy. In addition, fluorescent microscopy revealed the formation of long filaments consisting of cells that had not completed the cell division process, which indicates that cell wall synthesis was also affected. In contrast no antimicrobial effect or toxic effect was detected when the extract was used to treat Gram-negative bacteria or the yeast Saccharomyces cerevisiae. When applied to the surface of sliced deli meat, inhibition of Listeria growth was seen for up to 10 days, hence the extract has the potential as a natural food preservative and could be of use to improve the safety of this type of RTE product. To confirm that the extract was safe to use as a food preservative, the mutagenic potential of the extract was assessed using the Ames test for detecting carcinogens and mutagens by using different strains of Salmonella Typhimurium including TA100, TA102 and TA1535, no adverse results were gained. In addition in vitro cytotoxicity was measured using Caco-2 cells and the MTT cell viability assay. At the MIC concentration, some cytotoxicity was detected, and this requires further investigation but these results could indicate the potential for use of liquorice extract an anti-cancer agent. Overall the findings of this research suggest that the liquorice extract has a specific effect on Gram-positive bacteria and further work could lead to its use as an antimicrobial agent to control the growth of these bacteria either in food systems or in other settings