Antimicrobial Efficacy of Liposome Encapsulated Nisin and Nisin's Inhibition Against Listeria monocytogenes in Fluid Milk at Different Storage Temperatures

Nisin is a naturally occurring food antimicrobial that inhibits many Grampositive pathogens, including Listeria monocytogenes, a bacterial pathogen responsible for ~500 deaths in the U.S. annually. Factors known to counteract the nisin activity in a food matrix include: antimicrobial interaction with food components, insolubility, protease inactivation, and target cell-driven envelope modifications. Encapsulating nisin in liposomes can help protect nisin functionality by regulating its introduction to the external environment. The objectives of this study were to determine the encapsulation efficiency (%EE) of nisin within liposomes as a function of encapsulation method and the capacity of liposomal nisin to inhibit L. monocytogenes in fluid milk. Phosphatidylcholine (PC) and phosphotidyl-DL-glycerol (PG) were used to prepare three lipid molar formulations: PC, PC/PG 7:3, and PC/PG 6:4 (mol.%). Liposomes were formulated to entrap the self-quenching fluorophore calcein and nisin. Unencapsulated analyte was removed via size-exclusion chromatography, and percent EE was determined. To determine antilisterial activity of liposomes, fluid milk samples containing L. monocytogenes (4 log10 CFU/mL) in combination with liposomal or unencapsulated nisin at 50 IU/mL were mixed and aerobically stored at 5 degrees C and 20 degrees C. Surviving L. monocytogenes were enumerated via plating on a non-selective microbiological medium after 0, 1, 3, 6, 12, 24, 48, and 72 hours of incubation. Encapsulation of nisin via extrusion resulted in a mean EE% of 84.20%, 77.33% and 80.78% for PC, PC/PG 7:3, and PC/PG 6:4 liposomes, respectively. Freeze-thaw cycling formed liposomes without detectable fluorophore entrapment. L. monocytogenes populations grew to 5 log10 CFU/mL after 72 hours at 5 degrees C and 8 log10 CFU/mL at 20 degrees C after 48 hours. Unencapsulated nisin exerted statistically greater inhibition of Listeria in skim milk compared to liposomal nisin, regardless of incubation temperature. No statistically significant differences in Listeria populations exposed to free or encapsulated nisin in whole milk were observed at either incubation temperature. Results indicate storage temperature and presence of milk fat exert greater influence then nisin delivery (free vs. encapsulated) over Listeria inhibition. Further research is needed to confirm these findings and develop more effective means of liposome entrapment of nisin for the inhibition of foodborne bacterial pathogens.