Characteristics of enterocins of Enterococcus durans and their application in ham to inhibit Listeria monocytogenes

Our previous studies identified two lactic acid bacterium isolates, Lactococcus lactis subsp. lactis and Enterococcus durans with strong activity against L. monocytogenes growth in floor drains and E. durans with long survival ability.  Main products of nisin A and nisin B from isolate of L. lactis were identified.  Its bactericidal effect may be related with the production of nisin.  However, the bactericidal characteristic in E. durans was not identified.

The objectives of this project are: 1) to characterize the functions of this bacteriocin from the isolate of Enterococcus durans; 2) to determine its bactericidal specifics on Listeria monocytogenes and influence factors; 3) to validate its effects for protection in ready-to-eat meats from the contamination of L. monocytogenes.

 E. durans isolate #152 was grown in BHI medium and bacterial cells were washed and precipitated.  Plasmid DNA was extracted and its sequences were analyzed for enterocins.  The pellets were extracted in acidified ethanol and supernatant was dried in a vacuum shaker and freeze-dried.  The purity was analyzed by SDS-PAGE and the activity of separated bands were evaluated by loading the gel on BHI plate with 106 CFU/ml L. monocytogenes.  The bands separated by SDS-PAGE from partially purified enterocins were characterized for its amino-acid sequences. The enterocins were surface plated on the surface of hams and hams were inoculated with L. monocytogenes at different levels.  The contaminated hams were incubated at various temperatures for their efficacy from the contamination of L. monocytogenes at various temperatures.

By sequencing analysis, the plasmid isolated from strain #152 encodes at least two kinds of bacteriocins with high similarity to known L50A and L50A alignment revealed that two amino acids difference; and L50B and L50B alignment revealed that all amino acids were the same (100% similarity).  The purity and activity assays of the enterocins revealed that the molecular weight was around 5000 Daltons.  Inoculation studies of enterocins at 100 and 50 units/ml for reduction of L. monocytogenes in ham revealed that the antilisterial activity were temperature-related and both concentrations did not show significant difference.  At 8oC, its protection lasted more than 10 weeks; at 15oC, its protection lasted more than 33 days, and was much better than nisin.  However, its protection only lasted 12 days and similar to nisin at 21oC.

The biological activity of bacteriocins, LA50A and LA50B from E. durans #152 against growth of Listeria monocytogenes were revealed and characterized.  These determinations will enhance the application of strain, E. durans #152 for control of Listeria in floor drains at food processing facilities.