Determination of Bacteriocin Activity

To determine the bacteriocin antagonistic activity two methods has been performed. The first method was the plate activity assay and the second was well diffusion method.

(i) Plate Activity Assay:

MRS medium was prepared and plated under aseptic condition. The test organism (Salmonella typhi, E.coli, S.flexinariae) was swabbed over the respective MRS plate eventually. The swabbed plates were kept for 10mins to set. Using sterile inoculation loop, a loop full of direct Leuconostoc mesenteroides culture was touched over the swabbed plate and a small smear was made. The plates were incubated at 37oC for 24hrs.

(ii) Well Diffusion Method:

Mueller Hinton agar medium with 1.5% agar was prepared and plated under aseptic conditions. Using 6mm diameter well cutter, wells were made with equal distance, after the medium was set. A drop of the soft agar was dropped into the well to seal the bottom. The test organism Salmonella typhi, Escherichia coli, and Shigella flexinerrae were swabbed on the respective plates. After allowing for 10mins setting 100μl of the extracted bacteriocin was added into the well. The plates were incubated without inverting, at 37oC for 24hrs. Bacteriocin extracted by both the methods were loaded into their respective wells and checked for its antagonistic activity.

Result

(a) Well diffusion method – zone formed by bacteriocin against Salmonella typhi



(b) Well diffusion method – zone formed by bacteriocin against E.coli



(c) Plate Activity Assay






The bacteriocin was extracted and its antagonistic activity was studied against the indicator organisms by well diffusion method. The zone formed was measured and tabulated:

Salmonella typhi, zone formed – 11mm
Escherichia coli, zone formed – 6mm
Shigella flexinerrae – none

Discussion

Bacteriocin was extracted by cell free supernatant method and the crude supernatant was determined for its antagonistic activity. A well diffusion method was performed. In the activity it was observed that the bacteriocin produced by L.mesenteroides was effective against Salmonella typhi and Escherichia coli. But there was no effect against Shigella flexinerrae. The reviews say that the bacteriocins produced by L.mesenteroides are active against Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Bacillus cereus, L.monocytogenes has been considered as the major food borne pathogen and most activities were against them in food industries. Now these studies reveal the scope for bacteriocins, not only as preservatives but also as an antibiotic for many diseases and infections.