Extraction of Bacteriocin

Bacteriocin extraction from Leuconostoc was done by two methods.

- Bacterial cell lysis method
- Cell free extraction method.

The activity of the supernatant recovered from bacterial cell lysis method was not detectable. It is possible that much greater force is required to disrupt L. mesenteroides, although lactobacillus casei can be disrupted by this method, as shown by Arora et al, Whereas, cell free extraction method showed a great bacteriocin activity against salmonella typhi, and Escherichia coli.

The antibacterial activity appeared to be pronounced between early logarithmic and early stationary phase. Supplementation and/or replacement of nutrients demonstrated that larger quantities of bacteriocin could be produced by addition of yeast extracts (3.0%), NaCl (1.0-2.0%), glucose (1.0%) and tween 80 (0.5%). Maximal activity in composed medium was achieved at initial pH of 5.5 and incubation period of 48hrs at 30 – 37oC.

Procedure:

(i) Bacterial cell lysis method

An overnight culture of Leuconostoc mesenteroides was centrifuged at 10,000 rpm for 20mins at 4oC. The cell was suspended in 100ml of 0.1M phosphate buffer saline (pH 7). The centrifuge and washing was repeated and the cells were pelleted once more. Then, 10ml of PBS and 5g of glass beads were added to the cell pellet and centrifuged at 200rpm and 4oC for 1hr. the suspension was centrifuged and the supernatant was recovered for the determination of antagonistic activity.

(ii) Cell Free Extraction Method

The overnight culture of Leuconostoc mesenteroides was centrifuged at 10,000rpm for 20mins, at 4oC, to obtain a cell-free solution. The supernatant solution pH was adjusted to 7.0 to exclude antimicrobial effect of organic acids. The cell free solution obtained was stirred for 2hrs at 4oC and later centrifuged at 10,000rpm for 1hr at 4oC. The precipitate was resuspended in 25ml of 0.05M potassium phosphate buffer (pH 7.0). The new precipitate was collected and used in the bacteriocin activity assay.