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3.10.5 Purification of the bacteriocin from the bacterial culture
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Chapter III Material and methods
A cell-free solution (CFS) was obtained by centrifuging the 1 l culture at 12,000 x g
for 10 min. The pH of the CFS was then adjusted to 6.5 with 1 N NaOH. The proteins of 500
ml CFS was precipitated with ammonium sulphate (45% saturation) overnight at 4°C with
gentle stirring and centrifuged at 10,000 x g for 30 min. The precipitate fraction was
resuspended in 0.5 ml sodium phosphate buffer (pH 7.2).
3.11 Molecular characterization of Bacteriocins
3.11.1 Determination of molecular weight of bacteriocin
The molecular weight of the bacteriocin was determined by Tricine-SDS
polyacrylamide gel electrophoresis (Schagger and Jagow, 1987). After 20 h cultivation, the
cells were harvested by centrifugation (9,660 x g, 15 min at 4°C) and proteins were
precipitated from cell free supernatant with 70% saturated ammonium sulfate. The precipitate
was resuspended in 0.1 of 20 mM sodium phosphate buffer (pH 6.0), desalted against
distilled water by using a dialysis membrane (MwCO =1.2 kD, Sigma-Aldrich, MO, USA).
Desalted bacteriocin containing sample was separated by gel electrophoresis (Biorad, USA),
the low molecular weight size marker (2.5-45 kD) was used (Biogene, USA). After
electrophoresis, one half of the gel was stained with Coomassie Blue; the other unstained half
was used to determine the position of active antimicrobial peptide. The indicator strain
Listeria monocytogenes ATCC 19111 embedded in BHI agar (10 5 CFU/ml) was used for the
positioning of active band.
3.11.2 N-terminal amino acid sequence analysis
The activity of the purified bacteriocin was confirmed on the SDS-PAGE gel, and the
gel was then blotted onto polyvinylidene difluoride membranes and stained with CBB R-250
(Biorad, USA). The objective bands were cut out and analyzed, and the N terminal amino