from indigenous fermented foods and human gut ... - Thapar University

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Chapter III Material and methods
micro centrifuge tubes were frozen and the bottom of the tubes containing cell pellets was
removed. Cell pellets were digested with 1 ml of 3 M HNO3, vortexed vigorously and
incubated at an ambient temperature overnight. Concentrations of intracellular K + in the cell
pellets were determined by ICP (Perkin Elmer-Sciex Elan DRC Plus, USA).
3.12.4 Measurement of intracellular ATP content
Extracellular ATP levels of bacteriocins treated S. typhimurium ATCC 19585 was
determined as described previously (Bruno, 1993) with some modifications. Cells were
suspended in 2.5 mM sodium phosphate buffer (pH 7.0) with 10 mM glucose and 100 µg/ml
of bacteriocin (2,800 AU/ml). At various times, 20 ml samples were taken to determine the
extracellular ATP concentrations, respectively. 20 ml samples were immediately mixed with
80 ml of dimethyl sulfoxide. 50 ml samples were then spun down immediately for 2 min, and
20 ml of the supernatant was removed and mixed with 80 ml of dimethyl sulfoxide. All
samples were diluted with 5 ml of sterile deionised water. ATP concentrations were
determined by inactivation the luciferin-luciferase enzyme assay.
3.13 Inhibition of pathogen by bacteriocin in a simulated /laboratory prepared food
matrix
Fresh vegetable samples like cucumber, radish, carrot and tomato were purchased at
local supermarkets and kept under refrigeration for no longer than 24 h until use. These
vegetables were sliced (ca. 1.0 cm in size) and artificially contaminated (10 µl per piece)
with a sterile saline solution suspension of S. typhimurium ATCC 19585 (2.0 x10 8 CFU/ml)
previously grown overnight in brain heart infusion infusion broth at 37 °C. Following
inoculation, vegetables were allowed to dry for 1 h at room temperature and then were treated
by immersion for 5 min at room temperature in 5 ml of sterile distilled water (controls) or
distilled water containing bacteriocin (25-100 µg/ml). After immersion treatments, excess
immersion solution was drained on sterile filter paper, and samples were stored in sterile