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

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Chapter III Material and methods
addition of 0.1 N NaOH. The cells were removed by centrifugation (5500 rpm, 4°C, 20 min).
The cell-free culture supernatant was brought to a final ammonium sulphate concentration of
20 and 40% saturation by slow addition of the salt, and was stirred overnight at 4°C. Then,
the mixture was centrifuged (5500 x g, 4°C, 30 min) and the surface pellicles and bottom
pellets were harvested and resuspended in 10 ml of sodium phosphate buffer (pH 6·5). To one
volume of the resuspended product, 10 volumes of a methanol-chloroform mixture (1: 2, v/v)
were added, and the mixture was extracted at 4°C for 1 h. The sample was centrifuged (5500
rpm, 4°C, 30 min), supernatant fraction decanted and the pellet was air-dried. The pellet was
resuspended in 2 ml of milli-Q water. The partially purified bacteriocin was stored at 20°C.
Protein concentrations were determined by a modification of the method of Bradford (1976)
using a Protein Assay Kit from BioRad Laboratories (California, USA). Bovine serum
albumin was used as a standard.
3.10 Characterization of partially purified bacteriocin
3.10.1 Effect of enzymes and detergents
Sensitivity of the bacteriocin to enzymes: proteinase K, (1000 units mg/ml), α-
amylase (724 units mg/ml), DNase and RNase (470 units mg/ml), and pepsin (290 units
mg/ml) (Sigma, MO, USA) was tested using partially purified bacteriocin samples. Each
enzyme was dissolved in 10 mM sodium phosphate buffer (pH 7.0), and the solutions were
added to the bacteriocin solution for a final concentration of 1 mg/ml. Following incubation
at 37°C for 2 h, the mixture was heated at 100°C for 10 min to denature the enzymes. The
residual bacteriocin activity (AU/ml) was measured by bioassay, using Listeria
monocytogenes ATCC 19111 as an indicator strain. The effect of detergents on bacteriocin
activity was also determined by adding sodium dodecyl sulfate (SDS), Tween 80, Tween 20,
EDTA and urea at final concentration 0.1, 1, 2 or 5% to the cell-free supernatants in a
separate experiment. All detergent-treated supernatants were incubated at 37°C for 1 h, and