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

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Chapter III Material and methods
tested for residual bacteriocin activity. Untreated cell-free supernatants with above enzyme
and detergents were used as control.
3.10.2 Thermal stability
Thermal stability of the partially purified bacteriocin was determined by incubation of
partially purified bacteriocin solutions at 60°C for 20 min, at 100°C for 15 min, and at 121°C
for 15 min. Residual activity was determined for bacteriocin using sensitive strain as
indicator.
3.10.3 pH stability
The effect of pH on the activity of bacteriocin was tested by adjusting cell-free
supernatants from pH 2.0 to 12.0 (at increments of two pH units) with sterile 1 M NaOH or 1
M HCl. After 2 h incubation at 37°C, pH of all sample were readjusted to 6.0. Antimicrobial
activity was tested by using the well diffusion method as described above. Untreated samples
were used as the control.
3.10.4 Purification by chromatography
Bacteriocin from Lactobacilli casei LAM-1 was purified further by using gel filtration
chromatography Sephadex G-50 and 50 mM phosphate buffer (pH 6.5). Sephadex-G-50 (5 g)
was soaked in 200 ml of 50 mM phosphate buffer (pH 6.5) containing 0.1 g of sodium azide
and incubated for 72 h at room temperature. After soaking the gel was deaerated and poured
in a 0.9 x 60 cm column. Void volume was determined by passing blue dextran (2000 kDa)
through the column. The sample was loaded to the column 2.0 ml at a time. The above
mentioned buffer was used to elute the sample fractions each of 3.0 ml, which were collected
at a flow rate of 0.2 ml/min and read at 280 nm using spectrophotometer. Antimicrobial assay
was performed against indicator organism. The active fractions were pooled and subjected to
lyophilization (Modulyod-230, Thermo, USA). The lyophilized sample was used for further
analysis.