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Chapter III Material and methods
Plates were incubated at 37°C for 24 h and the antimicrobial activity titer was
determined by serial two-fold dilution method previously described (Kimura et al., 1998).
Bacteriocin activity was expressed as AU/ml and defined as the reciprocal of the highest
dilution showing a distinct zone of inhibition. To evaluate whether the antimicrobial activity
was due to peptides, culture supernatants were treated with 2 mg/ml pronase E (Sigma, USA)
for 30 min at 37°C before being tested for antimicrobial activity.
3.9.1 Batch fermentation for bacteriocin production by selected isolates
The isolated strains Lactobacilli casei LAM-1 strain was used for the production of
bacteriocin. Batch fermentation experiments using MRS broth (100 ml) were performed
under static conditions. Incubation time was optimized by performing the fermentation
experiment for 48 h at 37°C with 1% inoculum and the samples were collected upto 36 h of
incubation. Analysis of growth (CFU/ml) and bacteriocin activity (AU/ml) were performed at
each step.
3.9.2 Optimization of physical and chemical parameters for bacteriocin production by
selected Lactobacillus spp.
Parameters such as incubation time, media pH, incubation temperature, inoculum size
and media composition were optimized. Further the sets of experiments were performed at
different temperatures 25, 30, 35, 37, 40 and 45 °C. The flasks were incubated for 36 hrs and
samples were collected and analyzed. Optimization of media pH was done with MRS broth
adjusted to pH 4, 5.5, 6, 6.5, 7.0, 7.5 8, 9 and 10, inoculated with 1% of bacteriocin
producing culture grown overnight at 37°C. Inoculum size was optimized for 1, 2.5 and 5.0%
of inoculum at 37°C and constant pH 7.0.
3.9.3 Partial purification of bacteriocins
Bacteriocin was isolated from culture of the L. casei LAM-1 grown in MRS broth
medium at 37°C for 16 and 24 h. The pH of culture supernatant was adjusted to 6·5 by the