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

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Chapter III Material and methods
electrolyte solution mentioned above; containing 0.3% (w/v) pepsin and pH was adjusted to
2.5. If required, pH was readjusted to 2.5 with 5 M HCl. After 1 h of incubation at 37°C,
another 1ml aliquot was removed, serially diluted in QSR and plated onto MRS agar. To
simulate the dilution in the small intestine, the remaining volume was diluted 1:4 using an
artificial duodenal secretion (pH 7.2). One ml aliquots were again removed after 3 h, serially
diluted in QSR and spread-plated onto MRS agar to determine the CFU/ml in duplicate.
Experiments were conducted in triplicate. Only those strains which survived the simulated
gastrointestinal passage in detectable numbers were unequivocally identified and further
investigated.
3.4 Phenotypic characterization
3.4.1 Growth at different temperatures
Overnight cultures were inoculated (1%) in MRS broth and incubated in water bath at
15°C and 45°C for 5 days. Turbidity and growth was measured at 600 nm using
spectrophotometer (Hitachi U2900, Japan)
3.4.2 Production of gas from glucose
In order to determine the homofermentative and heterofermentative characterization
of isolates, CO2 production from glucose test was applied. Citrate lacking MRS broths and
inverted Durham tubes were prepared and inoculated with 1% overnight fresh cultures. Then
the test tubes were incubated at 37°C for 5 days. Gas production in Durham tubes was
observed during 5th day which is the evidence for CO2 production from glucose.
3.4.3 Hydrolysis of arginine
Overnight cultures were inoculated (1%) in MRS arginine broth at 37°C for 24-72 h.
For detection of ammonia, 100 μl samples were spotted onto a white spotting tile and an
equal volume of Nessler’s reagent was added. Immediate appearance of a dark orange colour