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

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Chapter III Material and methods
change from blue to yellow was recorded as positive fermentation results compared with the
positive and negative controls. Results were also compared with the absorbance of samples
read at 620 nm in an automated microplate reader (Bioscreen C, Oy Growth Curves Ab Ltd.,
Finland).
3.3.5 Strain selection and identification
Gram-positive, catalase-negative rods from traditional fermented nondairy products
and infant gut were selected for further studies to determine, if they were able to survive the
successive passage through solutions mimicking saliva, gastric juice and intestinal juice.
Strains with high survival rates were further identified using phenotypic and genotypic
characterization and identification techniques as described below.
3.3.5.1 Survival after the successive passages through artificial saliva, gastric and
intestinal juice
To test survival under gastrointestinal conditions, the protective effect of milk and the
effects of medium composition (lysozyme, pepsin, and pH of the medium) on bacterial
viability were assessed in vitro using the method was adopted by (Ehrmann et al., 2002;
Suskovic et al., 1997) in a model stomach/intestinal passage experiment in order to compare
the survival of potential probiotic strains. Reconstituted skim milk (15% w/v, Merck) was
inoculated with approximately 2 × 10 8 CFU/ml of an overnight culture of isolates. One ml
aliquot was removed, serially diluted in QSR and plated onto MRS agar to determine the
CFU/ml at 0 h.
To simulate the dilution and possible hydrolysis of bacteria in the human oral cavity,
the suspension was diluted to 1:1 ratio in a sterile electrolyte solution containing 6.2 g/l NaCl,
2.2 g/l KCl, 0.22 g/l CaCl2 and 1.2 g/l NaHCO3 to which lysozyme was added to a final
concentration of 100 ppm (parts per million), and incubated for 5 min at 37 °C. The sample
was subsequently diluted in 3:5 ratio with an artificial gastric fluid, consisting of the