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

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Chapter III Material and methods
indicates presence of ammonia. Cronobacter sakazakei MTCC 19111was used as an arginine
positive control strain.
3.4.4 Presence of meso-diaminopimelic acid (mDAP) in the cell walls
Overnight grown culture (1.5 ml) was centrifuged at 7,500 x g for 10 min in
eppendrof tubes and then resuspended in 200 μl of 4 N hydro chloric acid (HCl) and
hydrolysed at 100°C in a heating block overnight in tightly closed tubes. HCl was removed
by gently streaming nitrogen at 40 to 50°C into the solution for 1 h. The dry residue was
collected in a drop of water and spotted onto a thin layer chromatography (TLC) plate (pre-
coated cellulose plastic sheets 20 cm x 20 cm, Merck, no. 5577). A mDAP standard (5
mg/ml) was also spotted as a positive control. The ascending one-dimensional
chromatography was run in a solvent solution containing methanol: pyridine: 10 N HCl:
water in the ratio of 32:4:1:7. After drying, the chromatograms were developed with an acidic
ninhydrin spray (0.5 % w/v ninhydrin in 1-butanol:acetic acid [13:1] and heated at 100°C in
oven for 5 min. mDAP was characterised by a low R f (retention factor) and an olive green
colour which changes to yellow with time and light exposure.
3.4.5 Determination of lactic acid enantiomers produced
Lactic acid enantiomers were determined using an enzymatic kit (API System;
BioMerieux, Marcy I’Etoile, France) based on the oxidation of D-lactate or L-lactate into
pyruvate in the presence of NAD +
, which in turn reduces to NADH, by the corresponding
enzymes, D-lactate-dehydrogenase or L-lactate-dehydrogenase.
The equilibrium of these reactions lies on the side of lactate. By trapping pyruvate in a
subsequent reaction catalysed by glutamate pyruvate transaminase in the presence of L-
glutamate, the equilibrium can be shifted in favour of pyruvate and NADH. The amount of
NADH formed in the mentioned reactions is stoichiometric to the amounts of lactic acid
oxidized by the lactate-dehydrogenases. The increase of NADH was determined