Bile tolerance, Bile Deconjugation and Cholesterol reducing properties
was adjusted to pH 7.0 using 1.0 N NaOH and then the volume was made
to 25 ml with distilled water. The cells were removed by centrifugation at
5000 rpm for 20 min. Fifteen ml of the resultant supernatant was adjusted
to pH 1.0 with 1 N HCl and the volume was increased to 24 ml with
distilled water. Free cholic acid from 3 ml of this broth was extracted in 9
ml ethyl acetate. Three ml of ethyl acetate layer was separated in a fresh
tube and dried at 60°C using Thermomixer (Eppendorf). The residues were
dissolved in 1 ml of 1 N NaOH and 6 ml of 16 N sulphuric acid and 1 ml of
1 N NaOH furfuraldehyde to develop color. The tubes were heated for 15
min at 65°C and cooled to room temperature. The color development was
stopped by adding 5 ml of glacial acetic acid in each tube. The absorbance
was measured at 660 nm against reagent blank. The concentration of free
cholic acid was calculated with the help of a standard curve and expressed
as mM.
Table 3. Reduction in cholesterol content by different strains of lactobacilli
% Removal of cholesterol
Lactobacilli Strain
24 h
48 h
PD2
54.67
69.66
PC27
28.59
42.12
PSC6
13.65
22.68
PH5
52.34
76.85
PFC21
21.02
47.81
Cholesterol assimilating ability
Cholesterol assimilation by the culture was performed in 9 ml MRS broth
containing 0.2% Sodium thioglycollate and 0.3% Sodium taurocholate to
whichȱ1ȱmlȱofȱfilterȱsterilizedȱhumanȱplasmaȱasȱtheȱsourceȱofȱcholesterolȱ
was aseptically added. The extraction of cholesterol remaining after 24 and
48 h of incubation was done as per Gilliland & Walker (1990) with slight
modification.ȱCellsȱwereȱharvestedȱbyȱcentrifugationȱatȱ8000ȱrpmȱforȱ10ȱ
min at 4ºC. Spent broth was collected and used for cholesterol assay. The
estimation of the remaining cholesterol was performed as per Rudel and
Morrisȱ(1973)ȱwithȱlittleȱvariation.ȱTheȱtestȱabsorbanceȱwasȱreadȱagainstȱ
reagent blank at 550 nm. The concentration of cholesterol was determined
fromthestandardcurveandexpressedasμg/ml.theresultswereexpressed
in terms of percent reduction in cholesterol in spent broth as compared to
uninoculated broth.
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