The objective of this study was to evaluate the effect of several bacteria isolated from different sources on cholesterol levels in vitro. Continuous cultures inoculated with different samples of yoghurt and pickles from different sources with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these isolated bacteria was undertaken and their potential to be used as a probiotics was studied. The acid and bile tolerance of strains was also evaluated with the media composed to mimic the enteric conditions to be used as the suitable candidate to be used as a probiotic. The bacterium isolated from urine sample was able to maintain viability at low pH and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g
(dry weight) of cells from the culture medium and therefore was regarded as a better candidate to be used as a probiotic among its peers in the experiment.

Probiotic bacteria have multiple potential health effects, including blocking gastroenteric pathogens , neutralizing food mutagens produced in the colon ,enhancing the immune response , lowering serum cholesterol,improved lactose utilization, anticarcinogenic activity and stopping intestinal dysfunction. In general, probiotic bacteria must colonize the gastrointestinal tract (GIT) of the host, have acid- and bile salt-tolerance, and block putrefactive bacteria in the GIT. Lactic acid bacteria (LAB), especially Lactobacillus spp. and several other Streptococcus spp. and Enterococccus species are important gastro-intestinal residentsand are used as probiotic strains to improve health. Lactobacillus have been used in fermented foods for several centuries without adverse effects and are classified as Generally Recognized as Safe (GRAS) because of their long history of safe use, particularly in dairy foods.
Cholesterol (5-cholesten-3-ol) has been linked with the development of colon cancer and the potent angiotoxic effects of several cholesterol oxides have led researchers to hypothesize a likely role for them in cardiovascular diseases . Cholesterol is sensitive to free radical oxidation by atomic molecule oxygen (O2) in the air and its autooxidation has been extensively studied.
An increased supply of cholesterol can lead to hypercholesterolemia and it can accelerate the formation of atherosclerotic changes in the circulatory system.
In this study, we evaluated the in vitro cholesterol-lowering effects of strains of lactic acid bacteria and
Streptococcus and Enterococcus spp from different origin. Further, we have investigated the ability of the cultures to tolerate acid and bile concentrations typically found in the upper gastrointestinal tract of humans. We hope this study provides further background and new, improved strains for the understanding of the purported action of probiotic bacteria on cholesterol levels.

The bacteria were isolated from different samples of yoghurt, pickle, and pathological laboratory. Few
Lactobacillus,Streptococcus and Enterococcus spp. were identified and probiotic characteristics were tested. After isolation of pure culture, stock cultures were maintained at 4°C in the Laboratory refrigerator. Working cultures were frequently subcultured and maintained initially in respective selectively defined medium for the defined Genus.
a. Selective Media for Lactobaccilus spp
Table 1. Lactobacillu s MRS Broth
COMPONENTS
1) Enzymatic digest of casein
2) Yeast Extract
3) Monopotassium phosphate
4) Ammonium citrate
5) Dextrose
6) Sodium acetate hydrate
7) Magnesium sulphate
8) Manganese sulphate
9) Ferrous sulphate
10) Polysorbate 80
11) Agar
pH maintained at 5.5+/-0.2 at 25deg Celsius
QUANTITY(gm/ltr)
10
5
6
2
20
25
0.575
0.12
0.034
1
15 b. Selective Media for Streptococcus spp
Table 2.
COMPONENTS
1) Enzymatic digest of Casein
2) Beef Extract
3) Maltose
4) Nucleic acid
5) Sodium chloride
6) Neomycin sulphate
7) Polymixin B sulphate
8) Agar
pH maintained at 7.3+/-0.2 25deg Celsius c. Selective media for Enterococcus spp
QUANTITY (g/ltr)
10
6.7
0.25
6
5
0.002
0.024
15

Table 3.
COMPONENTS
1) Enzymatic digest of Casein
2) Peptone
3) Yeast extract
4) Dextrose
5) Di-potassium phosphate
6) Sodium Azide
7) Potassium di-chromate
8) Agar
pH maintained at 7.2+/-0.2 at 37deg Celsius
QUANTITY
15
5
5
2
4
0.4
0.1
10
10
Table 4. Nutrient Agar
COMPONENTS
1) Beef extract
2) Peptone
3) NaCl
4) Agar
QUANTITY(gm/Ltr)
3
10
5
4
Table 5. Modified MRS Broth
COMPONENTS
1) Peptone
2) Lab Lemco powder
3) Yeast extract
4) Tween 80
5) K2HPO4
6) Triammonium Citrate
7) Sodium Acetate
8) MgSO4.7H2O
9) MnSO4.H2O
10) Sodium Thioglycolate
11) Glucose
QUANTITY(gm/ltr)
10
8
4
1ml/ltr
2
2
3
0.2
0.04
2
10
The amounts of cholesterol removed by the test cultures and those in the control broth which had not been inoculated were determined by
Zak’s Method
.
The composition of the mMRS broth was (grams per liter unless otherwise indicated): peptone , 10.0; Beef extract, 8.0; yeast extract , 4.0; Tween 80, 1 ml per liter; K2HPO4, 2.0;

triammonium citrate, 2.0; sodium acetate, 3.0; MgSO4 _ 7H2O, 0.2; MnSO4 _ H2O, 0.04; sodium thioglycolate,
2.0; and glucose, 10.0. Two different bile concentrations were used, 0.2 and 0.4% (wt/vol) bile salt, to mimic approximate levels in the intestinal tract. The final cholesterol concentration in the medium was 100 mg per liter.
The seed culture was added at a 1% (vol/vol) inoculum size to test tubes containing 9.9-ml aliquots of the test medium. The cultures were statically fermented for 24 h at 37°C under anaerobic conditions. Uninoculated sterile broth was used as the control. Following incubation, bacterial cells were removed by centrifugation (6000 X g , 20 min, 4°C), and the spent broth and uninoculated control broths were then assayed for their cholesterol content. The dry weight of the cultures was determined after drying the centrifuged cells to a constant weight in an 80°C oven.
Strains were compared for cholesterol assimilation in terms of their specific cholesterol uptake after the 24-h incubation period, according to the following equation: where B is cholesterol content in the uninoculated control (milligrams per liter), T is cholesterol in the culture medium (milligrams per liter), and W is cells (dry weight [grams]) after 24 h of incubation.
Table 6.
Absorbance of selected bacterial strains
Blank Std Ctrl US 1 US 2 LP 1 LP 2 MP 1 MP 2 MP 3 HC 1 HC 2 HC 3 HC 4 MP 4 PS 1 Test samples
OD at
540nm
- 0.346 0.055 0.068 0.013 0.039 0.035 0.035 0.038 0.030 0.042 0.020 0.037 0.036 0.055 0.035
ABBREVIATIONS USED:
US: Urine sample, LP: Lemon Pickle, MP: Mango Pickle, HC: House Curd, PS: Pathological specimen
Table 7. Amount of residual cholesterol after determining OD at 540nm(Table 6) of in vitro incubation of selected bacterial strains:
Test
Samples
(Calculation) mg/10ml
Control US 2
6.35
Cholestrol % -
1.50
76.37
LP 1
4.50
LP 2
4.04
MP 1
4.04
MP 2
3.12
29.13 36.37 36.37 50.86
MP 3
4.39
HC 1
3.46
HC 2 HC 3
4.85
30.86 45.51 23.62
2.31
HC 4
4.27
MP4
4.16
PS
4.04
63.62 32.75 34.48 36.37

The studied isolated cultures showed various ability to assimilate cholesterol from culture medium. The results of these studies are presented in Table 7. In the case of cultures isolated from traditional yoghurt, pickle , the quantity of assimilated cholesterol did not exceed 76.37% of its initial contents.
The strain of bacteria Lactobacillus spp isolated from urine sample assimilates higher amount of cholesterol indicating its efficiency to be used as a candidate in the field of probiotics with its potential hypocholesterolmic benefits.

1.
Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content
Do Kyung Lee1, Seok Jang1, Eun Hye Baek1, Mi Jin Kim1, Kyung Soon Lee2, Hea Soon Shin3, Myung Jun
Chung4, Jin Eung Kim4, Kang Oh Lee5 and Nam Joo Ha
2.
Assimilation of cholesterol in broth, cream, and butter by probiotic bacteria. Hatice Alog˘ lu Zübeyde Öner
Suleyman Demirel University, Faculty of Agriculture, Department of Food Engineering, Isparta, Turkey.
3.
Degradation of cholesterol by Bacillus subtilis SFF34 isolated from Korean traditional fermented flatfish
K.P. Kim, C.H. Rhee and H.D. Park.
4. Cholesterol Assimilation by Lactic Acid Bacteria and Bifidobacteria Isolated from the Human Gut
Dora I. A. Pereira* and Glenn R. Gibson.
5. Laboratory Manual for Practical Biochemistry By Shivaraja Shankara YM
6. Assimilation of Cholesterol by Lactobacillus acidophilus. S. E. GILLILAND,* C. R. NELSON, AND C.
MAXWELL.
7.
Cholesterol Assimilation by Commertial Yoghurt Starter Cultures.
Małgorzata Ziarno, Ewa Sekul, Alvaro
Aguado Lafraya , Warsaw Agricultural University SGGW.