DEGRADATION OF CHOLESTROL BY SOME BACTERIA ISOLATE

FROM DIFFERENT SOURCES

INTRODUCTION:

CHOLESTEROL: Cholesterol is a compound of the steroid. It is the main sterol found in

animal and human tissues and in plasma lipoproteins. It is present as free cholesterol or
combined with long-chain fatty acids as cholesterol esters. It plays important role in production
of hormones , vitamin D and substance for digestion. It also needed to build healthy cells,
although too much cholesterol deposition in the body increases the risk of heart disease causing
cerebrovascular, coronary and peripheral vascular diseases (Steinberg, D.,et al.,2006). Many
bacteria have ability to degrade steroid compounds like cholesterol that includes members of the
genera Arthrobacte, Brevibacterium, Corenybacterium, Mycobacterium, Streptomyces,
Rhodococcus, Pseudomonas and Comamonas (Fernandez de las Heras,et al.,2011).

Fermented foods processed using lactic acid bacteria (LAB) are known for their beneficial
effects in human nutrition. So that LAB are considered as promising probiotic candidates and
studied to explore their desirable properties (Iranmanesh, et al., 2014). Cholesterol-rich foods
including chicken liver, turkey giblets, salmon, lamb, egg yolk, beef brain and shrimps are a rich
source of new interesting cholesterol degrading bacteria (Ouf et al., 2012).
MATERIALS AND METHODS:
The degradation of cholesterol by bacteria isolate from different sources are give below.
1.Isolation of cholesterol degrading bacteria from cow’s milk:
Thirty two fresh, raw cow’s milk sample were collected from fifteen farms.
Sample preparation:
Samples are incubated at 37ºC. The coagulated samples were then activated in MRS broth at
37°C for 24h in order to obtain enriched cultures. Enriched cultures were further streaked on
MRS agar medium and incubated under anaerobic condition using a candle extinction jar with a
moistened filter paper to provide a CO2-enriched, water-vapor saturated atmosphere at 37°Cfor
48 hours. Single colonies picked off the plates were sub cultured in MRS broth at 37°C for 24
hours. The isolated Lactobacillus species were transferred from MRS medium to cholesterol agar
(0.1 %) and incubated at 30° C for 12 hrs. The ability of the isolated bacteria to decompose
cholesterol was evaluated by measuring the zone of translucency around colonies on agarized
medium that contain cholesterol as a sole carbon source (Zanin,VA,1968). The developed
colonies were further enriched in cholesterol broth and incubated at 30°C for 24 hrs. The
enriched culture was homogenized to obtain cell free extract and was centrifuged at 7000 rpm for
10 minutes. The supernatant obtained was taken as a crude sourceof an enzyme cholesterol
oxidase (Salama, et.al,2012)
2.Isolation of cholesterol degrading bacteria from food:
Isolation of lactic acid bacteria (LAB) :Ten raw Lamb meat samples were collected from the
local market.
Sample preparation:
Samples were transferred to the laboratory and prepared. From meat samples, 25 g were taken
using to a depth of 2-3 mm, placed with 250 ml peptone water in a sterile plastic bag and
homogenized. These initial suspensions were incubated at 37ºC for 24 h in order to obtain
enriched cultures. The enriched cultures were further streaked on MRS agar and incubated under
anaerobic conditions at 37ºC for 48 h. Single colonies picked off and sub-cultured in MRS agar
at 37ºC for 24 h. The cultures were examined microscopically and re streaked on the MRS agar
medium for purification.
Screening of cholesterol degrading LAB: The pure isolated bacteria were transferred to
Minimal Salt Cholesterol (MSC) agar plates containing 0.2% cholesterol as a sole source of
carbon and energy (Nishiya, et al., 1997). The pH was adjusted to 7. For suspending cholesterol
and avoiding its coagulation, it was first dissolved in 10 ml solution of 20% isopropanol plus
10% of Tween 80 and then added to the medium. The ability of the isolated bacteria to
decompose cholesterol was confirmed by measuring the zone of translucency around colonies on
MSC agar after incubation at 37ºC for 7-12 days (Yazdi, et al., 2000).
Cholesterol degradation by cell-free supernatant: Fourteen cholesterol degrading strains
were subjected for preparation of cell-free supernatants (CFSs) as a crude source of extracellular
cholesterol degrading enzymes. The developed colonies were further enriched in MSC broth and
incubated at 37ºC for 7 days. The enriched culture was centrifuged at 10000 xg for 5 min. CFS
was then sterilized by using a 0.45-mm filter and stored at 80 ºC until needed. The cholesterol
degrading ability of CFS was measured by enzymatic colorimetric cholesterol oxidase
Peroxidase method (Kulkarni, et al.,2013)
Identification of the cholesterol degrading bacteria: According to the results of cholesterol
degrading ability of CFSs, isolates that showed high ability to degrading cholesterol were taken
for identification to species level. The carbohydrate fermentation profiles of the selected strains
were determined using API 50 CHL medium according to the manufacture’s instruction
(Iranmanesh, et al., 2012). In addition, standard tests were performed for identification of the
target strain in accordance with Bergey’s Manual of Determinative Bacteriology (Holt, 1994).
These tests included cell form and size, Gram staining, spore formation, motility, colony
pigmentation, production of UV-fluorescent pigments. Identification was confirmed by
determine partial sequence of 16S rRNA gene sequences.
Resistance of LAB to simulated gastric and intestinal juices Strains: Resistance to gastric
and intestinal juices simulated the in vivo environment as described by (Kos et al.,2000).
Simulated gastric juice (SGJ) was prepared by suspending pepsin in a sterile NaCl (0.5%) and
adjusting the pH to 1.5, 2.0, 2.5 and 3.0 with concentrated HCl. Simulated small intestinal juice
(SSIJ) was prepared by suspending pancreatin and bile salts (1.5, 2.0, 3.0 and 5.0 mg/mL in a
sterile NaCl (0.5%) and adjusting the pH to 8.0 with 0.1 mol/L NaOH. Bacterial strain was
activated in 10 mL of MRS broth at 37ºC with reciprocal shaking (120 rpm) for 48 h. Cells were
collected and washed twice with phosphate buffer of 0.1 M at pH 6.8. Cells were resuspended in
phosphate buffer to about 1x108 CFU/mL. Resuspended cells (0.2 mL) were vortexed with
gastric or small intestinal juice (1 mL). The total viable count was determined after treatment
with gastric or small intestinal juice for 2 and 4 hours respectively by using pour plate method
using MRS agar. Plates were incubated at 37 ºC for 48 hours. Results of log (CFU/mL) are
expressed as the mean of three replicates ± standard deviation.
Antibiotic sensitivity: The susceptibility to 15 antibiotics was determined using the disk
diffusion method on Mueller Hinton agar (C.L.S.I., 2007). The antibiotics used in the present
study were purchased from Bio-Rad, Laboratories, GmbH., Germany.
Antibiotic resistant pattern of three cholesterol degrading LAB isolated from raw meat
Antibiotic Lac . sakei Lac . rhamnosus Leu .
(concentration) GMK01 GMK02 mesenteroides
and mode of action GMK03
Inhibition of cell wall synthesis
Ampicillin (10 μg) - - -
Oxacillin (1 μg) R - R
Penicillin G (10 U) - - -
Bacitracin (10 U) R R R
Vancomycin (30 R R R
μg)
Cefixime (5 μg) R - R
Cloxacillin (5 μg) - - -
Inhibition of protein synthesis
Doxycycline (30 R R -
μg)
Clindamycin (2 - - -
μg)
Chloramphenicol - - -
(30 μg)
Erythromycin (15 - - -
μg)
Gentamycin (10 - - -
μg)
Kanamycin (30 - - -
μg)
Streptomycin (10 - - -
μg)
Tetracycline (30 R - -
μg)
R, resistant; (-), sensitive

Evaluation of cholesterol degrading ability:

The cholesterol degrading ability of the bacterial isolates was evaluated by enzymatic
colorimetric cholesterol oxidase (CHOD)-Peroxidase (POD) method. The assay was performed
using cholesterol estimation kit . The reagents were reconstituted as per the instructions provided
in kit and the cholesterol standard had concentration of 200 mg/dl. All the reagents were mixed
in the respective tubes labeled blank, standard and test as per the instruction,1 ml of supernatant
obtained from the bacterial isolates was used as source of cholesterol oxidase. The reaction
mixture was thoroughly mixed and incubated for 10min at 370C. Absorbance of standard and
test were read against the reagent blank at 505 nm. The concentration of cholesterol in test was
calculate dusing formula, Cholesterol (mg/dl) Absorbance of test/ Absorbance of standard X
Conc. of standard (mg/dl).Further the reduction in cholesterol (mg/dl) and percent reduction in
cholesterol were evaluated.
RESULT AND DISCUSSION:
In the present study total 11 cholesterol degrading bacterial isolates were obtained from milk. All
11 isolates showed a variable zone of translucency around colonies indicating the cholesterol
degrading ability. The quantitative determination of cholesterol degrading ability of the bacterial
isolates revealed that all bacterial samples showed a significant decrease incholesterol
concentration.
The maximum decrease level in cholesterol concentration (97.20 %) was shown by the isolate
no. 11, whereas, the minimum (42.88%) was recorded incase of isolate No 5. as compared to
standard cholesterol.

Screening and identifying cholesterol degrading LAB Lamb meat is well known as a one of
the most cholesterol-rich meat. Eighty-seven isolates collected from MRS agar were preliminary
identified as LAB based on their Gram reaction, morphology and catalase test. All isolates were
described as catalase-negative and Gram-positive bacilli in pairs or chains and cocobacilli . Out
of 87 isolates, only fourteen showed cholesterol degrading ability on mineral salt agar
supplemented with 0.2% cholesterol. Out of these isolates only three isolates showed strong
ability to degrading cholesterol in the MSC agar. Six isolates had moderate ability to degrade
cholesterol. The other isolates observed weak cholesterol degradation on MSC agar.
CONCLUSION:
In conclusion, the strains have high ability to degrading cholesterol even after exposure to 0.3%
bile salts and low acidity at pH 2.5. Moreover these strains had high ability to adhere to Caco-2
cells.So that the isolated Lactobacillus sakei, Lactobacillus rhamnosus and Leuconostoc
mesenteroides could be considered as potential probiotic strains for food industry and human
nutrition. Since cholest-4-en-3-one has positive uses against obesity, liver disease, and
keratinization (Wu, et al., 2015). Leu. mesenteroides isolated in the present study could be
considered as a promise probiotic strain.
The results in the present study are in accordance with the experimental findings of ( Sieladieet,
et al.2011). However they reported the probiotic properties of lactobacilli strains isolated from
raw cow milk in the Western highlands of Cameroon. The results indicate that microorganisms
present in milk can utilize cholesterol as their carbon source and can be exploited for the
development of pre/pro-biotic specially top revent the cardio artery diseases.
RECOMMENDATIONS:
Further studies are needed to understand the mechanism of cholesterol degradation using the
extracellular cholesterol oxidase especially by Leuconostoc mesenteroides.
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