Cholesterol reduction in vitro by novel probiotic lactic acid bacterial strains of Enterococcus isolated from healthy infants’ stool

The present study aimed to isolate and evaluate the potentiality of some probiotic lactic acid bacteria from infants stool to degrade and reduce cholesterol level in fermentation medium. Out of 50 lactic acid bacterial isolates recovered from healthy infants (3 to 18 months old) stool, two bacterial isolates W7 and Y1 showed highest cholesterol reduction percentage (68.38 and 71.6%, respectively). The cholesterol reduction potentiality by the two isolates W7 and Y1 were optimized to 94 and 98%, respectively, by studying the different cultural conditions; inoculum size, inoculum age, pH, incubation temperature, incubation period and cholesterol concentration. The gas chromatography analysis of the fermentation extract revealed that the resulted end product of cholesterol was 5á-cholestane-3à,25-diol (C27H48O2) of molecular weight 404. The selected bacterial isolate Y1 was identified phenotypically and genotypically as Enterococcus faecium and deposited in GenBank under the accession number KY788356.


INTRODUCTION
Cholesterol is a waxy steroid compound formed in all cells of the animal and human body and required for formation of steroid hormones (Young, 2001, Rigotti et al., 2003), a precursor for biosynthesis of bile acids in the liver that help the body to digest fat (Russell, 2003).Longstanding increased serum cholesterol levels may lead to atherosclerosis and as a result lead to development of cardiovascular diseases (CVDs) (Ngongang et al., 2016;Tsai et al., 2016).CVDs were reported to be responsible for 30% of deaths worldwide and predicted to remain the leading causes of death (WHO, 2013).Both pharmacologic and nonpharmacologic approaches, including follow healthy diet, behavior alteration, and regular practice, are common strategies to lower serum cholesterol levels (Dunn-Emke et al., 2001), Although they record cholesterol-lowering ability, but in some cases unwanted side effects can be produced, such as gastrointestinal disorder (Davidson et *Corresponding author.E-mail: am_rasmey@yahoo.com. Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

al., 1999).
There are some microorganisms that live as normal flora in animal and human gut, known as probiotics.The presence of these microorganisms in healthy form have different healthy benefits such as protection against gastrointestinal disorder (Vanderhoof and Young, 1998;Niv et al., 2005;Saxelin et al., 2005), reduction of cholesterol level in blood and lipids (Gilliland and Walker, 1990;Naidu et al., 1999), synthesizing extracellular bacteriocins as antimicrobial metabolites (Gill, 2003) and enhancing the immune system by production and circulation of serum antibodies (Maassen et al., 2000;Ezendam and van Loveren, 2006).An adequate quantity administration about 10 9 CFU per day of external probiotics especially lactic acid bacteria will be appropriate to maximize and balance the human gastrointestinal microbiota by replacing harmful pathogens and reinforcing the natural defence mechanisms (Casas and Dobrogosz, 2000;Ouwehand et al., 2002).Probiotics for human feeding are preferably isolated from the gastrointestinal (GI) tract of healthy human (Saarela et al., 2000).
Acid-producing probiotics have been extensively used for cholesterol reduction in human blood serum by applying theses bacterial strains in various fermented foods, milk products and beverages (Pereira and Gibson, 2002;Ouwehand and Vesterlund, 2004) and several studies reported the hypocholesterol ability of lactic acid probiotics in vitro or in vivo, especially the strains of genera Lactobacillus, Enterococcus and Bifidobacterium (Pan et al., 2011;Wang et al., 2012;Huang et al., 2013;Hu et al., 2013;Tsai et al., 2016).Lactic acid bacteria of cholesterol reduction potentiality are now available as probiotics to consumers in forms of milk products such as yoghurts and cheese, formulated tablets and lyophilized powders.However, long-term hypocholesterolemic effects requiring continuous research for novel health enhancing probiotic strains from healthy individual origin is still attracting area.
Different mechanisms for cholesterol removal by probiotics have been reported, such as deconjugation of bile salts by bile-salt hydrolase (BSH) (Ahn et al., 2003), cholesterol absorption into bacterial cell membranes (Kimoto et al., 2002), production of short-chain fatty acids (SCFAs) during the probiotics growth (Trautwein et al., 1998), and cholesterol transformation into coprostanol (Lye et al., 2010).Additionally, several cultural conditions such as media composition, pH, temperature, and inoculum size may change the metabolic pathways by altering pyruvate metabolism and external electron acceptors resulted in different end-products (Axelsson, 1998;Annuk et al., 2003).
The main objective of this study was to isolate new lactic acid probiotic strains of human origin with potential of highly cholesterol reduction and optimize the cultural condition for the cholesterol reduction process in addition to follow the resulted end products formed by the selected bacterial strain.

Isolation of microorganisms
A total of 50 different healthy infants (3 to 18 months old) stool samples were obtained from different baby centers located at Ismailia and Suez Governorates of Egypt, transferred in sterilized plastic bottles to the laboratory and immediately used for isolation of lactic acid bacteria.Ten grams of each infant stool sample were aseptically homogenized in 90 ml of sterile saline solution and sequential decimal dilutions were done.One milliliter of each dilution was inoculated into sterilized plates and about 20 ml of melted de Man, Rogosa and Sharpe (MRS) agar medium of pH 6.2 were poured in each plate.The used MRS agar (De Man et al., 1960) medium contained (g/L distilled water): glucose 10, peptone 10, beef extract 10, yeast extract 5, K2HPO4 2, sodium acetate 5, tri-ammonium citrate 2, MgSO4·7H2O 0.2, MnSO4• 4H2O 0.2 and Tween 80 (1 ml).The plates were incubated for 3 days at 37°C under anaerobic conditions (GasPak System -Oxoid, Basingstoke Hampshire, England).The separated colonies were picked, subcultured, maintained on MRS slants and stored at 4°C for further experiments.

Preparation of bacterial inoculum
A loopful of refreshed bacterial culture was inoculated and grown in bottles containing 20 ml of MRS broth medium and incubated for 24 h at 37°C under anaerobic conditions in CO2 incubator.

Cholesterol degradation in fermentation medium by the isolated bacteria
Cholesterol stock solution was prepared by dissolving 10 mg of cholesterol in 1 ml of 96% ethyl alcohol and filter sterilized.For each culture to be tested, 70 μl of cholesterol solution was added to 10 ml of MRS broth (final cholesterol concentration 70 μg/ml) and 1% (v/v) of freshly grown culture was added and incubated anaerobically at 37°C for 24 h.After incubation, the cells were removed by centrifugation at 10,000 g for 10 min at 4°C.

Determination of cholesterol concentration in fermentation medium
The cholesterol concentration was determined in the supernatant using a colorimetric method described by Rudel and Morris (1973).Three milliliters of supernatant, 2 ml of 33% (w/v) KOH and 3 ml 96% ethanol were placed in a capped test tube, vortexed for 20 s and incubated for 15 min at 60°C in a water bath.After incubation, the mixture was removed and cooled under tap water, then 5 ml of hexane and 3 ml of water were added and vortexed for one min.One milliliter of the hexane layer was transferred into a dry clean test tube and evaporated under nitrogen gas.One milliliter of cholesterol liquicolor enzymatic kit (Spinreact Kit, Spain) was added (Guo et al., 2011).The solution was mixed and left for 5 to 10 min at 37°C and absorbance was measured at 500 nm with a spectrophotometer.The cholesterol reduction was calculated as percentage from the following equation: A = 100 -(B/C)×100, where A = % of cholesterol removed, B = absorbance of the sample containing the cells and C = absorbance of the sample without cells.

Morphological characterization
The morphological growth characters of the selected isolate Y1 was conducted on MRS agar medium and the colony color, shape and texture were recorded.The cell shape and arrangement were determined by microscopic examination after Gram staining technique in accordance with Collins and Lyne (1985).

Physiological and biochemical characterization
The physiological and biochemical characteristics were estimated according to the standard methods.The selected bacterial isolate Y1 was examined for Catalase production (Wittenberg, 1964), carbohydrate utilization (Pridham and Gottlieb, 1948), growth at 6.5% NaCl, growth at different temperatures (15 and 45°C), production of CO2 from glucose and production of NH3 from arginine.

DNA extraction:
The extraction of bacterial DNA was performed according to the method of Kozaki et al. (1992) using DNA preparation kit (Jena Bioscience).

Polymerase chain reaction (PCR) amplification:
The PCR amplification was performed using the 16S primers: 16S F: 5′-GAGTTTGATCCTGGCTTAG-3′ and 16S R: 5′GGTTACCTTGTTACGACTT-3′ and Qiagen Proof-Start Tag Polymerase Kit (Qiagen, Hilden, Germany) according to standard PCR protocol.The following substrates were combined in a total volume of 25 µl containing about 20 ng of template DNA, 12.5 µl PCR Master Mix, 20 pmol (2 µl) of both forward and reverse primers and the reaction was completed by 8.5 µl of deionized water.The reaction conditions were: an initial denaturation at 94°C for 5 min, 37 cycles of denaturation at 94°C for 30 s, annealing at 51°C for 30 s, and extension at 72°C for 30 s.A final extension was performed at 72°C for 5 min.PCR products were purified from gel with the QIAquick gel extraction kit (Qiagen, Hilden, Germany).Then analyzed by electrophoresis on 1.5% agarose gel in 1X TAE buffer and finally observed and pictured under UV light.
DNA Sequencing: Sequence similarity was recorded by searching the homology in the Genbank DNA database using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
CLUSTALW program (http:// clustalw.ddbj.nig.ac.Jp/top-ehtml) was used for evaluating multiple sequence alignment and molecular phylogeny.The tree view program was applied for phylogenetic tree illustration.

Extraction of the remaining cholesterol and its derivatives from the bacterial culture
The LAB isolate (Y1) was grown in MRS broth medium supplemented with cholesterol (70 μg/ml) under the optimized cultural conditions and the obtained bacterial cells with the remaining broth medium were homogenized in chloroform (1:1, v/v) by sonication.The solvent-supernatant mixture was agitated and separated by separating funnel and filtered on sodium sulphate anhydrous to remove any water from the extract.All obtained chloroform extracts were dried using rotary evaporator at 50°C under vacuum and the crude extracts were then subjected to analysis by gas chromatography-mass spectra to clarify the structure of the resulted metabolites.

Determination of cholesterol degradation products using gas chromatography-mass spectra (GC-MS)
The GC-MS analysis of the crude extracts was carried out at the Central Laboratory, National Research Center of Egypt with the following specifications: Instrument, a TRACE GC Ultra Gas Chromatographs (THERMO Scientific Corp., USA), coupled with a THERMO mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer).The GC-MS system was equipped with a TG-5MS column (30 m × 0.32 mm i.d., 0.25 μm film thickness).Analyses were carried out using helium as carrier gas at a flow rate of 1.0 ml/min.Temperature program: 50°C for 3 min; rising at 5°C/min to 300°C and held for 5 min.The injector and detector were held at 220 and 200°C, respectively.Diluted samples (1:10 diethyl ether, v/v) of 1 μl of the mixtures were always injected.Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 50 to 500 (Said-AL Ahl and Omer, 2016).

Statistical analysis
Analysis of variance (ANOVA) was performed using CoStat V. 6.311 (CoHort software, Berkeley, CA94701).Cholesterol reduction mean values were compared at 5% significance level using Tukey's test.Least significant difference (LSD) test was used to test the significant differences between the whole means of different groups and compared with the critical difference at the 5% level.

RESULTS AND DISCUSSION
Fifty lactic acid bacterial isolates were recovered from the collected 50 infants stool samples based on criteria of lactic acid bacteria such as morphological shape, catalase negativity, Gram positive, and lactic acid formation.The selected bacterial isolates were tested for their potentiality to reduce or biodegrade cholesterol on basal cholesterol broth medium.A total of 10 out of 50 tested lactic acid bacterial isolates exhibited cholesterol reduction in high percentages (Table 1).Apparently, cholesterol reduction varied among the tested bacterial strains ranged from 48.22 to 71.6% reduction from the initial cholesterol concentration and the highest reduction mean values of 71.6 and 68.38% were obtained by the two isolates Y1 and W7, respectively.The results in the present study are in accordance with the experimental findings of Sieladie et al. (2011) who reported that about 11 lactobacilli strains isolated from raw cow milk exhibited cholesterol  Kimoto et al. (2002).It has been reported that cholesterol removal by lactic acid bacteria appeared not to be the only strain specific but also growth dependent, as shown by studies that have evaluated cholesterol removal by probiotic cells during different growth conditions (Kimoto et al., 2002).
The two bacterial isolates W7 and Y1 were selected as highly cholesterol reducing lactic acid bacteria in the present study and subjected to identification and further experiments.The bacterial isolate W7 was identified as Enterococcus faecalis and published in another study for production of lactic acid by the same authors (Aboseidah et al., 2017), while the bacterial isolate Y1 was identified phenotypically and genotypically in the present study.The results of physiological and biochemical characterizations of the isolate Y1 are shown in Table 2.The obtained results revealed that the bacterial isolate was Gram positive and catalase negative.This isolate was able to tolerate NaCl (6.5%).Also, the bacterial isolate was able to grow at 15 and 45°C.The results also indicated that the isolate was unable to produce CO 2 from glucose and NH 3 from arginine.The tested isolate had the ability to ferment glucose, mannose, galactose, xylose, maltose, mannitol, lactose and arabinose but was unable to use sucrose and glycerol as a carbon source.Based on the taxonomic characteristics described earlier, the isolate Y1 was assigned to the genus Enterococcus.
The bacterial isolate Y1 was identified using phylogenetic analysis of 16S rRNA gene sequences.The partial 16S rRNA gene sequences of tested isolate was matched with previously published bacterial 16S rRNA gene sequences available in National Center for Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST).Sequence analysis of the partial 16S rRNA gene sequence of 498 base pairs of the isolate Y1 revealed that this isolate had 96% similarity to Enterococcus faecium.A phylogenetic tree was constructed from a multiple sequences alignment of 16S rRNA gene sequences (Figure 1).The nucleotide sequences of the isolate Y1 was deposited in the Gen-Bank nucleotide sequence database under new accession number KY788356.
Figure 2 shows the effect of pH on cholesterol removal percentage by LAB isolates.The cholesterol reduction increased gradually with decreasing acidity of cultural medium until it reached maximum (65 and 78.5% for E. faecalis W7 and E. faecium Y1, respectively) at pH 6.5.At basic media the ability of bacteria to remove cholesterol decreased gradually with increasing alkalinity.Therefore, the optimum pH for maximum cholesterol reduction by the two selected isolates was pH 6.5.The study carried out by Kumar et al. (2013) suggested that a neutral pH is best for cholesterol reduction by L. casei LA-1.Also, Yazdi et al. (2001) reported that the optimal pH values for cholesterol decomposition are 7.2 for Streptomyces fradiae and 6.75 for Rhodococcus erythropolis ATCC 25544.The optimum production of enzymes required for metabolic process greatly affected by pH as enzymes, being proteins, contain ionizable groups; therefore, the pH of the culture medium affected their structure and function (Lekha and Lomane, 1997).
The effect of different incubation temperatures (25 to 45°C) on cholesterol reduction by the two tested bacterial isolates was studied and the obtained results are illustrated in Figure 3.The results revealed that the ability of bacterial isolates to remove cholesterol increased by increasing incubation temperature and reached to maximum values (80.7 and 85% for E. faecalis W7 and E. faecium Y1, respectively) at 37°C and then decreased by increasing temperature.Environmental and nutritional factors are known to influence the growth and decomposition of cholesterol by microorganisms.The optimum temperature for the decomposition of cholesterol in liquid medium by E. hirae was 37°C (Yehia et al., 2015).In contrast, several studies have reported other optimal incubation temperatures, such as 29°C for R. erythropolis ATCC 25544 (Sojo et al., 2002) and 30°C for Bacillus subtilis SFF34 (Lashkarian et al., 2010).The temperature range from 25 to 37°C is optimum for microbial growth and their enzymes activity and as a result maximum cholesterol removal was observed.
The effect of incubation time on cholesterol removal is observed in Figure 4.The maximum removal percentage was reported at 24 h of incubation.The bacterial isolate E. faecium Y1 removed 78.5% of cholesterol from the medium, while 65% of cholesterol was removed by isolate E. faecalis W7 at 24 h of growth.After that, the    ability of both isolates to remove cholesterol decrease gradually and finally reached stability at 48 h for W7 and 62 h for E. faecium Y1.Our findings is in accordance with the study of Kumar et al. (2013) who suggested that the ability of L. casei LA-1 to assimilate cholesterol was maximum after an incubation of 24 h.Hassanein et al.
(2013) also found that the maximum percentage of cholesterol reduction by L. lactis KF147 was achieved at 24 h.Additionally, Yehia et al. (2015) observed that the optimal cholesterol reduction (75.1%) in liquid medium was achieved after one day.However, Mahrous (2011) recorded maximum cholesterol reduction from liquid medium by probiotics bacteria (24.32 to 45.3%) at 20 h.The cholesterol reduction by the two highly degradable bacterial isolates E. faecalis W7 and E. faecium Y1 were studied at different concentrations from 50 to 150 µg/ml of cholesterol in culture broth medium (Figure 5) and the obtained results indicated that the highest reduction of cholesterol by both tested isolates was recorded at 100 µg/ml.The reduction of cholesterol by E. faecalis W7 in the cultural medium increased gradually with increasing cholesterol concentration until it reached the highest value of 68.8% at cholesterol concentration of 100 µg/ml.However, the maximum percentage of cholesterol removal by E. faecium Y1 was 90% at 100 µg/ml, then decreased at higher cholesterol concentrations.In a similar study, Mahrous (2011) found that maximum cholesterol reduction by Lactobacillus acidophilus P106 in liquid medium was obtained at 70 µg/ml cholesterol.While Hassanein et al. (2013) reported that L. lactis KF147 was able to remove 66.8% of cholesterol from MRS media supplemented with 100 µg/ml.Also, Pereira and Gibson (2002) reported high cholesterol removal ability (47%) by lactic acid bacteria and bifidobacteria isolated from human gut in broth media containing 100 µg/ml cholesterol.Guo et al. (2016) reported cholesterol reduction (41.29 to 56.61%) from liquid media containing 100 µg/ml of cholesterol by Enterococcus strains.Excessive cholesterol concentration may suppress bacterial ability of cholesterol reduction.Above a critical substrate concentration, a decreased water activity and onset of plasmolysis combine to cause a decrease in the rates of assimilation (Roukas, 1993).
The percentage of cholesterol removal at different growth stages of LAB were recorded in Figure 6.The maximum removal ability of E. faecalis W7 (93.5%) was observed at 42 h inoculum age, but in case of E. faecium Y1, the maximum removal value (94%) was reached at 18 h.In general, the results revealed that maximum cholesterol reduction was observed during the exponential growth phase and maximum biomass production when the cultures attained the stationary phase in which the medium nutrients is soon depleted and enriched with inhibitory products leading to decrease in bacterial biomass and rate of cholesterol removal (Kumar et al., 2013).
The effect of inoculum size on the cholesterol removal percentage is as shown in Figure 7.The highest value of cholesterol removal percentage by the two bacterial isolates E. faecalis W7 and E. faecium Y1 were 94 and 98%, respectively.In the case of isolate Y1, the ability of bacteria to remove cholesterol increased gradually with increasing inoculum size to reach maximum value of 98% at 3% inoculum size, while the maximum removal of cholesterol was reached at 2% inoculum size of isolate E. faecalis W7 then decreased slightly and became stable.The maximum removal percentage of cholesterol was recorded with inoculum size ranging from 2 to 3%.Our findings is in accordance with the study conducted by Kumar et al. (2013) who suggested that the ability of L. casei LA-1 to assimilate cholesterol is highly dependent on its growth, perhaps reflecting the growth of the inoculum used.Mahrous (2011) and Pereira and Gibson (2002) reported optimum cholesterol reduction with 1%   inoculum size of probiotic bacteria.
The results of GC-mass spectra analysis of E. faecium Y1 extract at interval incubation time is shown in Table 3.At zero time, the prominent peak was cholesterol which was observed at 51.08 min of retention time, while at 4 h of incubation cholest-5-ene-16, 22-dione, 3á,26-dihydroxy-, 3-acetate, was reported at 51.09 min of retention time and the peak of cholesterol became less predominant.At incubation time of 8 h, 5á-cholestane-3à,25-diol resulted in the peak at retention time 51.08 and the same result was observed at 24 h of incubation but by less amount (Figure 8).The results indicated that the bacterial isolate E. faecium Y1 transformed cholesterol to 5á-cholestane-3à,25-diol through oxidation and other chemical reactions whereas the reduced amount of cholesterol was utilized by the bacterial cells for its assimilation metabolism.The produced metabolites were ketonic derivatives of cholesterol and this due to transformation of some functional groups of cholesterol giving rise to ketones (Saranya, 2014).These results are in accordance with Suzuki et al. (1986), who reported that some intestinal bacteria such as Bifidobacterium, Eubacterium, Lactobacillus, Enterobacteriaceae, Clostridium and Enterococcus decompose cholesterol via the ChoX enzyme to cholest-4-en-3-one.
The reduction of cholesterol levels in vitro or in vivo by microorganisms may take place via enzymatic processes (Kovalenko et al., 2004) as the bacteria may decompose cholesterol due to their production of extracellular cholesterol oxidase (ChoX).ChOX attacks sterols at the 3β-hydroxyl positions to form 4-cholestenone and H 2 O 2 .This is the first step of microbial degradation of cholesterol in the oxidation of the 3β-hydroxyl group by ChOX (Sih et al., 1967) followed by the degradation of the side-chain (Shen et al., 1997;MacLachlan et al., 2000) by enzyme-complex possibly involving cytochrome P-450 and lyases leading to 4-androstene-3,17-dione, or 1,4-androstadiene-3 or 17-dione, and eventual breakdown of the steroid moiety with carbon dioxide and water being the final products.Also, the oxidation of cholesterol may results in the intermediate 3-ketosteroid as the final product (Motteran et al., 2001).Liu and Shan (2006) reported that cholesta-4,6-dien-3-ol, cholesta-4,6dien-3-one, and cholesta-3,5-dien-7-one are produced from the oxidation of cholesterol degradation by bacteria.Doukyu (2009) reported that the ChoX enzyme from Burkholderia cepacia strain ST-200 produces 6-betahydroperoxycholest-4-en-3-one from cholesterol.Liu and Shan (2006) reported that cholesta-4,6-dien-3-ol, cholesta-4,6-dien-3-one, and cholesta-3,5-dien-7-one are produced through the oxidation of cholesterol.The end products resulted from cholesterol degradation by microorganisms and their quantity varies between the different microbial strains and depends on the incubation period of the tested strains.

Conclusion
The use of fermented dairy products containing probiotic lactic acid bacteria is considered as one of attractive and effective treatment for hypercholestermia.Therefore, obtaining a novel probiotic strains with cholesterol reducing abilities is required and will be safe alternative for clinical drugs.Consequently, in the present study a total of 50 different probiotic lactic acid bacterial strains were isolated from healthy infants' stool which showed various cholesterol lowering abilities in culture media.The most active isolates E. faecalis W7 and E. faecium Y1 were selected to optimize cholesterol removal process under different condition.About 90% of cholesterol reduction was achieved by E. faecium Y1 under the optimized cultural conditions in the broth medium.In addition, the end products which resulted from the degradation process were also followed to study their impact when this probiotic strain was applied as cholesterol lowering agent in the dairy products.

Figure 1 .Figure 2 .
Figure 1.The neighbor-joining tree based on 16S rRNA gene sequences showing the position of Enterococcus faecium Y1 and related strains in GenBank.

Figure 3 .
Figure 3.Effect of incubation temperature on cholesterol reduction percentage by E. faecalis W7 and E. faecium Y1.

Figure 4 .Figure 5 .
Figure 4. Effect of incubation time on cholesterol removal percentage by E. faecalis W7 and E. faecium Y1.

Figure 6 .
Figure 6.Effect of inoculum age on cholesterol removal percentage by Enterococcus faecalis W7 and Enterococcus faecium Y1.

Figure 7 .
Figure 7. Effect of inoculum size on cholesterol removal percentage by E. faecalis W7 and E. faecium Y1.

Table 1 .
Mean values of cholesterol reduction percentage of the most active ten LAB isolates.
Yehia et al. (2015)lactis KF 147.In this coincidence,Yehia et al. (2015)recorded maximum cholesterol reduction of 75.3% in liquid medium by Enterococcus hirae isolated from milk.Also, the highest cholesterol reduction by L. lactis subsp.lactis N7 was 97.0% as recorded by

Table 2 .
Morphological and biochemical characterizations of the selected bacterial isolate Y1.

Table 3 .
Gas chromatography -Mass spectra analysis of cholesterol degradation products by Enterococcus faecium Y1.