Applied and Environmental Microbiology, August 2003, p . 4743-4752, Vol . 69, No . 8

An In Vitro Study of the Probiotic Potential of a Bile-Salt-Hydrolyzing Lactobacillus fermentum Strain, and Determination of Its Cholesterol-Lowering Properties

Dora I . A . Pereira,^* Anne L . McCartney, and Glenn R . Gibson

Food Microbial Sciences Unit, School of Food Biosciences, The University of Reading, Reading RG6 6BZ, United Kingdom

Received 12 March 2003/ Accepted 27 May 2003

One beneficial effect that has been suggested to result from human consumption of LAB is a reduction in serum cholesterol levels, as suggested by the results of several human and animal studies (reviewed by Pereira and Gibson [36]) . This effect can partially be ascribed to an enzymatic deconjugation of bile acids (21, 45-47) . Deconjugated bile salts are less soluble and less efficiently reabsorbed from the intestinal lumen than their conjugated counterparts, which results in excretion of larger amounts of free bile acids in feces (8, 10) . Also, free bile salts are less efficient in the solubilization and absorption of lipids in the gut (38) . Therefore, the deconjugation of bile acids by LAB bacteria could lead towards a reduction in serum cholesterol either by increasing the demand of cholesterol for de novo synthesis of bile acids to replace that lost in feces or by reducing cholesterol solubility and, thereby, absorption of cholesterol throughout the intestinal lumen . Moreover, Gilliland et al . (12) have observed a significant relationship between cholesterol assimilation by lactobacilli and their degree of bile deconjugation .

Bile salt hydrolase (BSH), the enzyme responsible for bile salt deconjugation during enterohepatic circulation, has been detected in several LAB species indigenous to the gastrointestinal tract (3, 9, 10, 11, 49) . It has also been suggested that BSH activity should be a requirement in the selection of probiotic organisms with cholesterol-lowering properties, as nondeconjugating organisms do not appear to be able to remove cholesterol from the culture medium to any significant extent (45, 46) .

Lactobacillus fermentum is a normal resident of the human gut microflora and is reported to be able to adhere to the epithelial cells, with a preference for the small intestine (17, 39) . It has also been shown to colonize the intestine after oral administration (37) . Moreover, it produces surface-active components which can inhibit the adhesion of uropathogenic bacteria (15, 16) .

In the present work, the in vitro BSH activity of a number of LAB strains was studied in view of their potential in vivo cholesterol-lowering effects through enhanced BSH activity . Furthermore, the in vitro effect of a BSH-active L . fermentum strain upon intestinal bacterial populations associated with the human gut and overall metabolic activity of this microbiota was evaluated . This study can be regarded as an in vitro assessment of the use of an indigenous L . fermentum strain as a probiotic with hypocholesterolemic potential for human use .

 
Screening of cultures for BSH activity.
Qualitative BSH activity of the cultures was evaluated using the procedure described by du Toit et al . (11) . Sterile filter disks were impregnated in an overnight culture of the test strain and placed on MRS agar plates supplemented with 0.5% (wt/vol) taurodeoxycholic acid sodium salt (TDCA; Sigma) and 0.37 g of CaCl₂ (Merck)/liter . The plates were incubated anaerobically at 37°C for 72 h, after which the diameters of the precipitation zones were measured . MRS agar plates without supplementation were used as controls . Each strain was tested in triplicate . The three strains that displayed the largest precipitation zones were selected for further study .

BSH assay.
A modification of the high-performance liquid chromatography (HPLC) method described by De Smet et al . (10) was used to determine quantitative BSH activity . A reverse-phase Ultrasphere ODS column (Hichrom Ltd.) (80 Å, 5 µm, 150 by 4.6 mm) was used . The HPLC system consisted of an HP series 1050 apparatus equipped with an autosampler and on-line vacuum degasser . Free and conjugated bile acids were eluted with a linear gradient of methanol aqueous buffer at a flow rate of 1.0 ml/min . The solvents used were solvent A, which consisted of a mixture of 65% methanol in 0.03 M sodium acetate adjusted to pH 4.3 with phosphoric acid, and HPLC-grade methanol (solvent B) . The elution program was as follows: isocratic elution was performed with 15% solvent B and 85% solvent A for 8 min and then a 17-min linear gradient to 85% solvent B, and the mobile phase composition was finally maintained at 85% solvent B for a further 5 min . The detector was set at 210 nm, and chromatography was performed at room temperature . The injection quantity was 10 µl . All bile acids used as standards (conjugated and free) had a purity of 97% or more and were purchased from Sigma .

Bile salt extracts.
To recover bile salts from the MRS broth cultures, cells were removed by centrifugation (6,000 x g for 10 min at 5°C) . The method of De Smet et al . (10) was modified to recover bile salts from the spent broth . Samples (1 ml) of the supernatants were acidified through the addition of 10 µl of 6 N HCl to stop BSH activity . Lithocholic acid was used as an internal standard and added to a final concentration of 8 mM . Isopropanol (4 ml) was used to extract the bile salts (1:4 [vol/vol]) . The samples were then mixed for 60 min at 420 rpm and centrifuged at 1,000 x g for 10 min . The isopropanol layer was transferred to a clean test tube and evaporated under an N₂ flow at 37°C . After complete isopropanol removal, the bile salt extract was redissolved in 800 µl of methanol and filtered through a 0.45-µm-pore-size polysulfone HPLC filter (Whatman) . Prior to injection in the HPLC filter, samples were stored at -20°C .

Quantitative BSH activity.
BSH activity of the cultures was determined using the HPLC procedure described above . Strains were compared for their abilities to deconjugate bile acids during growth . Dilution bottles of about 100-ml capacity were filled with 70-ml volumes of MRS broth, autoclaved at 121°C for 15 min, and cooled to around 50°C . The MRS broth was then supplemented with a filter-sterilized solution of sodium taurocholate (TCA) and sodium glycocholate (GCA) to give a final concentration of 1 mM for each bile acid . Overnight MRS broth cultures of the strains undergoing testing were inoculated (1% [vol/vol]) into the medium, and the mixtures were incubated anaerobically at 37°C for 24 h . Samples were taken aseptically at various time intervals during the incubation period . Growth was monitored through absorbance at 650 nm, and bacterial enumeration was determined by plate count on MRS agar (after inoculation and at the end of the 24-h incubation) . Samples were also analyzed for pH and bile acids . The experiment was performed in triplicate for each strain, and uninoculated MRS broth supplemented with TCA and GCA was used as a control . The BSH enzymatic activity was expressed as nanomoles of GCA and TCA deconjugated per minute . The strain with the highest BSH activity and simultaneously the highest probability of survival through the upper gastrointestinal tract was chosen for further studies .

Use of prebiotic substances to enhance growth of the probiotic strain.
Three different commercial prebiotic substances were tested for their ability to increase the growth and/or activity of the probiotic strain . The prebiotics used were galactooligosaccharides (GOS; Borculo Domo Ingredients, Zwolle, The Netherlands), fructooligosaccharides (FOS) (oligofructose P95; Orafti, Oreye, Belgium), and lactulose (Solvay Pharmaceuticals, Weesp, The Netherlands); glucose was used as a control . Sterile batch culture fermentors (280-ml capacity) were filled with 100 ml of a prereduced culture medium and maintained under a headspace of O₂-free N₂ . Carbohydrates (glucose, GOS, FOS, and lactulose) were added and filter sterilized (0.22-µm-pore-size polysulfone filter; Whatman) prior to inoculation to give a final concentration of 10 g/liter . The vessels were inoculated (1% [vol/vol]) with an overnight MRS culture of the bacterial strain (ca . 2 x 10⁹ CFU) .

The culture medium consisted of MRS broth without glucose (MRS-C) and contained the following components: peptone (Oxoid), 10.0 g liter^-1; Lab-Lemco powder (Oxoid), 8.0 g liter^-1; yeast extract (Oxoid), 4.0 g liter^-1; Tween 80, 1 ml liter^-1; K₂HPO₄, 2.0 g liter^-1; triammonium citrate, 2.0 g liter^-1; sodium acetate, 3.0 g liter^-1; MgSO₄ · 7 H₂O, 0.2 g liter^-1; MnSO₄ · H₂O, 0.04 g liter^-1; sodium thioglycolate, 2.0 g liter^-1; water-soluble cholesterol (polyoxyethanyl-cholesteryl sebacate; Sigma), 0.5 g liter^-1; and oxgall (Oxoid), 3.0 g liter^-1 . The final cholesterol concentration in the medium was ca . 100 mg/liter . All chemicals were obtained from Merck unless otherwise stated . The medium was autoclaved at 121°C for 15 min . The cultures were incubated at 37°C for 24 h, and the culture pH was maintained at 6.0 . Samples were taken aseptically every 2 h for 12 h and at the end of the 24-h incubation . Growth was monitored using absorbance values at 650 nm . Samples were also analyzed for cholesterol . The experiment was repeated in three different runs . The best prebiotic was chosen for further studies .

Cholesterol reduction during growth at pH 6.0.
The effect of different bile sources and concentrations on the growth and/or activity of the probiotic strain was evaluated using pH-controlled stirred batch culture fermentations . The different culture conditions are summarized in Table 2 . Unless otherwise stated, the fermentations were operated as described above . Two independent trials were carried out for each set of fermentations .

 
Three-stage continuous culture system.
To investigate the survival of the selected probiotic organism in a mixed intestinal microbial association, a multistage continuous culture simulation of the human colon was used (28) . The effect of the daily addition of the probiotic culture on bacterial populations, fermentation product formation, and cholesterol concentrations was evaluated at a retention time of 65 h . Briefly, the system consisted of three vessels, V1, V2, and V3, arranged in series, with respective working volumes of 0.28, 0.30, and 0.30 liters . Temperature was maintained at 37°C, and the pH was automatically controlled to maintain values of 5.5 (V1), 6.2 (V2), and 6.8 (V3) to reflect conditions in the proximal, transverse, and distal regions of the colon . All vessels were maintained under a headspace of oxygen-free nitrogen gas and continuously stirred . Retention times in the system were 20.8 h (V1) and 22.1 h (V2 and V3) .

The culture medium contained the following components: starch, 5.0 g liter^-1; pectin (citrus; Sigma), 2.0 g liter^-1; guar gum, 1.0 g liter^-1; mucin (porcine gastric type III; Sigma), 4.0 g liter^-1; xylan (oatspelt; Sigma), 2.0 g liter^-1; arabinogalactan (larch wood), 2.0 g liter^-1; casein (Sigma), 3.0 g liter^-1; peptone water (Oxoid), 5.0 g liter^-1; tryptone (Oxoid), 5.0 g liter^-1; bile salts no . 0.3 (Oxoid), 0.4 g liter^-1; yeast extract (Oxoid), 4.5 g liter^-1; FeSO₄ · 7 H₂O, 0.005 g liter^-1; NaCl, 4.5 g liter^-1; KCl, 4.5 g liter^-1; KH₂PO₄, 0.5 g liter^-1; MgSO₄ · 7 H₂O, 1.25 g liter^-1; CaCl₂ · 6 H₂O, 0.15 g liter^-1; NaHCO₃, 1.5 g liter^-1; cysteine-HCl (Sigma), 0.8 g liter^-1; hemin (Sigma), 0.05 g liter^-1; Tween 80, 1.0 ml liter^-1; vitamin K1 (Sigma), 10 µl liter^-1; and water-soluble cholesterol (Sigma), 0.5 g liter^-1 . The final cholesterol concentration in the medium was ca . 100 mg liter^-1 . All chemicals were obtained from Merck unless otherwise stated .

The sterile culture medium was continuously sparged with O₂-free N₂ (15 ml/min) and was fed by peristaltic pump to V1, which sequentially supplied V2 and V3 . Each vessel was inoculated with 100 ml of a 20% (wt/vol) fecal slurry from a healthy human donor . The slurries were prepared by homogenizing freshly collected feces in anoxic phosphate-buffered saline buffer at pH 7.3 (phosphate-buffered saline solution prepared according to the instructions of the manufacturer [Oxoid]) . The cultures were allowed to equilibrate overnight before fresh medium was added to the system (day 0) . A total of 1 ml of a washed probiotic preparation was added daily to V1 from day 0 to day 43 . Samples (8 ml) were collected from all three vessels at appropriate time intervals before the addition of the culture . Steady-state conditions were assessed by monitoring the production of short-chain fatty acids (SCFA) . After the first stabilization period (probiotic period; days 0 to 22), starch present in the culture medium was replaced by an equivalent amount of GOS and the system allowed to reach steady-state conditions once more (synbiotic period; days 22 to 44) . This was done to ascertain the effect of the prebiotic substance on survival and function of the probiotic strain . For each steady-state condition, two samples were taken 24 h apart for analyses . The experiment was repeated with three different donor inocula .

Preparation of the probiotic.
For detection of the probiotic throughout the multistage continuous culture system used, rifampin-resistant mutants of L . fermentum KC5b were isolated prior to the study . Briefly, the resistant variant of L . fermentum KC5b was selected by growing successive overnight, anaerobic cultures in MRS broth containing increasing amounts of rifampin (0.001 to 100 µg/ml) until growth was observed in the medium with the highest antibiotic concentration (data not shown) . Overnight cultures of the probiotic strain were grown in MRS broth supplemented with rifampin (100 µg ml^-1) under anaerobic conditions at 37°C . Cells (ca . 10⁹ CFU) were gently centrifuged (400 x g, 5 min) and resuspended in 1 ml of fresh MRS broth before being added to the fermentation vessel . Samples were taken periodically from the probiotic cultures for bacterial enumeration .

SCFA analysis.
To determine the SCFA contents, samples were centrifuged (6,000 x g, 10 min) to remove bacteria and solids and then filtered through a 0.2-µm-pore-size polysulfone HPLC filter . Then, 200 µl of each filtered supernatant was diluted with 800 µl of acetonitrile (1:4) containing 3.7 mM 2-ethylbutyric acid as the internal standard . Fatty acids were determined by gas-liquid chromatography with an HP 5890 series II GC system (Hewlett Packard, Palo Alto, Calif.) equipped with a capillary fused silica-packed column (Permabond FFAP; Macherey-Nagel, Düren, Germany) (25 m by 0.32 mm; film thickness, 0.25 µm) . Helium was used as the carrier gas at a flow rate of 2.42 ml/min . The column temperature was 140°C, and the temperature of the injector and detector was 240°C . At 5 min following injection of the sample, the temperature of the column was increased to 240°C in increments of 20°C/min and the system left to run for a further 5 min . The composition of the gases was analyzed using HP 3365 series II ChemStation Apg-top software, version A0.03.34 . The following acids were used as standards, with concentrations ranging from 0.5 to 20 mM each: acetic acid, propionic acid, N-butyric acid, DL-lactic acid (98%), N-valeric acid, isovaleric acid (98%; Fluka), isobutyric acid (Fluka), and N-caproic acid . Unless otherwise stated, all acids were purchased from Sigma and were more than 99% pure . SCFA concentrations were calculated using internal standard calibration and are expressed in millimoles per liter .

Bacteriology.
The bacterial numbers of the probiotic strain in the individual fermentation vessels were determined by plate counts in MRS agar supplemented with 100 µg of rifampin/ml . Samples (1 ml) taken from each vessel were serially 10-fold diluted (10^-1 to 10^-5) in anaerobic diluent (half-strength peptone water-0.5 g of L-cysteine HCl liter^-1, pH 7.0) . Triplicate plates (prereduced) were inoculated with 20-µl samples from the appropriate dilutions and incubated anaerobically at 37°C for 24 to 48 h .

Genus-specific 16S rRNA-targeted oligonucleotide probes labeled with the fluorescent dye Cy 3 were used for enumerating Bifidobacterium, Bacteroides, Lactobacillus/Enterococcus, and Clostridium subgroup histolyticum bacteria (40) . Samples (5 ml) from each fermentation vessel were placed into a 50-ml centrifuge tube containing glass beads (ca . 5 mm) . The samples were mixed in a vortex for 30 s and centrifuged for 2 min at 200 x g to remove particulate matter . The fluorescent in situ hybridization procedure described by Rycroft et al . (40) was used to obtain numbers of the different bacterial groups in the samples .

Cholesterol assay.
Cholesterol in the spent broths was first extracted using the procedure described by Gilliland et al . (12) . The total cholesterol content of the evaporated residues was then determined using the enzymatic assay described by Salè et al . (41) .

Statistical analysis.
The one-way analysis of variance procedure for SPSS (P . R . Kinner and C . D . Gray, SPSS for Windows made simple, release 10 ed., Psychology Press Ltd., Hove, United Kingdom, 2000) was used to determine whether significant (P < 0.05) variation occurred among means in each experiment . The least significant difference value (Bonferroni t test) was used to determine which means differed significantly . Pearson's correlation analysis was conducted on growth and GCA-TCA data . The standard deviation of the specific BSH activity rate was estimated from the standard deviations of measurements of optical density at 650 nm (OD₆₅₀) and GCA and TCA concentrations by the law of error propagation in arithmetic operations .

 
Additionally, the amount of bile acid deconjugated by S . bovis during the overall incubation period was higher than that deconjugated by the other two strains; however, this was not significant for GCA with E . faecalis (P > 0.05) . L . fermentum deconjugated significantly lower amounts of GCA than E . faecalis over the same period of time (P < 0.01) . Both L . fermentum and E . faecalis showed a significantly greater ability to deconjugate GCA than to deconjugate TCA (P < 0.05) . Statistical analysis of the growth and deconjugation data during the incubation period revealed a significant (P < 0.05) linear correlation between OD₆₅₀ values and concentrations of both TCA (-0.726) and GCA (-0.859) in the case of S . bovis . However, this was not observed for the other two strains tested . Specific bile-salt-hydrolyzing activities of the strains for GCA and TCA were estimated separately during the exponential and stationary phases of growth according to the ratio between the amount of bile acid deconjugated and the number of cells produced during those periods of time . The average overall specific bile-salt-hydrolyzing activities were determined according to the ratio between the sum of the partial activities and the overall incubation period (24 to 30 h) . These results are shown in Table 3 .

 
Although L . fermentum reached a higher cell density than the other two strains, the final amount of free cholic acid (CA) liberated was smaller; therefore, the specific BSH activity rates were lower . They were, however, in the same range as the BSH activities reported for other highly active lactobacilli species (10, 11) . The higher BSH activity of E . faecalis and S . bovis could play a detrimental role in the lower gastrointestinal tract, as such activity has been suggested to increase the risk for colon cancer due to the potential carcinogenic properties of some deconjugated bile acids (31, 42, 48) . Also, previous studies have shown that S . bovis ATCC 43143 has low acid tolerance, losing viability within 15 min at pH 2.0 (35) . Acid-tolerant strains have an advantage in surviving the low pH conditions in the stomach (pH 2.0 in most cases), where hydrochloric and gastric acids are secreted . Speculatively, the S . bovis strain might still survive passage through the stomach, although in lower numbers than the other two strains tested . Furthermore, E . faecalis strains are not the most suitable probiotic candidates, although they can exhibit good acid resistance and bile tolerance . Enterococcus faecalis is frequently isolated from intra-abdominal infections and is often implicated in postoperative infectious complications, particularly peritonitis (34, 44) . Therefore, the strain L . fermentum KC5b was chosen for further study as a good probiotic candidate adapted to tolerate the gastrointestinal tract acid and bile conditions .

Use of prebiotic substances to enhance growth of L . fermentum KC5b.
Figure 2 shows the growth of L . fermentum KC5b with three different prebiotic substances and glucose as the principal carbon source . The mean growth rate of the strain with GOS (0.34 ± 0.01 h^-1) was similar to that with glucose (0.323 ± 0.007 h^-1) but was significantly higher than with lactulose (0.297 ± 0.003 h^-1 [P < 0.05; n = 3]) . The growth rate of L . fermentum in MRS broth with FOS as the main carbon source was significantly lower than that with the other three carbohydrates (0.089 ± 0.003 h^-1 [P < 0.001, n = 3]) . This is in agreement with a previous study that reported that probiotic strains such as Lactobacillus rhamnosus GG and the yogurt starters Lactobacillus delbrueckii subsp . bulgaricus and Streptococcus thermophilus were FOS nonfermenters (20) . Moreover, the average reduction (expressed as percentage of total cholesterol added [ca . 100 mg/liter]) of the level of cholesterol in the medium with GOS (8%) was higher than that seen with both glucose (1.4%) and lactulose (4%); this was, however, not significant for the latter (P > 0.05) . No cholesterol reduction was observed when the strain was grown in the medium with FOS . The prebiotic GOS was therefore chosen for further studies with this strain .

 
Influence of oxgall concentration upon growth and cholesterol reduction.
Cholesterol was removed from the broth medium in the presence of oxgall, as previously observed in experiments without pH control (35) . Figure 3 shows a plot of the growth rate of the strain and the percentage of cholesterol removed against the oxgall concentration in the medium . The amount of bile in the fermentation medium influenced both the growth of the strain and the amount of cholesterol assimilated . Indeed, as the amount of oxgall in the medium increased, the growth rate of the strain decreased significantly (P < 0.05) . The percentage of cholesterol reduction in the culture medium seemed to be constant and small at oxgall concentrations lower than 0.5% . The amount of cholesterol measured in the supernatant fluids in the medium with 2% oxgall was significantly lower than for the other oxgall concentrations tested (P < 0.001) . This resulted in a significantly higher percentage of cholesterol reduction . No other significant differences in the amounts of cholesterol detected in the supernatant fluids were found .

 
Cholesterol reduction in the presence of different bile salts.
The L . fermentum strain was grown in cultures during 24 h in MRS-C broth containing 10 g of GOS/liter as the carbon source, 100 mg of cholesterol/liter, and 5 mM bile salts . Different bile sources were used, including oxgall, CA, deoxycholic acid (DCA), TCA, GCA, and TDCA . CA and DCA were used as sources of deconjugated bile, and the remaining bile acids were used as sources of conjugated bile . Oxgall is a mixture of conjugated (97%) and deconjugated (ca . 3%) bile . No significant differences were found in either the growth rate or maximum cell density achieved by the strain when oxgall, TCA, GCA, and TDCA were used as the sources of bile . The growth rate of the strain in CA was around five times lower than with the other bile sources, which was expected since deconjugated bile salts are more toxic to bacterial growth than those which are conjugated (33) . Growth of the strain in medium with DCA was not monitored due to a high initial turbidity of the medium caused by higher precipitation of this bile acid at pH 6.0 . Also, it was observed in this study that even before the bacterial strain was inoculated, a significant amount of cholesterol in the medium precipitated after deconjugated bile acids were added . This coprecipitation effect of cholesterol and deconjugated bile acids has been previously reported to occur only at pH values lower than 5.5 (21) . However, in the present study this effect was observed at pH 6.0 . No precipitation of cholesterol was observed in the presence of conjugated bile acids and oxgall . Results of experiments examining the cholesterol-reducing ability of L . fermentum KC5b in the presence of six sources of bile are shown in Table 4 . After 24 h of anaerobic growth at pH 6.0, 2.8 to 50% of cholesterol was removed from the medium . As shown in Table 4, maximum levels of cholesterol were reduced when CA and DCA were used as sources of bile . For the conjugated bile sources used, no significant difference (P > 0.05) was found between the amounts of cholesterol measured in the supernatant fluids .

 
Effect of L . fermentum upon microbial ecology and activity in a three-stage continuous culture system.
Fig . 4 shows the amount of L . fermentum KC5b present in each vessel of the three-stage fermentation system throughout the experiment . Each day from day 0 to day 43, 2.8 x 10⁹ ± 1.3 x 10⁹ CFU of the bacterial strain was added to V1 . In the synbiotic experiment, the addition of GOS to the fermentation medium did not cause profound changes in L . fermentum numbers in the three vessels . However, a significant increase (P < 0.005) in numbers (ranging from 0.6 to 0.8 log CFU ml^-1) of the probiotic strain was observed in all three vessels after starting addition of GOS . This was not sustained, and under both steady-state conditions no significant (P > 0.05) differences in L . fermentum numbers were detected . During the first stabilization period (in which only the probiotic strain was added to the system), numbers of L . fermentum in V3 were significantly lower (ca . 0.5 log unit) than in the other two vessels (P < 0.001) . When the second steady state was reached, however, no significant differences were found in the probiotic strain numbers among the three vessels . L . fermentum numbers in the three vessels significantly decreased (P < 0.05) by 2 days after addition of the probiotic to the system was stopped . However, after 10 days without addition of the strain to V1, numbers in the system were still ca . 10⁴ CFU ml^-1 .

 
Four major groups of intestinal bacteria were selected as marker populations to determine whether L . fermentum influenced the microbial composition of the three-stage continuous culture system . Table 5 gives results of bacterial enumerations in the three vessels of the system . No significant differences were found in numbers of each bacterial group among the three vessels of the system for the same period of time (P > 0.05) . Addition of the probiotic strain to the system caused a significant decrease (P < 0.05) in numbers of bifidobacteria (ca . 1 log unit) in all vessels . This decrease was maintained during the prebiotic supplementation period . At the start of the experiment, Bacteroides and bifidobacteria were numerically predominant populations detected in all vessels, with no significant differences in their numbers (P > 0.05) . However, through L . fermentum addition to the system the Bacteroides population became dominant in relation to the bifidobacteria, although this was only significant (P < 0.05) in V1 (ca . 0.9 log unit) . The Clostridium subgroup histolyticum numbers in the third run of the experiment were around 1 log unit lower at the start of the experiment in all the vessels in comparison to those in the other two runs carried out . In this case, Clostridium numbers significantly decreased (below 6 log CFU ml^-1) with the addition of L . fermentum in V3 and with the addition of both probiotic and prebiotic in all vessels (P < 0.001) . No significant differences in Clostridium numbers for the other two runs of the experiment, either between the three vessels or through different stabilization periods, were found . No significant differences were found in the remaining bacterial populations with the addition of L . fermentum either alone or in conjunction with the prebiotic GOS (P > 0.05) .

 
Acetate, propionate, and butyrate were the principal SCFA produced by colonic bacteria in the fermentation system (Table 6) . No significant differences in the concentrations of each SCFA among the three fermentation vessels at a given time period were observed . Measurements of SCFA formed showed that the addition of L . fermentum caused a decrease in acetate production in all three vessels; however, this was only significant in V2 (ca . 27% [P = 0.007]) . No significant differences in the acetate concentration with supplementation of GOS to the medium were found (P > 0.05) . Also, addition of the bacterial strain to the fermentation system caused a considerable increase in both propionate (50 to 90%), the increase of which was significant for all three vessels (P < 0.005), and butyrate (52 to 158%), the increase of which was significant for V3 only (P < 0.01) . Supplementation of the prebiotic to the system did not influence propionate concentrations in the system but increased those of butyrate, although not significantly (P > 0.05) . However, butyrate levels in the three vessels at the end of the second stabilization period were significantly higher than at the beginning of the experiment (P < 0.05) . Similarly, the addition of L . fermentum to the system increased the concentrations of isobutyrate and isovalerate by one- to twofold in all vessels (P < 0.05), and this result was maintained during the GOS supplementation period . No significant differences were observed in the concentrations of valerate and caproate throughout the three vessels during the experimental period .

 
Following the addition of L . fermentum, there was a general increase in lactate production, an effect that was most marked in the second run of the experiment (Table 7) . In this case lactate production in V3 was significantly higher than in V2 (P = 0.003), which in turn was significantly higher than in V1 (P = 0.002) . In the third run the addition of GOS resulted in increased lactate production compared to that seen in the period in which probiotic alone was added, and this increase was significant for V1 (P < 0.001) .

 
Cholesterol was removed from the fermentation medium in the presence of L . fermentum in all three vessels of the system (11 to 25% of the total) . The maximum level of cholesterol reduction was achieved in V3; however, the difference between this result and those seen with the other vessels was not significant .

As observed for L . acidophilus (12) and bifidobacteria (46), a low amount of cholesterol was removed from the medium by the L . fermentum strain in the absence of bile salts . In the presence of 2% oxgall, in contrast, 14% of cholesterol was removed from the culture medium even with growth being partially inhibited . Furthermore, the [cholesterol assimilated (micrograms/milliliter)/maximum OD_{650 nm}] ratio was from 3- to 28-fold higher in the presence of 0.1 to 2% oxgall, respectively, than that for the medium without bile salts . Thus, the presence of bile salts seemed to be a prerequisite for cholesterol assimilation by L . fermentum KC5b . Maximum levels of cholesterol removal were obtained when CA and DCA were used as sources of bile (Table 4) . However, this was attributed to the coprecipitation of cholesterol observed in these cases and not to assimilation by the bacterial strain . This coprecipitation effect would not be of relevance in vivo, since the physiological pH in the small intestine is usually neutral to alkaline . Nevertheless, (lower) cholesterol reduction was also observed in the presence of TCA, GCA, and TDCA, although no significant precipitation occurred . This indicates that the cholesterol removed was in some way associated with the L . fermentum cells, as observed for L . acidophilus (12, 24) and Bifidobacterium longum (6) .

When L . fermentum was grown in the presence of bile (0.3% oxgall) and 10 g of GOS liter^-1, cholesterol was measured in supernatant fluids, washing buffers, and cell extracts (data not shown) . The concentrations of cholesterol in the cell extracts contributed to approximately 20% of the total cholesterol removed from the medium . This is in agreement with the findings from Dambekodi and Gilliland for cells of B . longum (6) . Around 35% of the total cholesterol removed was found in the washing buffers . This gives a further indication that at least part of the cholesterol removed was associated with the cells of L . fermentum KC5b (either through attachment to the cell membrane or through assimilation) .

In addition, this study evaluated the effect of administration of high levels of L . fermentum KC5b cells (ca . 10⁶ CFU ml^-1) upon representative bacterial groups of the gastrointestinal tract (Table 5) . A probiotic concentration of 10⁶ CFU/g of fecal contents is considered sufficient to produce physiologically relevant benefits in vivo (14) . A 1-log-unit decrease in the numbers of bifidobacteria was observed with the addition of L . fermentum to the system, and this was maintained during the GOS supplementation period . This was not surprising and confirmed an antagonistic effect of the cell-free neutralized supernatant of L . fermentum KC5b upon some bifidobacterial species (data not shown) . It should also be noted that lactobacillus numbers did not increase significantly through administration of L . fermentum . This can probably be attributed to the fact that some indigenous lactobacillus species were present in low numbers at the start of the experiment and therefore were partially out-competed .

The principal organic acids produced in the fermentation system were acetate, propionate, and butyrate; this resembles what happens in vivo during degradation of carbohydrates (26) . Linear-chain fatty acids were investigated as markers of glycolytic fermentation, and branched-chain fatty acids were investigated as markers of a proteolytic fermentation (27, 30) . The considerable increase in the production of butyrate observed during the administration of L . fermentum to the system may be of particular importance in relation to colonic cancer . Butyrate is the principal energy source for normal colonocytes and has been shown to reduce the growth rate of human colonic cancer cells and to facilitate DNA repair in vitro (7, 22, 29) . Butyrate is not a major end product of Bacteroides, bifidobacteria, or lactobacilli, whereas it is for eubacteria, fusobacteria, and clostridia (1) . Possibly, a reduction in the bifidobacterial population allowed butyrate producers like eubacteria and fusobacteria to increase in the system . Furthermore, addition of the L . fermentum strain originated a significant increase in the production of propionate, which could also be considered beneficial . Propionate is cleared by the liver and has been suggested to affect cholesterol metabolism (50) . Furthermore, propionate (but not acetate) has also been shown to be effective in inhibiting cell proliferation with carcinoma cell lines, although it is two- to threefold less potent than butyrate (7) . Acetate is the major acid product of nearly all species of Bacteroides and bifidobacteria (1) . The decrease observed in acetate production provides further evidence of lower bifidobacterial metabolism .

A production of lactic acid indicates saccharolytic activity, but lactic acid is an electron sink product (normally further oxidized to other volatile fatty acids in the gut) and does not accumulate to any significant extent in colonic contents (26) . Thus, the observed increase on lactate production could be an indication of incomplete or rapid fermentation .

The increase in overall volatile fatty acid production (26 to 57%) denotes a higher fermentation rate after the addition of L . fermentum . The production of SCFA and lactic acid leads to a drop in the pH of the large intestine (5), which can limit the development of pathogenic bacteria .

One major risk for coronary heart disease is elevated serum cholesterol levels (19, 23) . The one-to-two rule applies, which states that a 1% reduction of serum cholesterol causes a 2% reduction of the risk for coronary heart disease (18) . LAB with active BSH (or products containing them) are suggested to lower cholesterol levels through an interaction with host bile salt metabolism (9, 10) . The main proposed mechanism of cholesterol reduction was comparable to that of bile salt sequestrants (e.g., cholestyramine and colestipol) that bind to bile acids and prevent their being reabsorbed into the enterohepatic circulation, thereby resulting in increased demand for cholesterol as a precursor of bile salt synthesis . Also, deconjugated bile salts do not function as well as conjugated forms in the solubilization of cholesterol and therefore prevent it from being absorbed (38), which could then reduce serum cholesterol concentrations .

During this in vitro study, the L . fermentum strain did not cause any potentially undesirable microbial-metabolic characteristics which could hamper its use as a probiotic for human consumption . On the contrary, L . fermentum showed a positive effect upon SCFA production, increasing the production of propionate and butyrate . The colon is the second commonest site of cancer in developed countries; therefore, the possible biological properties of butyrate directed towards colon cancer are of relevance, and it would be desirable to promote its production in the colon . The supplementation of L . fermentum KC5b to the system increased butyrate production in all three fermentation vessels, and this was even more significant during supplementation in conjunction with GOS .

Finally, bile tolerance is considered to be an important characteristic for a probiotic that enables it to survive and then grow and exert its action in the small intestine . The L . fermentum strain used in this study was found previously to have a high level of bile tolerance and acid resistance (35) . Therefore, as with other L . fermentum strains, this strain can be considered intrinsically resistant to human upper gastrointestinal transit (2) .

In conclusion, this in vitro study should be regarded as of indicative value in the investigation of the hypocholesterolemic effect of the probiotic strain L . fermentum KC5b .

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