Cloning and analysis of bile salt hydrolase genes from Lactobacillus plantarum CGMCC No. 8198

Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20 % in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids.     

Functional and Probiotic Attributes of an Indigenous Isolate of Lactobacillus plantarum

Background

Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.

Methods and Findings

An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.

Conclusion

The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.     

Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394

Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni⁺²-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 °C while showed its maximum activity at 37 °C. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.     

A New Insight into the Physiological Role of Bile Salt Hydrolase among Intestinal Bacteria from the Genus Bifidobacterium

This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.     

Probiotic Properties of Lactobacillus Strains Isolated from Tibetan Kefir Grains

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) isolated from Tibetan kefir grains. Three Lactobacillus isolates identified as Lactobacillus acidophilus LA15, Lactobacillus plantarum B23 and Lactobacillus kefiri D17 that showed resistance to acid and bile salts were selected for further evaluation of their probiotic properties. The 3 selected strains expressed high in vitro adherence to Caco-2 cells. They were sensitive to gentamicin, erythromycin and chloramphenicol and resistant to vancomycin with MIC values of 26 µg/ml. All 3 strains showed potential bile salt hydrolase (BSH) activity, cholesterol assimilation and cholesterol co-precipitation ability. Additionally, the potential effect of these strains on plasma cholesterol levels was evaluated in Sprague-Dawley (SD) rats. Rats in 4 treatment groups were fed the following experimental diets for 4 weeks: a high-cholesterol diet, a high-cholesterol diet plus LA15, a high-cholesterol diet plus B23 or a high-cholesterol diet plus D17. The total cholesterol, triglyceride and low-density lipoprotein cholesterol levels in the serum were significantly (P<0.05) decreased in the LAB-treated rats compared with rats fed a high-cholesterol diet without LAB supplementation. The high-density lipoprotein cholesterol levels in groups B23 and D17 were significantly (P<0.05) higher than those in the control and LA15 groups. Additionally, both fecal cholesterol and bile acid levels were significantly (P<0.05) increased after LAB administration. Fecal lactobacilli counts were significantly (P<0.05) higher in the LAB treatment groups than in the control groups. Furthermore, the 3 strains were detected in the rat small intestine, colon and feces during the feeding trial. The bacteria levels remained high even after the LAB administration had been stopped for 2 weeks. These results suggest that these strains may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.     

The effect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92

Bile tolerance is an important criterion in the selection of microbial strains for probiotic use. The survival and morphological changes of a potential probiotic strain, Lactobacillus acidophilus M92, in the presence of bile salts were examined. Lactobacillus acidophilus M92 has shown a satisfactory degree of tolerance against oxgall and individual bile salts tested, especially to taurocholate. The higher resistance of L. acidophilus M92 against taurine-conjugated bile salts relative to deconjugated and glycine-conjugated bile salts was attributed to its reaction to the stronger acidity of the former. Furthermore, bile salt hydrolase (BSH) was active when L. acidophilus M92 was grown in the presence of sodium taurocholate. The rate of BSH activity was highest at the exponential growth phase. It was hypothesised that BSH activity may be important for the bile salt resistance of this strain. The colonial and cellular morphology may also be a valuable parameter in the selection of bile salt-resistant Lactobacillus strains for probiotic use. Smooth (S) and rough (R) colonies, appeared in the original L. acidophilus M92 bacterial culture and demonstrated a different degree of bile tolerance. Rough colonies were more sensitive to bile salts than smooth ones. The R colony cells assumed a round form, probably induced by gaps in the cell wall caused by the cytotoxicity of glycodeoxycholate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantitative Appraisal of the Probiotic Attributes and In Vitro Adhesion Potential of Anti-listerial Bacteriocin-producing Lactic Acid Bacteria

Estimation of bile tolerance, endurance to gastric and intestinal environment and adhesion potential to intestinal cells are significant selection criteria for probiotic lactic acid bacteria (LAB). In this paper, the probiotic potential of native bacteriocin-producing LAB isolated previously from indigenous source has been determined through quantitative approaches. Among fifteen anti-listerial bacteriocin-producing native LAB, ten strains were found to be bile tolerant. The presence of bile salt hydrolase (bsh) gene in native Lactobacillus plantarum strains was detected by PCR and confirmed by nucleic acid sequencing of a representative amplicon. Interestingly, three native LAB strains exhibited significant viability in simulated gastric fluid, analogous to the standard LAB Lactobacillus rhamnosus GG, while an overwhelming majority of the native LAB strains demonstrated the ability to survive and remain viable in simulated intestinal fluid. Quantitative adhesion assays based on conventional plating method and a fluorescence-based method revealed that the LAB isolates obtained from dried fish displayed significant in vitro adhesion potential to human adenocarcinoma HT-29 cells, and the adhesion level was comparable to some of the standard probiotic LAB strains. The present study unravels putative probiotic attributes in certain bacteriocin-producing LAB strains of non-human origin, which on further in vivo characterization could find specific applications in probiotic food formulations targeted for health benefits.     

In Vitro Evaluation of the Probiotic and Safety Properties of Bacteriocinogenic and Non-Bacteriocinogenic Lactic Acid Bacteria from the Intestines of Nile Tilapia and Common Carp for Their Use as Probiotics in Aquaculture

In this study, seven bacteriocinogenic and non-bacteriocinogenic LAB strains previously isolated from the intestines of Nile tilapia and common carp and that showed potent antibacterial activity against host-derived and non-host-derived fish pathogens were assayed for their probiotic and safety properties so as to select promising candidates for in vivo application as probiotic in aquaculture. All the strains were investigated for acid and bile tolerances, transit tolerance in simulated gastrointestinal conditions, for cell surface characteristics including hydrophobicity, co-aggregation and auto-aggregation, and for bile salt hydrolase activity. Moreover, haemolytic, gelatinase and biogenic amine-producing abilities were investigated for safety assessment. The strains were found to be tolerant at low pH (two strains at pH 2.0 and all the strains at pH 3.0). All of them could also survive in the presence of bile salts (0.3% oxgall) and in simulated gastric and intestinal juices conditions. Besides, three of them were found to harbour the gtf gene involved in pH and bile salt survival. The strains also showed remarkable cell surface characteristics, and 57.14% exhibited the ability to deconjugate bile salts. When assayed for their safety properties, the strains prove to be free from haemolytic activity, gelatinase activity and they could neither produce biogenic amines nor harbour the hdc gene. They did not also show antibiotic resistance, thus confirming to be safe for application as probiotics. Among them, Lactobacillus brevis 1BT and Lactobacillus plantarum 1KMT exhibited the best probiotic potentials, making them the most promising candidates.     

Partial Characterization of Bacteriocins Produced by Two New Enterococcus faecium Isolated from Human Intestine

This study aimed at characterizing two novel bacteriocin-producing enterococcal strains isolated from human intestine. A total of 200 lactic acid bacteria were isolated from a woman stool sample. Two of them were selected for characterization due to their high antimicrobial activity against five strains of Listeria monocytogenes. The selected bacteria were identified as two different strains of Enterococcus faecium and designated MT 104 and MT 162. The bacteriocins produced by MT 104 and MT 162 were stable at different pH ranging from 2 to 11 and were active after different treatments such as heat, enzymes, detergents, and γ-irradiation. The two isolated strains exhibited some probiotic properties such as survival in simulated gastric fluid and intestinal fluid, lack of expression of bile salt hydrolase or hemolytic activity, adhesion to Caco-2 cells efficiently, and sensitivity to clinical antimicrobial agents. Thus, the two isolated strains of E. faecium could become new probiotic bacteria and their bacteriocins could be used for controlling L. monocytogenes in combination with irradiation for food preservation.     

Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains from breast-fed infant and investigation of their cholesterol-reducing effects

Hypercholesterolemia has been reported to be the main cause of cardiovascular diseases and the leading cause of death. Therefore, decreasing serum cholesterol level is very important for preventing the cardiovascular diseases. It has been supposed that probiotics in human gastrointestinal tract have the ability to decrease serum cholesterol level by reducing the absorption of cholesterol from the intestinal tract and the bile salt deconjugation. In this study, 28 strains of Lactobacillus spp., isolated from breast-fed infant’s feces, were identified and investigated for their bile salt deconjugation ability. The deconjugation ability of the strains was determined by the release of cholic acid resulting from the deconjugation of conjugated bile salts. Research results showed that four of the strains had bile salt deconjugation ability. The strains with deconjugation ability have been identified in species level by using biochemical test, and molecular techniques, API 50CHL test and 16S rRNA gene sequence analysis respectively. LP1, E3, and E9 strains with deconjugation activity were identified as Lactobacillus rhamnosus and GD2 strain as Lactobacillus plantarum. Even if oxgall decreases the viability of bacteria, the highest amount of cholesterol precipitation (42%) was performed by GD2 strain in the presence of 0.3% (w/v) bile. This study demonstrated that the identified Lactobacillus strains had an excellent ability to survive at low pH, a high bile deconjugation ability, and hypocholesterolemic effect in in vitro conditions.     

Bile Salt Hydrolase (Bsh) Activity Screening of Lactobacilli: In Vitro Selection of Indigenous Lactobacillus Strains with Potential Bile Salt Hydrolysing and Cholesterol-Lowering Ability

The bile salt hydrolase (Bsh) activity of probiotic bacterium residing in gastrointestinal tract has often being associated with its cholesterol-lowering effects. Hence, Bsh activity was explored in this study as the criterion for the selection of most potential Bsh-active and cholesterol-lowering indigenous Lactobacillus strains. Forty lactobacilli were adjudged Bsh active after a preliminary screening of 102 lactobacilli and occurrence of Bsh activity correlated well with their natural habitats. Of the 40 shortlisted lactobacilli, fifteen putative Lactobacillus strains were selected and further tested for their comparative Bsh activity. In the end, indigenous Lactobacillus plantarum strains Lp91 and Lp21 were emerged as the promising Bsh-active lactobacilli with their substrate preference inclined more towards glycocholate than other bile acid amino conjugates. In addition, strains Lp91 and Lp21 also exhibited significantly high bile salt deconjugation, cholesterol assimilation and cholesterol co-precipitation ability in vitro. In conclusion, indigenous L. plantarum strains Lp91 and Lp21 may be the promising candidate probiotics to elucidate the ecological significance of probiotic Bsh activity in vivo.     

Genomic analysis of Enterococcus durans LAB18S,a potential probiotic strain isolated from cheese

Gut microbiota exerts a fundamental role in human health and increased evidence supports the beneficial role of probiotic microorganisms in the maintenance of intestinal health. Enterococcus durans LAB18S was previously isolated from soft cheese and showed some desirable in vitro probiotic properties, for that reason its genome was sequenced and evaluated for genes that can be relevant for probiotic activity and are involved in selenium metabolism. Genome sequencing was performed using the Illumina MiSeq System. A variety of genes potentially associated with probiotic properties, including adhesion capability, viability at low pH, bile salt resistance, antimicrobial activity, and utilization of prebiotic fructooligosaccharides (FOS) were identified. The strain showed tolerance to acid pH and bile salts, exhibited antimicrobial activity and thrived on prebiotic oligosaccharides. Six genes involved in selenium metabolism were predicted. Analysis of the SECIS element showed twelve known selenoprotein candidates. E. durans LAB18S was the only food isolate showing absence of plasmids, virulence and antimicrobial resistance genes, when compared with other 30 E. durans genomes. The results of this study provide evidence supporting the potential of E. durans LAB18S as alternative for probiotic formulations.     

Comparison of Lactobacillus strains with respect to bile salt hydrolase activity, colonization of the gastrointestinal tract, and growth rate of the murine host.

The significance of bile salt hydrolase production by lactobacilli in the microecology of the murine intestinal tract has not been extensively studied previously. Assays of bile salt hydrolase (sodium taurocholate as substrate) associated with cell extracts of five Lactobacillus strains of murine origin gave a range of activities (from 915 nmol of cholate released per mg of protein per 30 min to none detected). All of the strains tested colonized the murine gastrointestinal tract equally well. The growth rates of mice were not affected by colonization of their intestinal tracts by lactobacilli whether or not the bacteria produced bile salt hydrolase.     

Potential probiotic properties of lactic acid bacteria isolated from the intestinal mucosa of healthy piglets

In the present study, the probiotic properties of 52 lactic acid bacteria strains, isolated from the intestinal mucosa of 60-day-old healthy piglets, were evaluated in vitro in order to acquire probiotics of potential application. Based on acidic and bile salt resistance, 11 lactic acid bacteria strains were selected, among which 1 was identified as Pediococcus acidilactici, 3 as Enterococcus faecium, 3 as Lactobacillus rhamnosus, 2 as Lactobacillus brevis, and 2 as Lactobacillus plantarum by 16S rRNA gene sequencing. All selected strains were further investigated for transit tolerance in simulated upper gastrointestinal tract, for adhesion capacity to swine intestinal epithelial cells J2 (IPEC-J2), for cell surface characteristics including hydrophobicity, co-aggregation and auto-aggregation, and for antimicrobial activities. Moreover, hemolytic, bile salt hydrolase and biogenic amine-producing abilities were investigated for safety assessment. Two E. faecium (WEI-9 and WEI-10) and one L. plantarum (WEI-51) exhibited good simulated upper gastrointestinal tract tolerance, and showed high auto-aggregation and co-aggregation with Escherichia coli 1570. The strains WEI-9 and WEI-10 demonstrated the highest adherence capacity. The 11 selected strains mentioned above exhibited strong antimicrobial activity against E. coli CVCC1570, Staphylococcus aureus CVCC1882 and Salmonella pullorum AS1.1859. None of the 11 selected strains, except WEI-9 and WEI-33, exhibited bile salt hydrolase, hemolytic or biogenic amine-producing abilities. This work showed that the E. faecium WEI-10 and L. plantarum WEI-51were found to have the probiotic properties required for use as potential probiotics in animal feed supplements.     

Polysorbate 80 improves the adhesion and survival of yogurt starters with cholesterol uptake abilities

The goal of this study is to improve the adhesion and survival of yogurt bacteria with probiotic traits by using polysorbate 80, a food additive emulsifier commonly found in milk derivative products. Polysorbate 80 was used at 1% (w/v), and its effects on yogurt bacteria's survival under simulated digestive conditions, cholesterol uptake activities, bile salt hydrolase (BSH) activity, and adhesion to HT-29 culture were studied. In the presence of 1% polysorbate 80, both starters demonstrated better cholesterol uptake and BSH activities, as well as higher bacterial survival at pH 2.5, particularly in associated cultures. In the presence of 0.3 % bile or cholic acid, polysorbate 80 reduced the drop in L. bulgaricus's survival load. However, the carbon source had a greater impact on S. thermophilus bile tolerance than the food additive emulsifier. Oleic acid was incorporated into both bacterial membranes when grown in the presence of bile and polysorbate 80, resulting in a higher unsaturated/saturated fatty acid ratio. In the presence of polysorbate 80, S. thermophilus adhered to HT-29 cells 2.3-fold better, while L. bulgaricus's adhesion remained unchanged. We suggest that polysorbate 80 may have a protective effect on cell survival under simulated digestive stress as well as a role in yogurt bacteria adhesion to the intestines, giving these bacteria more opportunities to exert their purported cholesterol-removal activities.     

Lactobacillus plantarum LRCC 5273 isolated from Kimchi ameliorates diet-induced hypercholesterolemia in C57BL/6 mice

ABSTRACT

This study was designed to select potent cholesterol-lowering probiotic strains on HepG2 cell and investigate the effect of selected strain, Lactobacillus plantarum LRCC 5273 and LRCC 5279 in hypercholesterolemic mice. In the results, LP5273 group showed significantly reduced total and LDL cholesterol compared to HCD group. In addition to significantly up-regulated hepatic mRNA expression of LXR-α and CYP7A1, intestinal LXR-α and ABCG5 were significantly up-regulated in LP5273 group. With activation of hepatic and intestinal LXR-α and its target genes, fecal cholesterol and bile acid excretion were increased in LP5273 fed mice. These results suggest that LP5273 ameliorates hypercholesterolemia in mice through the activation of hepatic and intestinal LXR-α, resulting in enhancement of fecal cholesterol and bile acids excretion in the small intestine. The results of present study suggest mechanistic evidences for hypocholesterolemic effects of L. plantarum spp., and may contribute to future researches for prevention of hypercholesterolemia and cardiovascular disease.     

Bile salt hydrolase and cholesterol removal effect by Bifidobacterium bifidum NRRL 1976

Summary Growing cells of Bifidobacterum bifidum NRRL 1976 exhibited an ability to remove cholesterol in the presence of bile salts. The cholesterol removal by Bifidobacterium bifidum was due to a co-precipitation together with unconjugated bile acids, which was linked to the bile salt hydrolase (BSH) activity of the cells at pH values lower than 5.0 and the cholesterol removed was partially recovered when the cells were washed with phosphate buffer at pH 7, while the remaining cholesterol was extracted from the cells. It is concluded that the removal of cholesterol from the growth medium by Bifidobacterium bifidum strain is due to both bacterial assimilation and precipitation of cholesterol.     

Characterization of GP21 and GP12: Two Potential Probiotic Bacteria Isolated from the Gastrointestinal Tract of Atlantic Cod

This study evaluated the probiotic potential of GP21 (Pseudomonas sp.) and GP12 (Psychrobacter sp.), two bacteria isolated from the intestinal tract of a cold-water fish, Atlantic cod. The antagonistic activity of the two intestinal bacteria against two fish pathogens (Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida) was studied under different physical conditions. Further, their resistance to physiological barriers and their ability to form biofilms were examined. In addition, a test was conducted to confirm that the isolates were not pathogenic to the host fish. The two bacteria exhibited differences in their antagonism to the pathogens. Both were active against V. anguillarum at mildly acidic conditions over a 5-day period. The activity of GP21 against A. salmonicida was greater at pH 7–8. The maximum antagonistic activity was observed at a temperature of 15°C and at a salt concentration of 15 ppt for both the isolates. They did not produce acids, could release siderophores and tolerated both the acidic environment and the bile salts. Their ability to form biofilms was high around 15°C and when iron was supplemented in the medium at 5 μmol l^?1. There was no mortality of fish during the pathogenicity experiment, confirming the safety of both isolates for further applications. Considering the favorable characteristics identified here, it could be concluded that GP21 and GP12 isolated from the gastrointestinal tract of Atlantic cod are potential probiotic candidates.     

Bile salt biotransformations by human intestinal bacteria

Secondary bile acids, produced solely by intestinal bacteria, can accumulate to high levels in the enterohepatic circulation of some individuals and may contribute to the pathogenesis of colon cancer, gallstones, and other gastrointestinal (GI) diseases. Bile salt hydrolysis and hydroxy group dehydrogenation reactions are carried out by a broad spectrum of intestinal anaerobic bacteria, whereas bile acid 7-dehydroxylation appears restricted to a limited number of intestinal anaerobes representing a small fraction of the total colonic flora. Microbial enzymes modifying bile salts differ between species with respect to pH optima, enzyme kinetics, substrate specificity, cellular location, and possibly physiological function. Crystallization, site-directed mutagenesis, and comparisons of protein secondary structure have provided insight into the mechanisms of several bile acid-biotransforming enzymatic reactions. Molecular cloning of genes encoding bile salt-modifying enzymes has facilitated the understanding of the genetic organization of these pathways and is a means of developing probes for the detection of bile salt-modifying bacteria. The potential exists for altering the bile acid pool by targeting key enzymes in the 7alpha/beta-dehydroxylation pathway through the development of pharmaceuticals or sequestering bile acids biologically in probiotic bacteria, which may result in their effective removal from the host after excretion.