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Diversity of bile salt hydrolase activities in different lactobacilli toward human bile salts
Abstract
This study was conducted to evaluate the diversity of bile salt hydrolase (BSH) activities in eight species of lactobacilli.
BSH activities were quantified based on the amount of taurine or glycine liberated from six main human bile sodium salts [glycocholic,
glycodeoxycholic, glycochenodeoxycholic, taurocholic (TC), taurochenodeoxycholic, taurodeoxycholic] and a mixture of bile
salts that resembled human bile. The eight species differed in their BSH activities. Specifically, Lactobacillus helveticus, Lactobacillus fermentum and Lactobacillus gallinarum had the ability to deconjugate taurine-conjugated bile salts, but not glycine-conjugated bile salts, which suggested that
microbial BSHs recognize bile salts on both the cholate steroid nucleus and the amino acid moiety. Of the eight species evaluated,
Lactobacillus acidophilus strains exhibited the highest specific BSH activity toward human bile salts, with the exception of TC. In addition, the L. acidophilus specific BSH activity toward glycine-conjugated bile salts was ten times higher than that toward taurine-conjugated bile
salts. Moreover, the specific BSH activity of Lactobacillus plantarum did not vary significantly toward different bile salts, and Lactobacillus gasseri Am1 exhibited higher specific BSH activity toward TC than other lactobacilli. A comparison of bsh genes indicated that the LA-bshA, LA-bshB, LG-bsh and LP-bsh1 genes that encode the BSH enzymes are highly homologous (higher than 45%), while the LP-bsh2, LP-bsh3 and LP-bsh4 genes, which might not encode BSH enzymes, had lower similarity (lower than 26.3%).
KeywordsLactobacillus-Bile salt hydrolase-Human bile salts-Secondary bile acid
... The reports of the presence of BSH in L. casei strains have been controversial. There are some studies where the authors have not found the activity of this enzyme in their L. casei strains [10,22]. However, there are others (including this study) in which good BSH activities have been detected [9,[23][24][25]. ...
... It has the ability to absorb cholesterol and is reported to have good BSH activity [14]. There are many reports dealing with the presence of BSH in L. fermentum strains [9,22]. The substrate specificity varies with the strain. ...
... Several studies on BSH substrate specificity have shown substrate preference toward glycineconjugated bile salts [10,12,37,38]. There are also a few reports indicating a preference for taurine-conjugated bile salts [28] and others indicating that for the strains Lp80 [12] and Lp-onlly, Lp-529, and Lp501 [22] the substrate preference is unclear since the strains have good activity in the two kinds of conjugated bile acids. Table 3 shows that in this study, the results for BSH activity in the strain 299v indicate a preference (as in most studies) for the glycine-conjugated bile salts (85% and 100% deconjugation for sodium glycocholate and glycodeoxycholate, respectively, compared to 65% and 31% deconjugation for sodium taurocholate and taurodeoxycholate, respectively). ...
Bile salt hydrolase (BSH) activity in probiotic strains is usually correlated with the ability to lower serum cholesterol levels in hypercholesterolemic patients. The objective of this study was the evaluation of BSH in five probiotic strains of lactic acid bacteria (LAB) and a probiotic yeast. The activity was assessed using a qualitative direct plate test and a quantitative high-performance thin- layer chromatography assay. The six strains differed in their BSH substrate preference and activity. Lactobacillus plantarum DGIA1, a potentially probiotic strain isolated from a double cream cheese from Chiapas, Mexico, showed excellent deconjugation activities in the four tested bile acids (69, 100, 81, and 92% for sodium glycocholate, glycodeoxycholate, taurocholate, and taurodeoxycholate, respectively). In the case of the commercial probiotic yeast Saccharomyces boulardii, the deconjugation activities were good against sodium glycodeoxycholate, taurocholate, and taurodeoxycholate (100, 57, and 63%, respectively). These last two results are part of the novelty of the work. A weak deconjugative activity (5%) was observed in the case of sodium glycocholate. This is the first time that the BSH activity has been detected in this yeast.
... The ninhydrin biochemical method described previously by Liong and Shah [34] was used with a few modifications to determine specific BSH activity. It is based on determining the amount of amino acids released from conjugated bile salts [36,37]. Since BSH in this study was secreted into the media, cell disruption steps were omitted and 0.1 ml of cell-free supernatant was mixed with 0.8 ml 0.1 M sodium phosphate buffer (pH 6.0) and 0.1 ml of 50 mM/l of respective conjugated bile salt (GC, TC, and TDC) and incubated at 37°C for 30 min. ...
... Values were found to be identical with a specific activity of BSH from L. fermentum NCDO 394 previously reported by Kumar et al. [36] against the listed bile salts. Since BSH enzymes extracted from different sources tend to have different substrate specificities [34,37,40], both the strains showed a high specific enzyme activity towards glycine-conjugated bile salts as compared to tauro-conjugated salts. P. ...
... This also obviates the need for use of any other selective pressure for the maintenance of the plasmid vector. Specific BSH activity revealed that substrate preference of BSH enzyme is more inclined towards glycine-conjugated bile substrates as compared to taurine-conjugated salts, consistent with the data already reported in literature for other LAB BSH enzymes [36,37,54,59,[69][70][71]. BSH from L. fermentum NCDO 394 has already been well characterized by Kumar et al. [36] and found to be stable and functional in a range of pH and temperature values identical to the human body. ...
Genetic engineering has emerged as an effective means to improve and enhance the potential of commercially important bacterial strains. However, the biosafety of recombinant systems is an important concern during the implementation of such technologies on an industrial scale. In order to overcome this issue, cloning and expression systems have been developed preferably from fully characterized and annotated LAB plasmids encoding genes with known functions. The developed shuttle vector pPBT-GFP contains two theta-type replicons with a copy number of 4.4 and 2.8 in Pediococcus acidilactici MTCC 5101 and Lactobacillus brevis MTCC 1750, respectively. Antimicrobial "pediocin" produced by P. acidilactici MTCC 5101 and green fluorescent protein (GFP) of Aequorea victoria were successfully expressed as selectable markers. Heterologous bile salt hydrolase (BSH) from Lactobacillus fermentum NCDO 394 has been efficiently expressed in the host strains showing high specific activity of 126.12 ± 10.62 in P. acidilactici MTCC 5101 and 95.43 ± 4.26 in the case of L. brevis MTCC 1750, towards glycine-conjugated bile salts preferably as compared to taurine-conjugated salts. The present article details the development of a LAB/LAB shuttle expression vector pPBT-GFP, capable of replication in LAB hosts, P. acidilactici MTCC 5101, and L. brevis MTCC 1750. Pediocin and GFP have been used as selectable markers with the efficient production of heterologous extracellular bile salt hydrolase. Thus, the constructed vector pPBT-GFP, with its ability to replicate in multiple hosts, low copy number, and stability in host cells, may serve as an ideal tool for improving LAB strains of commercial value using genetic engineering.
... 36 The contents of conjugated bile acids in human are as follows: 9% TCA, 9% TDCA, 8% TCDCA, 26% GCA, 22% GDCA, and 26% GCDCA. 37 Thus, the ratio of glycine to taurine conjugates is approximately 3:1. 10,38 BSH enzymes have been identified from commensal gut microbiota, pathogen, and free-living bacteria. ...
... 64 Because glyco-conjugated bile acids are far more toxic for bacteria than the tauro-conjugates, the higher affinity of BSHs for glyco-conjugates may be of great importance in the ecology of gut microbe. 37,65 Seven BSH enzymes preferentially hydrolyze tauroconjugates, whereas other seven BSHs hydrolyze both glyco-and tauro-conjugated bile acids, displaying a broad spectrum of specificity. ...
... However, in each group, BSHs exhibit different substrate preferences, which may be because evolution tends to broaden substrate selectivity. 37 Multiple sequence alignment shows that the pairwise sequence identity of these BSHs ranges from 20.33% to 99.69%, with an average of 40.88%. BSH enzymes from L. johnsonii PF01 and L. johnsonii 100-100 have a high degree of similarities to those from other species, such as L. acidophilus and L. plantarum, because they are acquired through horizontal gene transfer. ...
The worldwide trend of limiting the use of antibiotic growth promoters (AGPs) in animal production creates challenges for the animal feed industry, thus necessitating the development of effective non-antibiotic alternatives to improve animal performance. Increasing evidence has shown that the growth-promoting effect of AGPs is highly correlated with the reduced activity of bile salt hydro-lase (BSH, EC 3.5.1.24), an intestinal bacteria-producing enzyme that has a negative impact on host fat digestion and energy harvest. Therefore, BSH inhibitors may become novel, attractive alternatives to AGPs. Detailed knowledge of BSH substrate preferences and the wealth of structural data on BSHs provide a solid foundation for rationally tailored BSH inhibitor design. This review focuses on the relationship between structure and function of BSHs based on the crystal structure, kinetic data, molecular docking and comparative structural analyses. The molecular basis for BSH substrate recognition is also discussed. Finally, recent advances and future prospectives in the development of potent, safe, and cost-effective BSH inhibitors are described.
... The ninhydrin biochemical method described previously by Liong and Shah [34] was used with a few modifications to determine specific BSH activity. It is based on determining the amount of amino acids released from conjugated bile salts [36,37]. Since BSH in this study was secreted into the media, cell disruption steps were omitted and 0.1 ml of cell-free supernatant was mixed with 0.8 ml 0.1 M sodium phosphate buffer (pH 6.0) and 0.1 ml of 50 mM/l of respective conjugated bile salt (GC, TC, and TDC) and incubated at 37°C for 30 min. ...
... Values were found to be identical with a specific activity of BSH from L. fermentum NCDO 394 previously reported by Kumar et al. [36] against the listed bile salts. Since BSH enzymes extracted from different sources tend to have different substrate specificities [34,37,40], both the strains showed a high specific enzyme activity towards glycine-conjugated bile salts as compared to tauro-conjugated salts. P. ...
... This also obviates the need for use of any other selective pressure for the maintenance of the plasmid vector. Specific BSH activity revealed that substrate preference of BSH enzyme is more inclined towards glycine-conjugated bile substrates as compared to taurine-conjugated salts, consistent with the data already reported in literature for other LAB BSH enzymes [36,37,54,59,[69][70][71]. BSH from L. fermentum NCDO 394 has already been well characterized by Kumar et al. [36] and found to be stable and functional in a range of pH and temperature values identical to the human body. ...
Background:
Lactic acid bacteria (LAB) are a diverse group of Gram-positive bacteria, which are widely distributed in various diverse natural habitats. These are used in a variety of industrial food fermentations and carry numerous traits with utmost relevance to the food industry. Genetic engineering has emerged as an effective means to improve and enhance the potential of commercially important bacterial strains. However, the biosafety of recombinant systems is an important concern during the implementation of such technologies on an industrial scale. In order to overcome this issue, cloning and expression systems have been developed preferably from fully characterized and annotated LAB plasmids encoding genes with known functions.
Results:
The developed shuttle vector pPBT-GFP contains two theta-type replicons with a copy number of 4.4 and 2.8 in Pediococcus acidilactici MTCC 5101 and Lactobacillus brevis MTCC 1750, respectively. Antimicrobial "pediocin" produced by P. acidilactici MTCC 5101 and green fluorescent protein (GFP) of Aequorea victoria were successfully expressed as selectable markers. Heterologous bile salt hydrolase (BSH) from Lactobacillus fermentum NCDO 394 has been efficiently expressed in the host strains showing high specific activity of 126.12 ± 10.62 in P. acidilactici MTCC 5101 and 95.43 ± 4.26 in the case of L. brevis MTCC 1750, towards glycine-conjugated bile salts preferably as compared to taurine-conjugated salts.
Conclusion:
The present article details the development of a LAB/LAB shuttle expression vector pPBT-GFP, capable of replication in LAB hosts, P. acidilactici MTCC 5101, and L. brevis MTCC 1750. Pediocin and GFP have been used as selectable markers with the efficient production of heterologous extracellular bile salt hydrolase. Thus, the constructed vector pPBT-GFP, with its ability to replicate in multiple hosts, low copy number, and stability in host cells, may serve as an ideal tool for improving LAB strains of commercial value using genetic engineering.
... 36 The contents of conjugated bile acids in human are as follows: 9% TCA, 9% TDCA, 8% TCDCA, 26% GCA, 22% GDCA, and 26% GCDCA. 37 Thus, the ratio of glycine to taurine conjugates is approximately 3:1. 10,38 BSH enzymes have been identified from commensal gut microbiota, pathogen, and free-living bacteria. ...
... 64 Because glyco-conjugated bile acids are far more toxic for bacteria than the tauro-conjugates, the higher affinity of BSHs for glyco-conjugates may be of great importance in the ecology of gut microbe. 37,65 Seven BSH enzymes preferentially hydrolyze tauroconjugates, whereas other seven BSHs hydrolyze both glyco-and tauro-conjugated bile acids, displaying a broad spectrum of specificity. ...
... However, in each group, BSHs exhibit different substrate preferences, which may be because evolution tends to broaden substrate selectivity. 37 Multiple sequence alignment shows that the pairwise sequence identity of these BSHs ranges from 20.33% to 99.69%, with an average of 40.88%. BSH enzymes from L. johnsonii PF01 and L. johnsonii 100-100 have a high degree of similarities to those from other species, such as L. acidophilus and L. plantarum, because they are acquired through horizontal gene transfer. ...
... Because of the strong relationship between BSH and negative or positive health consequences, various BSH genes have been cloned and characterized from Lactobacillus species such as Lb. acidophilus (Corzo and Gilliland, 1999;Jiang et al., 2010), Lb. johnsonii (Chae et al., 2012), Lb. fermentum (Kumar et al., 2013), Lb. salivarus (Bi et al., 2013), and Lb. plantarum (Lambert et al., 2008;Dong et al., 2012;Gu et al., 2014;Chang-qing and Rong, 2015). ...
... Therefore, further genotypic-phenotypic characterization, functional studies of BSHs from different strains, and comparisons with other characterized BSHs are necessary. The Lb. gasseri is a good candidate for these targets since this organism exhibited higher specific BSH activity toward TC than other species of lactobacilli (Jiang et al., 2010). Even though Lb. gasseri is probably one of the most beneficial probiotics on the market, there is little to no information on the mechanism of action of the catalytic activity and substrate preferences of its BSH. ...
... The activities and characteristics of BSHs show variation between species and sometimes even between the strains of the same species. It has been suggested that most evolution occurs to broaden substrate specificity (Jiang et al., 2010). Begley et al.(2006) and Patel et al. (2010) emphasized that substrate specificity may occur at amino acid or steroid cholyl moieties. ...
Bile salt hydrolase (BSH) active probiotic strains are being used in the treatment of hypercholesterolemia related diseases. Understanding the mechanism of action of the BSH of probiotics is crucial. Even though Lactobacillus gasseri is probably one of the most beneficial probiotics on the market, there is no information on the catalytic activity and substrate preferences of its BSH. This study aims to detect the substrate specificity of the BSH of Lb. gasseri ATCC 33323 strain, to determine the optimum temperature and pH of the BSH’s activity by ninhydrin assay. To do so, the BSH gene from Lb. gasseri ATCC 33323 was cloned into Escherichia coli XL1-Blue strain, expressed and characterized in E. coli BLR(DE3). Results indicated that even though the recombinant BSH (rBSH) was able to hydrolyze the six major human bile salts, there was an obvious preference for the glycine-conjugated bile salts and the maximum activities of the rBSH were recorded.
... The bile salt hydrolase activities (Bsh activities) of the isolates were assessed by determining the amount of amino acids released from the hydrolysis of 160 conjugated bile salts [Oxgall and taurodeoxycholate (TDC)] by the cell extracts of the LAB isolates using Ninhydrin assay. The experiment was performed following the protocol of Jiang et al. (2010), and the results were expressed as specific activity of the enzyme (U/mg). One unit of Bsh activity was defined as the amount of the enzyme that liberated 1 μmol of 165 amino acids from the substrate per minute. ...
... It consisted of three steps: Table 1. List of primers used for Bsh gene screening (Jiang et al., 2010). 185 denaturation at 94°C for 1 min, annealing at 42°C for 20 s, and elongation at 72°C for 2 min. ...
... This last statement could explain the low representation of isolates with TDC preference in this study. The greatest Bsh activity recorded, 93 U/mg, obtained with isolate 86L in Oxgall 420 presence, is higher than the one (ranged 10.5 ± 0.6 to 41.7 ± 3 U/mg) obtained in a bile salts mixture by Jiang et al. (2010) with L. plantarum, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus salivarius, and Lactobacillus casei strains isolated from human intestine. ...
The present study aimed to evaluate selected probiotic properties of Lactobacilli isolated from the water of submerged cassava fermentation. Following Lactobacilli isolation, isolates were screened for their antimicrobial activity. Acid and bile tolerances, bile salt hydrolase (Bsh) activity spectrum were assessed. Among the 113 isolates obtained, 16 showed a broad antimicrobial activity spectrum against the indicator microorganisms. From these 16, 12 were found acid and bile resistant. They hydrolyzed glycoconjugated or tauroconjugated bile salts. From the four bile Bsh genes screened, only Bsh-Lp1 was found in five isolates. They identified as Lactobacillus paraplantarum, Lactobacillus paracasei, Lactobacillus brevis, and Lactobacillus plantarum. Based on the principal component analysis, L. paracasei 62L, L. plantarum 85L and 86L were selected as the most promising strains. These results suggest that water from submerged cassava fermentation can be a source of Lactobacilli with high probiotic potential.
... Considering the implications of deconjugation of tauro-conjugated salts for negative health consequences (Ridlon et al., 2006;Jiang et al., 2010), further studies characterizing the BSH enzymes from different bacterial species are beneficial. The activities and characteristics of such important BSH enzymes show variation between species and sometimes even between the strains of the same species. ...
... The activities and characteristics of such important BSH enzymes show variation between species and sometimes even between the strains of the same species. Many bsh genes from different species and strains of lactic acid bacteria have been cloned and characterized, including those from Bifidobacterium strains (Kim et al., 2005;Jarocki et al., 2014), L. plantarum (Ha et al., 2006;Gu et al., 2014), L. acidophilus (Jiang et al., 2010), L. johnsonii (Chae et al., 2012) and L. fermentum (Kumar et al., 2013). However, the substrate specificity and activity mechanism of BSH enzyme in probiotic strains still remains unclear. ...
... Although the BSHs of L. rhamnosus E9 have the same catalytic sites to other Lactobacillus species, their substrate-hydrolyzing capabilities are different. It has been suggested that most evolution occurs to broaden substrate specificity (Jiang et al., 2010). Begley et al. (2006) and Patel et al. (2010) emphasized that substrate specificity may occur at amino acid or steroid cholyl moieties. ...
Bile salt hydrolase (BSH) enzyme, commonly found in probiotic bacteria of gut origin, catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts allowing for colonization of the bacteria in the gut and contributing to a decrease in levels of cholesterol. However an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid can have harmful side-effects. The aim of this study was to characterize the activity of BSH enzymes from Lactobacillus rhamnosus E9, a popular probiotic strain. The bsh gene was cloned, expressed, purified and characterized in Escherichia coli BLR(DE3) strain. The hydrolysis activities and substrate specificities of the recombinant BSH (rBSH) enzyme were examined using six different bile acids. Nucleotide sequence analysis results indicated that the bsh of E9 contained an open reading frame (ORF) of 1014 and nucleotides encoding a 338-amino acid protein with a molecular weight of 37 kDa. Five catalytically important amino acids and the amino acid motifs located around the active site were highly conserved. The rBSH showed a slight preference towards glycine-conjugated to tauro-conjugated bile salts. This confirms that it is a safe strain for probiotics and its preference for glycine-conjugated bile salts should be further investigated.
... Abs 570 was measured after 30 min in an Epoch TM Spectrophotometer (Biotek, Winooski, VT, United States). BSH activity was expressed in U/ml, since one unit of BSH activity was defined as the amount of enzyme that liberated 1 µmol glycine from GDCA per minute (Jiang et al., 2010). ...
... However, in the six strains, the level of BSH was lower than the values reported in literature for other Lactobacillus species (Liong and Shah, 2005) and Bifidobacterium longum (Tanaka et al., 2000). These findings suggest weak BSH activity and contrast with the results reported by Jiang et al. (2010). In the latter study, BSH activity associated with deconjugation of GDC in L. helveticus Lh1 was not detected. ...
Lactobacillus helveticus belongs to the large group of lactic acid bacteria (LAB), which are the major players in the fermentation of a wide range of foods. LAB are also present in the human gut, which has often been exploited as a reservoir of potential novel probiotic strains, but several parameters need to be assessed before establishing their safety and potential use for human consumption. In the present study, six L. helveticus strains isolated from natural whey cultures were analyzed for their phenotype and genotype in exopolysaccharide (EPS) production, low pH and bile salt tolerance, bile salt hydrolase (BSH) activity, and antibiotic resistance profile. In addition, a comparative genomic investigation was performed between the six newly sequenced strains and the 51 publicly available genomes of L. helveticus to define the pangenome structure. The results indicate that the newly sequenced strain UC1267 and the deposited strain DSM 20075 can be considered good candidates for gut-adapted strains due to their ability to survive in the presence of 0.2% glycocholic acid (GCA) and 1% taurocholic and taurodeoxycholic acid (TDCA). Moreover, these strains had the highest bile salt deconjugation activity among the tested L. helveticus strains. Considering the safety profile, none of these strains presented antibiotic resistance phenotypically and/or at the genome level. The pangenome analysis revealed genes specific to the new isolates, such as enzymes related to folate biosynthesis in strains UC1266 and UC1267 and an integrated phage in strain UC1035. Finally, the presence of maltose-degrading enzymes and multiple copies of 6-phospho-β-glucosidase genes in our strains indicates the capability to metabolize sugars other than lactose, which is related solely to dairy niches.
... Recent investigations have shown that bile salt hydrolyzing Lactobacillus are suggested to manage hypercholesterolaemia through interaction with host bile salt metabolism, Bile salts are synthesized from cholesterol, conjugated with glycine or taurine in the liver, and secreted into the small intestine .The gastrointestinal microbiota have evolved BSH activity to degrade bile salts, playing an essential role in bile detoxification and reducing serum cholesterol levels [19]. Study done by Jiang et al., (2010) [20] demonstrated the Lactobacillus fermentum capable only of deconjugating taurine-conjugated bile salts, and did not exhibit any BSH activity to glycoconjugated bile salt. As well, Lactobacillus paracasi showed activity when grown on MRS agar containing 0.5% (w/v) taurodeoxycholic acid sodium salt [21]. ...
... Recent investigations have shown that bile salt hydrolyzing Lactobacillus are suggested to manage hypercholesterolaemia through interaction with host bile salt metabolism, Bile salts are synthesized from cholesterol, conjugated with glycine or taurine in the liver, and secreted into the small intestine .The gastrointestinal microbiota have evolved BSH activity to degrade bile salts, playing an essential role in bile detoxification and reducing serum cholesterol levels [19]. Study done by Jiang et al., (2010) [20] demonstrated the Lactobacillus fermentum capable only of deconjugating taurine-conjugated bile salts, and did not exhibit any BSH activity to glycoconjugated bile salt. As well, Lactobacillus paracasi showed activity when grown on MRS agar containing 0.5% (w/v) taurodeoxycholic acid sodium salt [21]. ...
Bile salt hydrolase (BSH) activity of intestinal bacteria including (lactobacilli) is one of the indirect ways of decreasing a cholesterol level in human body. Tested Lactobacillus strains were isolated from faeces of fully breast-fed infants, identified by phenotypic methods and biochemical tests screening for production BSH by direct plate assay method, and quantitative method.Among12 lactobacillus isolates ,only 3 strains were shown to be positive for BSH activity by agar plates supplemented with 0.5 % (w/v) Na-taurocholate (TCA) and 0.37 g/L of CaCl2. While 10 isolates showed activity in a quantitative assay. The results shown quantitative assay was more sensitive compared with the plate assay (qualitative method) for BSH activity determination.
... Microbial enzymes could also play an important role in bile-acid metabolism. First, gut bacteria release the bile salt hydrolase (BSH), which acts on the amide bonds of bile salts to produce glycine or taurine and free bile acids [22]. Furthermore, gut bacteria can catalyze dehydroxylation reactions of free cholic acid [23,24]. ...
... Bacterial BSH has two classes of steroid and amino acid recognition. In addition, it has been reported that the addition of a strain with BSH activity can effectively reduce serum cholesterol levels in mice and dogs [22]. It is therefore evident that these unique enzymes in the gut flora can influence drug metabolism in humans [25]. ...
Background:
Modern features of drug development such as low permeability, low solubility, and improved release affect the interplay of the gut microbiota and drug metabolism. In recent years, studies have established the impact of plateau hypoxia on gut microbiota, where drug use by plateau populations is affected by hypoxia-induced changes in intestinal microflora-mediated drug metabolism.
Methods:
In this review, we summarized the effects of gut microbiota on drug metabolism, and of plateau hypoxia on the intestinal flora, with the aim of providing guidance for the rational use of drugs in high-altitude populations.
Results:
The evidence clearly shows that alterations in gut microbiota can affect pro-drug activation, drug inactivation, and the biotransformation of xenobiotics. Additionally, plateau hypoxia alters drug metabolism by affecting intestinal flora.
Conclusion:
This review provides an overview of the effects of gut microbiota on drug metabolism and provides guidance for rational drug use under hypoxic conditions at high altitudes.
... Specifically, BSH activity deconjugates bile salts to generate free bile acids, which can further be converted into secondary bile acids and metabolites such as hydrogen sulfide. These have been linked to the development of colorectal cancer, gallstones, and other gastrointestinal diseases [14]. Secondary bile acids reduce the reabsorption of bile acids in the intestine. ...
Gallstone disease is a common and complex condition, strongly associated with abnormal cholesterol metabolism, changes in bile composition, and impaired gallbladder motility. Recent studies have suggested that the gut microbiota, particularly probiotics like lactic acid bacteria, may play a significant role in the prevention and treatment of cholesterol gallstones. This study aims to optimize the cholesterol gallstone model in C57BL/6 mice and evaluate the effects of Lactobacillus intervention on gallstone formation induced by a high-fat diet. In this study, 8-week-old male C57BL/6 mice were randomly divided into four groups: a high-fat diet + saline group (HF-S), a high-fat diet + probiotic group (HF-P), a normal diet + saline group (ND-S), and a normal diet + probiotic group (ND-P), to assess the effect of probiotics on gallstone formation. The results showed significant differences among the four groups in body weight gain, liver weight, gallstone formation, and histopathology. Based on these preliminary findings, we added two more experimental groups: a 2-week probiotic pretreatment + high-fat diet group (Pre2w-HF) and a 4-week probiotic pretreatment + high-fat diet group (Pre4w-HF), to further investigate the dose-dependence and efficacy of probiotic pretreatment. The results indicated that probiotic intervention significantly reduced the incidence and severity of gallstones induced by a high-fat diet, with the pretreatment groups showing more pronounced effects. Histological analysis also revealed that probiotic intervention reduced inflammation and pathological changes in the liver and gallbladder. This study suggests that probiotics have potential therapeutic value in the prevention and treatment of cholesterol gallstones. Future research should explore the effects of different strains and doses, as well as the underlying mechanisms involved.
... as well as two alpha/beta hydrolases. Studies have shown that BSHs from L. acidophilus exhibit specificity for deconjugating taurine-conjugated bile acids [60], supporting the prediction that L. acidophilus is capable of consuming TC. ...
Background: The human gut microbiome is critical for host health by facilitating essential metabolic processes. Our study presents a data-driven analysis across 312 bacterial species and 154 unique metabolites to enhance the understanding of underlying metabolic processes in gut bacteria. The focus of the study was to create a strategy to generate a theoretical (negative) set for binary classification models to predict the consumption and production of metabolites in the human gut microbiome. Results: Our models achieved median balanced accuracies of 0.74 for consumption predictions and 0.95 for production predictions, highlighting the effectiveness of this approach in generating reliable negative sets. Additionally, we applied a kernel principal component analysis for dimensionality reduction. The consumption model with a polynomial kernel, and the production model with a radial basis function with 32 reduced features, showed median accuracies of 0.58 and 0.67, respectively. This demonstrates that biological information can still be captured, albeit with some loss, even after reducing the number of features. Furthermore, our models were validated on six previously unseen cases, achieving five correct predictions for consumption and four for production, demonstrating alignment with known biological outcomes. Conclusions: These findings highlight the potential of integrating data-driven approaches with machine learning techniques to enhance our understanding of gut microbiome metabolism. This work provides a foundation for creating bacteria–metabolite datasets to enhance machine learning-based predictive tools, with potential applications in developing therapeutic methods targeting gut microbes.
... Previous studies show that germ-free rodents have more total BAs but fewer types of BAs compared to normal counterparts [80]. The conversion of BAs in the intestine is mainly mediated by anaerobic bacteria in the intestines possessing bile salt hydrolase: Bacteroides, Eubacterium, Lactobacillus, and Clostridium [81]. They unbind cholate and glycine from bile salt through the action of bile salt hydrolase, forming free BAs. ...
The gut microbiota constitutes a complex ecosystem, comprising trillions of microbes that have co-evolved with their host over hundreds of millions of years. Over the past decade, a growing body of knowledge has underscored the intricate connections among diet, gut microbiota, and human health. Bioactive polysaccharides (BPs) from natural sources like medicinal plants, sea-weeds, and fungi have diverse biological functions, including antioxidant, immunoregulatory, and metabolic activities. Their effects are closely tied to the gut microbiota, which metabolizes BPs into health-influencing compounds. Understanding how BPs and gut microbiota interact is critical for harnessing their potential health benefits. This review provides an overview of the human gut microbiota, focusing on its role in metabolic diseases like obesity, type II diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases (CVD). It explores the basic characteristics of several BPs and their impact on gut microbiota. Given their significance for human health, we summarize the biological functions of these BPs, particularly in terms of im-munoregulatory activities, blood sugar, and hypolipidemic effect, providing a valuable reference for understanding the potential benefits of natural BPs in treating metabolic diseases. These properties make BPs promising agents for preventing and treating metabolic diseases. The comprehensive understanding of the mechanisms by which BPs exert their effects through gut microbiota opens new avenues for developing targeted therapies to improve metabolic health.
... In the current study, unlike that reported by Corzo and Gilliland (1999), oxgall, a mixture of taurocholic acid and glycocholic acid (in addition to cholesterol and lecithin), instead of sodium glycolate or sodium taurocholate, was the substrate for BSH assay. In a previous study investigating the activities of different microbial BSHs towards human bile salts, Jiang et al. (2010) had observed a great drop in BSH activity when oxgall was included in the growth medium of some strains of Lactobacillus (L. gasseri and L. acidophilus), but not in others, suggesting the possibility that components of bile (oxgall, in the current instance) may exert some complex as yet unknown regulatory process on the manner in which different BSHs process their bile acid components. ...
Bile salt hydrolases (BSH) results in microbes releasing enzymes conferring physiological functions for both the producing organisms and the host and subsequently lower serum cholesterol. In recent years, these attributes have generated interest towards screening of lactic acid bacteria (LAB) for functional food production. In this present study, lactobacilli (n=34) (13 L. casei, 6 L. paracasei, 11 L. brevis, 3 L. pentosus and 1 L. plantarum) from varied sources viz. Nigerian fermented foods and human specimens were screened qualitatively and quantitatively for BSH activity using the direct plate assay and cell free extracts (CFE) of overnight cultures. To establish a correlation between BSH and cholesterol reduction, 12 isolates displaying high precipitation over bile salt agar plates, and high BSH enzyme activity in CFE were selected for in vitro cholesterol reduction assay. Results showed that 58% of the isolates from humans, and 36% from fermented food origin showed larger zones of precipitation. The highest BSH activity was exhibited by Lactobacillus casei GR4 (76mM) after 10 min of incubation, an isolate from fermented food. Isolates from human specimens expressed higher enzyme activity compared to those from fermented foods with significant increase (P<0.05) after 30 min incubation. Cholesterol level was variable as L. pentosus 8ST5 and 8ST7 had 2 and 4% cholesterol levels in the medium; while L. casei and paracasei group (Lactobacillus casei group, LCG) 3MB4, 8BM6, BK4 and L. brevis GR29 had 7%/ml each proving their application in food formulation, reduction of hypercholesterolemia, and as prophylaxis for controlling serum cholesterol levels.
... A prerequisite for these transformations is deconjugation (33,34). To further investigate the role of bacterial transformation of BAs by gut bacteria in HFD-induced upregulation of Rspo3 gene expression, we inoculated germ-free mice with either Lactobacillus plantarum (10 8 CFU), which contains BSH for bile salt deconjugation (35), or Clostridium scindens (10 8 CFU), which contains 7α-dehydroxylates for BA transformation (36), or a combination of both L. plantarum and C. scindens (double inoculations), and fed the mice an HFD for 2 weeks. The successful colonization of L. plantarum, C. scindens, or both in germ-free mouse gut was validated at the end of 2 weeks by qPCR using bacterial species-specific primers and mouse fecal DNA. ...
High-fat diet (HFD) contributes to the increased incidence of colorectal cancer; but the mechanisms are unclear. We found that R-spondin 3 (Rspo3), a ligand for LGR4 and LGR5, was the major subtype and produced by myofibroblasts beneath the crypts in the intestine; HFD upregulated colonic Rspo3, LGR4, LGR5 and β-catenin gene expressions in specific pathogen free rodents, but not in germfree mice, and the upregulations were prevented by bile acids (BA) binder, cholestyramine (CHO) or antibiotic treatment, indicating mediating by both BA and gut microbiota. CHO or antibiotic treatments prevented HFD-induced enrichment of Lachnospiraceae and Rumincoccaceae, which are capable of transforming 10 into 20 BA. Oral administration of deoxycholic acid (DCA), or inoculation of a combination of BA deconjugator Lactobacillus plantarum and 7-α-dehydroxylase-containing Clostridium scindens with HFD to germfree mice increased colonic Rspo3 mRNA, indicating that formation of 20 BA by gut microbiota is responsible for HFD-induced upregulation of Rspo3. In primary myofibroblasts DCA increased Rpso3 mRNA via TGR5. Finally, we showed that CHO or conditional deletion of Rspo3 prevented HFD- or DCA- induced intestinal proliferation. We conclude that secondary BA is responsible for HFD-induced upregulation of Rspo3, which in turn mediates HFD-induced intestinal epithelial proliferation.
... Of 70 Lactobacillaceae strains tested against tauroconjugated and glyco-conjugated cholic acids, 18 showed BSH activity for either one or the other form of bile salts or both. This is in agreement with other studies demonstrating that BSH are not ubiquitous among lactobacilli [48,49] and that their substrate specificity varies greatly [50,51]. In this study, most BSH-positive strains exhibited specificity for taurocholic acid. ...
Dyslipidemia, specifically abnormal levels of low-density lipoprotein cholesterol (LDL-C), is an important risk factor of cardiovascular disease. Evidence showing the promising abilities of probiotics to lower total cholesterol or LDL-C has, however, not yet convinced experts to recommend probiotic bacteria as treatment for blood lipid management. Therefore, there are opportunities for the development of new efficient cholesterol-lowering probiotics. Bile salt hydrolase (BSH) and feruloyl esterase (FAE) are bacterial enzymes proposed to explain the cholesterol-lowering capacity of some bacteria and have both been shown to be responsible for lipid reduction in vivo. Here, in order to select for cholesterol-lowering bacteria, 70 strains related to Lactobacillaceae were screened for BSH and FAE activities. Based on this two-way screening approach, two bacteria were selected and assessed for their capacity to assimilate cholesterol in vitro, another suggested mechanism. Lactobacillus acidophilus CL1285 showed BSH and FAE activity as well as capacity to assimilate cholesterol in vitro. Lactiplantibacillus plantarum CHOL-200 exhibited BSH activity and ability to assimilate cholesterol. These properties observed in vitro make both strains good probiotic candidates for the management of dyslipidemia. Further investigation is needed to assess their ability to reduce blood cholesterol in human trial.
... The inhibition of FXR in the small intestine increases insulin sensitivity and glucose tolerance in rats [195,196]. Surprisingly enough, a high-fat diet in mice decreases Lactobacillus Gasseri species in the gut microbiome, the same species that expresses a bile salt hydrolase capable of transforming the dangerous TCDCA in chenodeoxycholic acid, a bile acid with unknown effects on insulin activities [197]. ...
Overweight and obesity in children and adolescents are overwhelming problems in western countries. Adipocytes, far from being only fat deposits, are capable of endocrine functions, and the endocrine activity of adipose tissue, resumable in adipokines production, seems to be a key modulator of central nervous system function, suggesting the existence of an “adipo-cerebral axis.” This connection exerts a key role in children growth and puberty development, and it is exemplified by the leptin–kisspeptin interaction. The aim of this review was to describe recent advances in the knowledge of adipose tissue endocrine functions and their relations with nutrition and growth. The peculiarities of major adipokines are briefly summarized in the first paragraph; leptin and its interaction with kisspeptin are focused on in the second paragraph; the third paragraph deals with the regulation of the GH-IGF axis, with a special focus on the model represented by growth hormone deficiency (GHD); finally, old and new nutritional aspects are described in the last paragraph.
... Finally, we investigated whether upper small intestinal healthy microbiome transplantation in HF rats induces microbial bile salt hydrolasedependent changes in the USI and/or ileum to alter upper small intestinal and/or ileal TCDCA-FXR axis and nutrient sensing glucoregulation. The Lactobacillus genus is the dominant microbiota in the USI of rats and mice [9,27], and many Lactobacillus species deconjugate taurineconjugated bile acids (i.e., TCDCA) via their respective bile salt hydrolase [28]. Given the current short term 3-d HF vs. chow-fed feeding rat protocol decreases L. gasseri in the USI and increases USI and ileal TCDCA levels, and that these HF-induced effects are prevented by upper small intestinal healthy microbiome transplantation [7,9], we reasoned that inhibiting bile salt hydrolase in the USI of HF rats with healthy microbiome transplant would prevent the inhibition of the TCDCA-FXR axis and negate nutrient sensing in the USI and ileum ( Figure 4A). ...
Objective
The mechanism of nutrient sensing in the upper small intestine (USI) and ileum that regulates glucose homeostasis remains elusive. Short-term high-fat (HF) feeding increases taurochenodeoxycholic acid (TCDCA; an agonist of farnesoid X receptor (FXR)) in the USI and ileum of rats, and the increase of TCDCA is prevented by transplantation of microbiota obtained from the USI of healthy donors into the USI of HF rats. However, whether changes of TCDCA-FXR axis in the USI and ileum alter nutrient sensing remains unknown.
Methods
Intravenous glucose tolerance test was performed in rats that received USI or ileal infusion of nutrients (i.e., oleic acids or glucose) via catheters placed toward the lumen of USI and/or ileum, while mechanistic gain- and loss-of-function studies targeting the TCDCA-FXR axis or bile salt hydrolase activity in USI and ileum were performed.
Results
USI or ileum infusion of nutrients increased glucose tolerance in healthy but not HF rats. Transplantation of healthy microbiome obtained from USI into the USI of HF rats restored nutrient sensing and inhibited FXR via a reduction of TCDCA in the USI and ileum. Further, inhibition of USI and ileal FXR enhanced nutrient sensing in HF rats, while inhibiting USI (but not ileal) bile salt hydrolase of HF rats transplanted with healthy microbiome activated FXR and disrupted nutrient sensing in the USI and ileum.
Conclusions
We reveal a TCDCA-FXR axis in both the USI and ileum that is necessary for the upper small intestinal microbiome to govern local nutrient-sensing glucoregulatory pathways in rats.
... Lactobacillus species are effective at deconjugating bile acids, particularly those conjugated to taurine. 50 These species were more abundant in fructose-chow compared to glucose-and normal-chow groups. Moreover, bile acid deconjugation by Lactobacillus can be further stimulated by high glycolytic activity. ...
Increased sugar intake is implicated in Type-2 diabetes and fatty liver disease. Mechanisms by which glucose and fructose components promote these conditions are unclear. We hypothesize that alterations in intestinal metabolite and microbiota profiles specific to each monosaccharide are involved. Two groups of six adult C57BL/6 mice were fed for 10-weeks with a diet where either glucose or fructose was the sole carbohydrate component (G and F, respectively). A third group was fed with normal chow (N). Fecal metabolites were profiled every 2-weeks by 1H NMR and microbial composition was analysed by real-time PCR (qPCR). Glucose tolerance was also periodically assessed. N, G and F mice had similar weight gains and glucose tolerance. Multivariate analysis of NMR profiles indicated that F mice were separated from both N and G, with decreased butyrate and glutamate and increased fructose, succinate, taurine, tyrosine and xylose. Compared to N and G, F mice showed a shift in microbe populations from gram-positive Lactobacillus spp. to gram-negative Enterobacteria species. Substitution of normal chow carbohydrate mixture by either pure glucose or fructose for 10 weeks did not alter adiposity or glucose tolerance. However, F G and N mice generated distinctive fecal metabolite signatures with incomplete fructose absorption as a dominant feature of F mice.
... In addition, bacteria with BSH activity are proved to be functional in bile detoxification, which will increase the intestinal persistence and survival of these strains [12]. Consequently, BSH activity has become a desirable trait when selecting probiotics [13] and has been reported in many species such as Lactobacillus, Bifidobacterium, Clostridium, Enterococcus, Bacteroids, Brevibacillus sp., and Brucella obortus [14]. However, few reports show that probiotics have no significant cholesterol-lowering effects on the host, which disputes the hypocholesterolemic claims [15]. ...
Bile salt hydrolase (BSH, EC 3.5.1.24) is considered as an ideal way with lower cost and less side effects to release the risk of coronary heart disease caused by hypercholes-terolemia. As bile salt hydrolase from Lactobacillus plantarum BBE7 could not be efficiently exported by PelB signal peptide of the general secretory (Sec) pathway, three twin-arginine signal peptides from twin-arginine translocation (Tat) pathway were synthesized, fused with bsh gene, inserted into expression vectors pET-20b(+) and pET-22b(+), and transformed into four different Escherichia coli hosts, respectively. Among the 24 recombinant bacteria obtained , E. coli BL21 (DE3) pLysS (pET-20b(+)-dmsA-bsh) showed the highest BSH activity in periplasmic fraction, which was further increased to 1.21±0.03 U/mL by orthogonal experimental design. And, signal peptide dimethyl sulfoxide reductase subunit DmsA (DMSA) had the best activity of exported BSH. More importantly, the presence of BSH in the periplasm had proven to be caused by the export rather than cell leakage. For the first time, we report the periplasmic expression of BSH by signal peptides from the Tat pathway. This will lay a solid foundation for the purification and biochemical characterization of BSH from the supernatant, and strategies adopted here could be used for the periplasmic expression of other proteins in E. coli.
... Recently, BSHs of different Lactobacillus species Chae et al. 2013;Chang-qing and Rong 2015;Gu et al. 2014;Jiang et al. 2010;Kaya, Kök, and Öztürk 2017) were identified and characterized. Furthermore, several BSH enzymes from various bacterial species including C. perfringens (Rossocha Figure 5. Measurement of the enzyme activities of partially purified rBSH and mrBSHs obtained from pCON2/BLR(DE3), pCON2F129I/BLR(DE3) and pCON2L138E/BLR(DE3) clones, respectively. ...
Bile salt hydrolase (BSH) is a gut-bacterial enzyme that influences human health by altering the host fat digestion and cellular energy generation. BSH is essential for deconjugation of the glycine or taurine-conjugated bile salts in the small intestine of humans. Therefore, BSH may be a key microbiome target for the designing of new measures to control some diseases in humans. BSHs, a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, exhibit higher variation in substrate specificity. The phenylalanine-129 (F129) and leucine-138 (L138) in loop III of BSH, thought to be responsible for substrate specificity, are partially conserved in this superfamily. In this study, the aromatic-hydrophobic F129 and aliphatic-hydrophobic L138 of C-terminally His-tagged BSH from Lactobacillus plantarum B14 (LbBSH) was substituted for aliphatic-hydrophobic isoleucine (I) and negatively charged polar glutamate (E) amino acid, respectively, by site-directed mutagenesis and characterized using an Escherichia coli BLR(DE3) expression system. Although both mutations resulted in an assembled and stable recombinant BSHs (rBSHs), they altered the catalytic activity and substrate specificity of rBSH. This is the first experimental finding which confirmed that F129 and L138 were critical amino acids for the catalytic activity and substrate specificity turnover of BSH.
... Glycine-conjugated bile salts are more toxic than taurine-conjugated bile salt, which could be explained with the difference in dissociation constant of taurine and glycine conjugates (Lye et al. 2010;Ramasamy et al. 2010). Jiang et al. (2010) reported that high BSH activity against taurine-conjugated bile salts is an unadmitted feature for probiotic strains since it results in increased hydrogen sulphide production, which is highly toxic to the host after deconjugation. For this reason, Propionibacterium spp. ...
The present study investigated the deconjugation of bile salts and co‐precipitation of cholesterol with deconjugated bile salts by seven Propionibacterium spp. strains in vitro. Propionibacterium spp. could deconjugate sodium glycocholate (1.53–5.31 mM) and sodium taurocholate (0.08–0.25 mM) bile salts. The highest cholesterol precipitation (47.8 µg/mL) was determined with Propionibacterium freudenreichii subsp. freudenreichii SP3 strain in a 0.15% oxgall‐containing medium. Significant (P < 0.05) correlations were observed between cholesterol co‐precipitation and deconjugation of sodium glycocholate among the strains. In vitro bile salt deconjugation activity studies of P. freudenreichii subsp. freudenreichii SP3 strain revealed that this strain may have potential as a probiotic strain for deconjugation of bile salts in vivo studies.
... During the past several decades, probiotic bacteria with BSH activity were used to alleviate cholesterol levels in humans and animals [18]. Because of the implication between BSH and human health, to date, many bsh genes were identified and characterized from different strains of L. plantarum [11,[19][20][21][22], L. acidophilus [22,23], L. johnsonii [24], L. salivarus [7], L. rhamnosus [25] and L. gasseri [14]. At least nine different types of BSH were predicted from those organisms [26] and these were different in size, genetic organization and regulation, substrate specificity and pH optima [27]. ...
Microbial bile salt hydrolases (BSHs), a member of cholylglycine hydrolase (CGH) family, catalyze the hydrolysis of glycine and taurine-linked bile salts in the small intestine of human. BSH is evolutionarily related to penicillin V acylase (PVA) which hydrolyses a penicillin V and is also a member of CGH family. Although, five of the six amino acids, C2, R16, D19, N170, N79 and R223, supposed to be responsible for catalytic activity of BSH enzyme, are strictly conserved in all CGH family members, N79 is partially conserved in this family. In this study, in order to analyze the correlation between N79 and catalytic activity or substrate specificity of BSH, the polar and acidic N79 was substituted for the aliphatic and hydrophobic V79 by PCR-based site directed mutagenesis and mutant recombinant BSH was expressed in E. coli BLR(DE3). While the effects of the mutation on catalytic activity and substrate specificity of BSH were detected by ninhydrin assay. The effect of this mutation on the stability of the BSH was observed by SDS-PAGE analysis. Although V79 mutation resulted in stable BSH, it reduced the catalytic activity and altered substrate specificity of BSH. The results suggested that N79 might be important for substrate binding and catalytic turnover of BSH.
... For example, high fecal DCA concentrations and a high DCA to LCA ratio have been associated with an increased risk of the CRC (Owen 1997). Considering the implications between deconjugation of conjugated salts and negative or positive health consequences (Ridlon, Kang, and Hylemon 2006;Jiang et al. 2010), further functional studies of BSH proteins and site-directed mutagenesis of some strictly conserved amino acids are required. ...
Bile salt hydrolase (BSH), found commonly in intestinal species of Lactobacillus and Bifidobacterium, catalyzes the hydrolysis of glycine or taurine-conjugated bile acids into the amino acids and free bile acids. Deconjugated bile acids potentially play an important role in the reduction of blood cholesterol level and formation of some gastrointestinal diseases such as cholestasis, gallstone formation, and colon cancer. Although the crystal and three-dimensional structures of BSH enzyme are known, the working mechanism of catalytic activity of such an important BSH enzyme is not known very well. Previous in silico analysis of multiple BSH has identified that Arginine-16 (R16) and Aspartate-19 (D19) were catalytically important residues in the active site of BSH. To confirm the function of these amino acids, in this study, BSH enzyme from Lactobacillus plantarum B14 strain was cloned into Escherichia coli and strictly conserved polar R16 and D19 amino acids of BSH enzyme were substituted for hydrophobic Phenylalanine-16 (F16) and Leucine-19 (L19) amino acids, respectively, by polymerase chain reaction (PCR)-based site-directed mutagenesis. The effects of the mutations on catalytic activity and structure of the BSH enzymes were detected by ninhidrin assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, respectively. Research results showed that although F16 mutation led to loss of enzyme activity completely, L19 mutation led to abolishment of the synthesis of BSH enzyme. These results indicated that R16 and D19 amino acids located in loop I of the BSH enzyme might be critical for catalytic activity and assembly of the BSH enzyme respectively.
... BSH in microorganisms may have the ability to recognize steroids and amino acids. In addition, it has been reported that the addition of BSH-active bacterias can effectively reduce serum and cholesterol levels in mice and dogs [58]. HFD-induced mice treated with antibiotics or tempol modifi ed their microbiota resulting in a reduction in poor metabolic phenotypes, which was due to decreased activity of BSH and decreased levels of Lactobacillus. ...
p>Bile acid (BA) plays an important role in the absorption and translocation of fat and fat-soluble vitamins. In addition, it can also act as a signaling molecule to influence the energy metabolism of organisms, glucose metabolism, and the development of liver and intestinal diseases by activating receptor. Gut microbiota participates in the metabolism and transport of BA, which changes the BA associated with the occurrence and development of a variety of diseases. This is achieved through a variety of regulatory processes and is intrinsically linked to host physiology. In recent years, many scholars have used 16S rRNA gene sequencing in conjunction with serum, urine, and fecal metabolomics methods to study the mechanisms underlying the occurrence and development of disease associated with BA and gut microbiota, or to evaluate the protective action of drugs on the metabolic phenotype in rats with gut microbiota disorder. On the one hand, the gut microbiota regulates BA by activating receptors such as FXR, TGR5, and FGF15, and can regulate BA synthesis through enzyme reaction. In addition, gut microbiota can effectively hydrolyze bound parasites or heterogenous organisms that have been cleared by BA. On the other hand, BA can alter the composition of the gut microbiota by inhibiting the growth of bacteria in the intestine. These studies provide new ideas for further elucidating the relationship between gut microbiota and BA and treatment for related disease.</p
... BSH in microorganisms may have the ability to recognize steroids and amino acids. In addition, it has been reported that the addition of BSH-active bacterias can effectively reduce serum and cholesterol levels in mice and dogs [58]. HFD-induced mice treated with antibiotics or tempol modifi ed their microbiota resulting in a reduction in poor metabolic phenotypes, which was due to decreased activity of BSH and decreased levels of Lactobacillus. ...
Bile acid (BA) plays an important role in the absorption and translocation of fat and fat-soluble vitamins. In addition, it can also act as a signaling molecule to infl uence the energy metabolism of organisms, glucose metabolism, and the development of liver and intestinal diseases by activating receptor. Gut microbiota participates in the metabolism and transport of BA, which changes the BA associated with the occurrence and development of a variety of diseases. This is achieved through a variety of regulatory processes and is intrinsically linked to host physiology. In recent years, many scholars have used 16S rRNA gene sequencing in conjunction with serum, urine, and fecal metabolomics methods to study the mechanisms underlying the occurrence and development of disease associated with BA and gut microbiota, or to evaluate the protective action of drugs on the metabolic phenotype in rats with gut microbiota disorder. On the one hand, the gut microbiota regulates BA by activating receptors such as FXR, TGR5, and FGF15, and can regulate BA synthesis through enzyme reaction. In addition, gut microbiota can effectively hydrolyze bound parasites or heterogenous organisms that have been cleared by BA. On the other hand, BA can alter the composition of the gut microbiota by inhibiting the growth of bacteria in the intestine. These studies provide new ideas for further elucidating the relationship between gut microbiota and BA and treatment for related disease.
... Because of the strong implications between bsh and negative or positive health consequences, various bsh genes have been cloned and characterized from Lactobacillus species such as Lb. plantarum (Chang-Qing and Rong 2015; Dong et al. 2012;Gu et al. 2014;Lambert et al. 2008), Lb. johnsonii (Chae et al. 2012), Lb. acidophilus (Corzo and Gilliland 1999;Jiang et al. 2010), Lb. salivarus (Bi et al. 2013), Lb. fermentum (Kumar et al. 2013), and Bifidobacterium species (Kim and Lee 2005;Kim et al. 2004). However, the catalytic mechanism and substrate specificity of such an important bsh enzyme still remain unclear. ...
A bile salt hydrolase (bsh) enzyme deconjugate taurine and glycine-linked bile salts. Because of the strong implications between deconjugation of tauro- or gluco-conjugated salts and its positive or negative health consequences, the characterization of the bsh enzymes is important. bshs from different lactobacilli species, even strains, exhibit higher variation in sequence, kinetic properties, and substrate specificity. In the present study four bsh related genes from Lactobacillus plantarum GD2 strain were cloned and expressed in Escherichia coli BLR (DE3) strain. Amino acid residues of recombinant bshs were analyzed and their deconjugation abilities were tested with six human conjugated bile salts. Results indicated that the genetic distance among four related bsh genes in Lb. plantarum is far from each other and bsh2-4 enzymes share significant sequence homology specifically with penicillin V acylase (PVA) family members. Biochemical and the in silico analysis suggest that the bsh1 enzyme is a member of the bsh family while bsh2-4 enzymes are members of the PVA family.
... Lactobacillus species are effective at deconjugating bile acids, particularly those conjugated to taurine. 50 These species were more abundant in fructose-chow compared to glucose-and normal-chow groups. Moreover, bile acid deconjugation by Lactobacillus can be further stimulated by high glycolytic activity. ...
Increased sugar intake is implicated in Type-2 diabetes and fatty liver disease, however, the mechanisms through which glucose and fructose promote these conditions are unclear. We hypothesize that alterations in intestinal metabolite and microbiota profiles specific to each monosaccharide are involved. Two groups of six adult C57BL/6 mice were fed for 10-weeks with diets with glucose (G) or fructose (F) as sole carbohydrates, and a third group was fed with a normal chow carbohydrate mixture (N). Fecal metabolites were profiled by nuclear magnetic resonance (NMR) and microbial composition by real-time polymerase chain reaction (qPCR). Although N, G and F mice exhibited similar weight gains (with slight slower gains for F) and glucose tolerance, multivariate analysis of NMR data indicated that F mice were separated from N and G, with decreased butyrate and glutamate and increased fructose, succinate, taurine, tyrosine and xylose. The different sugar diets also resulted in distinct intestinal microbiota profiles. That associated with fructose seemed to hold more potential to induce host metabolic disturbances compared to glucose, mainly by promoting bile acid deconjugation and taurine release and compromising intestinal barrier integrity. This may reflect the noted non-quantitative intestinal fructose absorption hence increasing its availability for microbial metabolism, a subject for further investigation.
... Specifically, DDS-1 maintained higher concentrations followed by UALp-05, UABla- 12, and UASt-09. This phenomenon may be linked to the ability of these strains to produce bile salt hydrolase (BSH) and previous studies have demonstrated BSH genes in lactobacilli strains which are believed to be responsible for their resistance [32,33,34,35]. Adhesion capacity to colonic cells also plays a role in the selection of probiotic bacteria [17]. ...
Background: The health benefits of probiotics are well established and known to be strain-specific. However, the role of probiotics obtained from different origins and their efficacy largely remains unexplored. The aim of this study is to investigate the in vitro efficacy of probiotics from different origins.
Methods: Probiotic strains utilized in this study include Lactobacillus acidophilus DDS-1 (human origin), Bifidobacterium animalis ssp. lactis UABla-12 (human origin), L. plantarum UALp-05 (plant origin) and Streptococcus thermophilus UASt-09 (dairy origin). Screening assays such as in vitro digestion simulation, adhesion, cell viability and cytokine release were used to evaluate the probiotic potential.
Results: All strains showed good resistance in the digestion simulation process, especially DDS-1 and UALp-05, which survived up to a range of 107 to 108 CFU/mL from an initial concentration of 109 CFU/mL. Two human colonic mucus-secreting cells, HT-29 and LS174T, were used to assess the adhesion capacity, cytotoxicity/viability, and cytokine quantification. All strains exhibited good adhesion capacity. No significant cellular cytotoxicity or loss in cell viability was observed. DDS-1 and UALp-05 significantly upregulated anti-inflammatory IL-10 and downregulated pro-inflammatory TNF-α cytokine production. All the strains were able to downregulate IL-8 cytokine levels.
Conclusion: Of the 4 strains tested, DDS-1 demonstrated superior survival rates, good adhesion capacity and strong immunomodulatory effect under different experimental conditions.
... Specifically, DDS-1 maintained higher concentrations followed by UALp-05, UABla-12, and UASt-09. This phenomenon may be linked to the ability of these strains to produce bile salt hydrolase (BSH) and previous studies have demonstrated BSH genes in lactobacilli strains which are believed to be responsible for their resistance [32,33,34,35]. Adhesion capacity to colonic cells also plays a role in the selection of probiotic bacteria [17]. ...
Background: The health benefits of probiotics are well established and known to be strain-specific. However, the role of probiotics obtained from different origins and their efficacy largely remains unexplored. The aim of this study is to investigate the in vitro efficacy of probiotics from different origins.
Methods: Probiotic strains utilized in this study include Lactobacillus acidophilus DDS-1 (human origin), Bifidobacterium animalis ssp. lactis UABla-12 (human origin), L. plantarum UALp-05 (plant origin) and Streptococcus thermophilus UASt-09 (dairy origin). Screening assays such as in vitro digestion simulation, adhesion, cell viability and cytokine release were used to evaluate the probiotic potential.
Results: All strains showed good resistance in the digestion simulation process, especially DDS-1 and UALp-05, which survived up to a range of 10⁷ to 10⁸ CFU/mL from an initial concentration of 10⁹ CFU/mL. Two human colonic mucus-secreting cells, HT-29 and LS174T, were used to assess the adhesion capacity, cytotoxicity/viability, and cytokine quantification. All strains exhibited good adhesion capacity. No significant cellular cytotoxicity or loss in cell viability was observed. DDS-1 and UALp-05 significantly upregulated anti-inflammatory IL-10 and downregulated pro-inflammatory TNF-α cytokine production. All the strains were able to downregulate IL-8 cytokine levels.
Conclusion: Of the 4 strains tested, DDS-1 demonstrated superior survival rates, good adhesion capacity and strong immunomodulatory effect under different experimental conditions.
... Although the L. plantarum 86L BSHs have the same catalytic sites to other bacterial genera or Lactobacilli species, their substrate-hydrolyzing capabilities are not the same [12]. It has been suggested that most evolution occurs to broaden substrate specificity [47]. Thus, as well as BSHs from most bacterial species, our L. plantarum86L BSH seems to have substrate preferences toward glyco conjugated bile salts rather than tauroconjugated ones [12]. ...
... This indicates that the use of active cells, with no need for enzyme extraction, is better for cholesterol reduction and will facilitate cholesterol reduction by LAB when used in vivo. The results of Jiang et al. (2010) showed that some strains of L. casei and L. salivarius did not exhibit any BSH activity, whereas L. helveticus, L. fermentum and L. gallinarum strains only deconjugated taurine-conjugated bile salts. As such, their BSH activity was very low, ranging from 8.7 to 32.3 U/mg. ...
EIGHT lactic acid bacterial (LAB) isolates were obtained from food and non-food sources
and identified by 16S rRNA gene sequence analysis. Based on the sequencing results,
the isolates belong to two species of lactobacilli, Lactobacillus plantarum and L. rhamnosus.
These strains were then compared with a reference strain, Lactobacillus casei, to assess various
probiotic properties, such as haemolytic activity, histamine formation ability, stress tolerance
under certain stress conditions, antibiotic susceptibility and in vitro adhesion ability. Moreover,
bile salt hydrolase (BSH) activity was evaluated both qualitatively and quantitatively. The
results showed that none of the isolates demonstrated any haemolytic activity or histamine
formation. The isolated strains were also tolerant to acidic and alkaline conditions (pH 2.5, 3.5
and 9) for 3 and 6 h, as well as osmotic (3 M NaCl) and heat (55 and 70°C) stress, but were
more responsive to oxidative and bile stress. The bacterial isolates also expressed high amounts
of BSH, ranging from 90 to 142 U/mg in active cells, compared to L. casei (74 U/mg), which
may be useful in cholesterol reduction. All bacterial isolates were resistant to vancomycin and
susceptible to amoxicillin, cloxacillin and penicillin. All isolates were also highly hydrophobic
(>70%), indicating that they are not easily flushed from the intestines.
... However, many Bifidobacterium and Lactobacillus species have been found to have bile salt hydrolase (BSH) genes, some strains having more than one BSH homolog, which are not identical (Chae et al., 2013). The BSH genes have been cloned and characterized ( Jiang et al., 2010;Lee, 2005, 2008;Kim et al., 2004). Blast search analysis of the BSH DNA sequences showed that these enzymes are part of the N-terminal nucleophile hydrolase superfamily and therefore have an N-terminal catalytic nucleophile that can cleave amide bonds (Chae et al., 2013). ...
Composed of nearly a thousand different types of micro-organisms, some beneficial, others not, the human gut microbiota plays an important role in health and disease. This is due to the presence of probiotic or beneficial microbes, or due to the feeding of prebiotics that stimulate the endogenous beneficial microbes: these promote health by stimulating the immune system, improving the digestion and absorption of nutrients, and inhibiting the growth of pathogens. The notable health benefits of probiotic organisms have stimulated much commercial interest, which in turn has led to a plethora of research initiatives in this area; these range from studies to elucidate the efficacy of the various health benefits to analyses of the diet-microbe interaction as a means of modulating the gut microbiota composition. Research in this area is at a very exciting stage.
With state-of-the-art commentaries on all aspects of probiotics and prebiotics research, this book provides an authoritative and timely overview of the field. Written by leading international researchers, each chapter affords a critical insight to a particular topic, reviews current research, discusses future direction and aims to stimulate discussion. Topics range from the different microorganisms used as probiotics (lactobacilli, bifidobacteria, yeast, etc) and techniques and approaches used (metagenomics, etc) to the reviews of the clinical and medical aspects. The provision of extensive reference sections positively encourages readers to pursue each subject in greater detail.
Containing 33 chapters, the book is an invaluable source of information and essential reading for everyone working with probiotics, prebiotics and the gut microbiota, from the PhD student to the experienced scientist, in academia, the pharmaceutical or biotechnology industries and working in clinical environments.
... Many identified BSH enzymes have a narrow substrate spectrum and display much higher activity in hydrolyzing glycineconjugated bile salts than taurine-conjugated bile salts (Coleman and Hudson, 1995;Smet et al., 1995;Tanaka et al., 2000;Kim et al., 2004;Liong and Shah, 2005;Pavlović et al., 2012). However, some BSH enzymes show a preference for taurine-conjugated bile salts, such as two BSH enzymes in Lactobacillus jonsonii PF01 (Chae et al., 2013) and the BSH enzymes from five lactobacilli strains (Jiang et al., 2010). Recently, a potent BSH enzyme was identified and characterized from a chicken Lactobacillus salivarius strain; this BSH displayed potent hydrolysis activity towards both glycol-conjugated and taurine-conjugated bile salts (Wang et al., 2012). ...
To effectively mitigate antimicrobial resistance in the agricultural ecosystem, there is an increasing pressure
to reduce and eliminate the use of in-feed antibiotics for growth promotion and disease prevention in
food animals. However, limiting antibiotic use could compromise animal production efficiency and
health. Thus, there is an urgent need to develop effective alternatives to antibiotic growth promoters
(AGPs). Increasing evidence has shown that the growth-promoting effect of AGPs was highly correlated
with the reduced activity of bile salt hydrolase (BSH), an intestinal bacterial enzyme that has negative impact
on host fat digestion and energy harvest; consistent with this finding, the population of Lactobacillus
species, the major intestinal BSH-producer, was significantly reduced in response to AGP use. Thus, BSH
is a key mechanistic microbiome target for developing novel alternatives to AGPs. Despite recent significant
progress in the characterization of diverse BSH enzymes, research on BSH is still in its infancy.
This review is focused on the function of BSH and its significant impacts on host physiology in humans,
laboratory animals as well as food animals. The gaps in BSH-based translational microbiome research for
enhanced animal health are also identified and discussed.
... L. helveticus strains of dairy and vegetable origin did not possess BSH activity [45]. However, BSH activity was observed in L. helveticus strains of human fecal origin [30]. It has been reported that the bsh gene could be transferred between commensal bacteria in the intestine via horizontal gene transfer [8,31]. ...
We characterized the probiotic properties of Lactobacillus helveticus strains KII13 and KHI1 isolated from fermented cow milk by in vitro and in vivo studies. The strains exhibited tolerance to simulated oro-gastrointestinal (OGT) condition, adherence to Caco-2 cells, and antimicrobial activity. Both the L. helveticus strains produced bioactive tripeptides isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP) during fermentation of milk. KII13 showed higher in vitro cholesterol-lowering activity (47%) compared to KHI1 (28%) and L. helveticus ATCC 15009 (22%), hence, it was selected for in vivo study of cholesterol-lowering activity in atherogenic diet-fed hypercholesterolemic mice. For the study, mice were divided into 4 groups viz., normal diet-control group (NCD), atherogenic diet-control group (HCD), KII13-atherogenic diet (HCD-KII13), and Lactobacillus acidophilus ATCC 43121-atherogenic diet (HCD-L.ac) as positive control. Serum total cholesterol (T-CHO) level was significantly decreased by 8.6% and 7.78% in the HCD-KII13 and HCD-L.ac group (P<0.05), respectively, compared with HCD group. Low density lipoprotein (LDL) cholesterol levels in both HCD-KII13 and HCD-L.ac groups were decreased by 13% and 11%, respectively, compared to HCD group (both, P<0.05). Analysis of cholesterol metabolism related gene expression in mice liver showed increased expression of LDLR and SREBF2 genes in mice fed with KII13. By comparing all the results, we conclude that L. helveticus KII13 could be used as potential probiotic strain to produce anti-hypertensive peptides and reduce serum cholesterol.
... L. helveticus strains of dairy and vegetable origin did not possess BSH activity [45]. However, BSH activity was observed in L. helveticus strains of human fecal origin [30]. It has been reported that the bsh gene could be transferred between commensal bacteria in the intestine via horizontal gene transfer [8,31]. ...
We characterized the probiotic properties of Lactobacillus helveticus strains KII13 and KHI1 isolated from fermented cow milk by in vitro and in vivo studies. The strains exhibited tolerance to simulated oro-gastrointestinal (OGT) condition, adherence to Caco-2 cells, and antimicrobial activity. Both the L. helveticus strains produced bioactive tripeptides isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP) during fermentation of milk. KII13 showed higher in vitro cholesterol-lowering activity (47%) compared to KHI1 (28%) and L. helveticus ATCC 15009 (22%), hence, it was selected for in vivo study of cholesterol-lowering activity atherogenic diet-fed hypercholesterolemic mice. For the study, mice were divided into 4 groups viz., normal diet-control group (NCD), atherogenic diet-control group (HCD), KII13-atherogenic diet (HCD-KII13), and Lactobacillus acidophilus ATCC 43121-atherogenic diet (HCD-L.ac) as positive control. Serum total cholesterol (T-CHO) level was significantly decreased by 8.6% and 7.78% in the HCD-KII13 and HCD-L.ac group (P<0.05), respectively, compared with HCD group. Low density lipoprotein (LDL) cholesterol levels in both HCD-KII13 and HCD-L.ac groups were decreased by 13% and 11%, respectively, compared to HCD group (both, P<0.05). By comparing all the results, we conclude that L. helveticus KII13 could be used as potential probiotic strain to produce anti-hypertensive peptides and reduce serum cholesterol.
... However, many Bifidobacterium and Lactobacillus species have been found to have bile salt hydrolase (BSH) genes, some strains having more than one BSH homolog, which are not identical (Chae et al., 2013). The BSH genes have been cloned and characterized ( Jiang et al., 2010;Lee, 2005, 2008;Kim et al., 2004). Blast search analysis of the BSH DNA sequences showed that these enzymes are part of the N-terminal nucleophile hydrolase superfamily and therefore have an N-terminal catalytic nucleophile that can cleave amide bonds (Chae et al., 2013). ...
... Within the Firmicutes phylum, the genus Lactobacillus was significantly reduced (Fig. 3). Multiple members of the Lactobacillus genus encode bile salt hydrolases, which produce free bile acids by deconjugating taurine-conjugated bile acids 86 . Consistent with these findings, tempol increases the intestinal concentration of multiple taurine-conjugated bile acids, including tauro-β-muricholic acid (T-β-MCA). ...
Although the importance of human genetic polymorphisms in therapeutic outcomes is well established, the role of our 'second genome' (the microbiome) has been largely overlooked. In this Review, we highlight recent studies that have shed light on the mechanisms that link the human gut microbiome to the efficacy and toxicity of xenobiotics, including drugs, dietary compounds and environmental toxins. Continued progress in this area could enable more precise tools for predicting patient responses and for the development of a new generation of therapeutics based on, or targeted at, the gut microbiome. Indeed, the admirable goal of precision medicine may require us to first understand the microbial pharmacists within.
The gut microbiota constitutes a complex ecosystem, comprising trillions of microbes that have co-evolved with their host over hundreds of millions of years. Over the past decade, a growing body of knowledge has underscored the intricate connections among diet, gut microbiota, and human health. Bioactive polysaccharides (BPs) from natural sources like medicinal plants, seaweeds, and fungi have diverse biological functions including antioxidant, immunoregulatory, and metabolic activities. Their effects are closely tied to the gut microbiota, which metabolizes BPs into health-influencing compounds. Understanding how BPs and gut microbiota interact is critical for harnessing their potential health benefits. This review provides an overview of the human gut microbiota, focusing on its role in metabolic diseases like obesity, type II diabetes mellitus, non-alcoholic fatty liver disease, and cardiovascular diseases. It explores the basic characteristics of several BPs and their impact on gut microbiota. Given their significance for human health, we summarize the biological functions of these BPs, particularly in terms of immunoregulatory activities, blood sugar, and hypolipidemic effect, thus providing a valuable reference for understanding the potential benefits of natural BPs in treating metabolic diseases. These properties make BPs promising agents for preventing and treating metabolic diseases. The comprehensive understanding of the mechanisms by which BPs exert their effects through gut microbiota opens new avenues for developing targeted therapies to improve metabolic health.
The bile acids (BAs) de-conjugation is catalyzed by bile salt hydrolase (BSH) enzyme, that is an intestinal bacterial product and a member of the cholylglycine hydrolase (CGH) family. De-conjugated BAs alter BA-mediated signaling pathways such as glucose metabolism, energy homeostasis and lipid absorption and this makes the BSH clinically important. However, BSHs from different sources have a variable substrate preference to eight different bile salts. Although BSH is a well-studied enzyme, its molecular investigations based on BSH substrate recognition are not very well known. In this study, the relationship between substrate specificity of BSH from Lactobacillus plantarum B14 (LpBSH) and its loop II, the aliphatic-hydrophobic V58 and aromatic-hydrophobic Y65 residues in this loop was mutated and analyzed. While PCR-based site-directed mutagenesis was used to substitute V58 and Y65 amino acids for N58, F58, M58, C65, F65 and L65 amino acids, respectively, the BLR (DE3) strain of E. coli was used to express mutant recombinant LpBSHs (mrLpBSHs). Site-directed mutagenesis of LpBSH showed reduced activity of mrLpBSHs against six different BAs. Our results indicated that the V58 and mostly Y65 residues in loop II might be critical for the structural site that is involved in substrate specificity and catalysis. These findings suggested that V58 and Y65 residues of LpBSH might participate in substrate specificity and BSH substrate specificity may be dependent upon the collate group, rather than amino acid moieties. However, more mutagenesis-based investigation on other CGH family members are needed in order to understand the structure and substrate specificity relations of BSHs.
The current trend in starter selection is to combine both technological and probiotic properties to standardise and make functional artisanal fermented beverages such as Sha'a whose properties are very variable due to the lack of a known starter. The objective of this work was to study technological and probiotic properties of yeasts isolated from Sha'a sold in Bamenda, Bafoussam, Bonabérie, Dschang, Foumbot, Mbouda and Njombé (Cameroon). The isolated yeasts were studied for their ability to produce CO2 from glucose, to grow in the presence of 8% ethanol, 20% glucose and pH 3, to assimilate maltose and to produce ethanol. Then, the survival of the pre-selected isolates was assessed in simulated gastric (pH 2 and 3) and intestinal juices, followed by self-aggregation, co-aggregation, hydrophobicity, haemolysin, gelatinase, biogenic amine production, antibiotic and antifungal susceptibility, bile salt hydrolase and antiradical activity. The selected isolates were identified by sequencing the 5.8S/28S rRNA gene. From the 98 isolates obtained, 66 produced CO2 from glucose and 16 were then selected for their ability to grow in the presence of 8% ethanol, 20% glucose, pH 3 and maltose. The overall survival of isolates ranged from 4.12 ± 1.63 to 104.25 ± 0.19% (LT16) and from 0.56 ± 0.20 to 96.74 ± 1.60% (LT66) at pH 3 and pH 2 respectively. All of them have remarkable surface hydrophobicity properties. Based on principal component analysis, 5 isolates were selected as the best. However, only 3 of them, LT16 (the most promising), LT25 identified as Saccharomyces cerevisiae and LT80 as Nakaseomyces delphensis, do not produce a virulence factor. The latter can deconjugate bile salts with a maximum percentage of 60.54 ± 0.12% (LT16) and the highest inhibition of DPPH° radicals was 55.94 ± 1.14% (LT25). In summary, the yeast flora of Sha'a contains yeasts capable of fermenting and producing ethanol while producing bioactive compounds that would benefit the consumer.
Complex carbohydrates, proteins, and other food components require a longer digestion process to be absorbed by the lining of the alimentary canal. In addition to the enzymes of the gastrointestinal tract, gut microbiota, comprising a large range of bacteria and fungi, has complementary action on the production of digestive enzymes. Within this universe of "hidden soldiers", lactobacilli are extensively studied because of their ability to produce lactase, proteases, peptidases, fructanases, amylases, bile salt hydrolases, phytases, and esterases. The administration of living lactobacilli cells has been shown to increase nutrient digestibility. However, it is still little known how these microbial-derived enzymes act in the human body. Enzyme secretion may be affected by variations in temperature, pH, and other extreme conditions faced by the bacterial cells in the human body. Besides, lactobacilli administration cannot itself be considered the only factor interfering with enzyme secretion, human diet (microbial substrate) being determinant in their metabolism. This review highlights the potential of lactobacilli to release functional enzymes associated with the digestive process and how this complex metabolism can be explored to contribute to the human diet. Enzymatic activity of lactobacilli is exerted in a strain-dependent manner, i.e., within the same lactobacilli species, there are different enzyme contents, leading to a large variety of enzymatic activities. Thus, we report current methods to select the most promising lactobacilli strains as sources of bioactive enzymes. Finally, a patent landscape and commercial products are described to provide the state of art of the transfer of knowledge from the scientific sphere to the industrial application.
The metabolism of bile acid by the gut microbiota is associated with host health. Bile salt hydrolases (BSHs) play a crucial role in controlling microbial bile acid metabolism. Herein, we conducted a comparative study to investigate the alterations in the abundance of BSHs using data from three human studies involving dietary interventions, which included a ketogenetic diet (KD) versus baseline diet (BD), overfeeding diet (OFD) versus underfeeding diet, and low-carbohydrate diet (LCD) versus BD. The KD increased BSH abundance compared to the BD, while the OFD and LCD did not change the total abundance of BSHs in the human gut. BSHs can be classified into seven clusters; Clusters 1 to 4 are relatively abundant in the gut. In the KD cohort, the levels of BSHs from Clusters 1, 3, and 4 increased significantly, whereas there was no notable change in the levels of BSHs from the clusters in the OFD and LCD cohorts. Taxonomic studies showed that members of the phyla Bacteroidetes, Firmicutes, and Actinobacteria predominantly produced BSHs. The KD altered the community structure of BSH-active bacteria, causing an increase in the abundance of Bacteroidetes and decrease in Actinobacteria. In contrast, the abundance of BSH-active Bacteroidetes decreased in the OFD cohort, and no significant change was observed in the LCD cohort. These results highlight that dietary patterns are associated with the abundance of BSHs and community structure of BSH-active bacteria and demonstrate the possibility of manipulating the composition of BSHs in the gut through dietary interventions to impact human health.
Extibacter muris is a newly described mouse gut bacterium which metabolizes cholic acid (CA) to deoxycholic acid (DCA) via 7α-dehydroxylation. Although bile acids influence metabolic and inflammatory responses, few in vivo models exist for studying their metabolism and impact on the host. Mice were colonized from birth with the simplified community Oligo-MM¹² with or without E. muris. As the metabolism of bile acids is known to affect lipid homeostasis, mice were fed either a low- or high-fat diet for eight weeks before sampling and analyses targeting the gut and liver. Multiple Oligo-MM¹² strains were capable of deconjugating primary bile acids in vitro. E. muris produced DCA from CA either as pure compound or in mouse bile. This production was inducible by CA in vitro. Ursodeoxycholic, chenodeoxycholic, and β-muricholic acid were not metabolized under the conditions tested. All gnotobiotic mice were stably colonized with E. muris, which showed higher relative abundances after HF diet feeding. The presence of E. muris had minor, diet-dependent effects on Oligo-MM¹² communities. The secondary bile acids DCA and surprisingly LCA and their taurine conjugates were detected exclusively in E. muris-colonized mice. E. muris colonization did not influence body weight, white adipose tissue mass, liver histopathology, hepatic aspartate aminotransferase, or blood levels of cholesterol, insulin, and paralytic peptide (PP). However, proteomics revealed shifts in hepatic pathways involved in amino acid, glucose, lipid, energy, and drug metabolism in E. muris-colonized mice. Liver fatty acid composition was substantially altered by dietary fat but not by E. muris.In summary, E. muris stably colonized the gut of mice harboring a simplified community and produced secondary bile acids, which affected proteomes in the liver. This new gnotobiotic mouse model can now be used to study the pathophysiological role of secondary bile acids in vivo.
Thirteen strains of Lactobacillus acidophilus of various origins were tested for bile salt hydrolase (BSH) activity, growth characteristics and surface properties during cultivation in the presence of ox bile. Seven strains were BSH positive, three strains were found to hydrolyse both sodium glycocholate and taurocholate and four hydrolysed only glycocholate. The addition of ox bile (3; 5 or 10 g/l) to MRS growth medium affected the growth rate of both BSH-positive and BSH-negative strains. The lag phases were increased when 5 or 10 g/l of ox bile were added. Growth rates and lag phases were significantly different between BSH-positive and BSH-negative strains in MRS medium with bile. BSH-positive strains were better able to adapt to increasing bile concentrations. The concentration of 2 g/l sodium cholate completely inhibited growth of all lactobacilli tested. Its toxicity was influenced by pH of medium. The addition of 3 g/l ox bile affected the properties related to ability of lactobacilli to adhere to surfaces––autoaggregation increased in all strains and zeta potential only in BSH-negative strains. In all strains tested, bshA and bshB genes were identified with the exception of L. acidophilus Lafti-L10. Repeated cultivation in MRS with bile did not cause the expression of genes in BSH-negative strains.
An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.
When bile salt hydrolase (bsh) genes from Lactobacillus plantarum are expressed intracellularly and extracellularly by Escherichia coli, they usually form inclusion bodies. Therefore, Pichia pastoris, an efficient expression system, was used to achieve the secretory expression of bsh. Firstly, signal peptide α-factor was ligated with bsh by fusion PCR, and the fragment obtained was then cloned into vector pPIC9K, resulting in a recombinant plasmid pPIC9K-α-factor-BSH. Finally, this plasmid was linearized by Sal I and transformed into Pichia pastoris GS115. As analyzed by SDS-PAGE and BSH assay, the molecular weight and extracellular activity of recombinant BSH was determined to be 37.0 × 103 and 1.08 U mL-1, respectively. After optimization of fermentation conditions using orthogonal experimental design (L9 (34)), the extracellular BSH activity was 2.69 U mL-1, 1.49 times higher than before. This lays a solid foundation for purification, characterization and large-scale production of BSH.
We have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving [14C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal, 1.41 and 1.53 mumol/min per mg of protein, respectively, whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Both had similar substrate specificities, highest on taurodeoxycholic and glycocholic acid, and pH optima between 3.8 and 4.5. The kinetic properties were also similar, with Vmaxs of 17 and 53 micromoles/min per mg of protein and Kms of 0.76 and 0.95 mM taurocholic acid for A and B, respectively. Therefore, whether the enzyme exists in two forms in the cells remains to be determined.
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.
The effects of bile salts on the survival of lactobacilli were investigated using glycocholic acid, cholic acid and deoxycholic acid as model compounds and the bile salt hydrolase active Lactobacillus plantarum 80 (BSH+) and its BSH negative mutant. The detrimental effects of cholic acid, i.e. growth inhibition and cytotoxicity at a concentration of 1 and 5 mmol l-1, respectively, were considered to be due to the hydrophobic protonated form of the molecule, which brings about membrane damage. The conversion of glycocholic acid to cholic acid by the BSH active L. plantarum 80 caused a growth inhibition which was comparable with the inhibition observed in the broth supplemented with 1 mmol l-1 cholic acid. Deoxycholic acid caused toxicity through membrane damage when the compound was in solution. Its toxicity disappeared in the culture broth as the molecule precipitated. In case of cholic acid, the toxicity could be removed by buffering the solution at pH 7.0. It was calculated that at this pH most of the cholic acid molecules were ionized. The results led to the formulation of an extended hypothesis about the ecological significance of bile salt transformations. Primary deconjugation is carried out to counteract intracellular acidification. Yet, the deconjugated molecule can be harmful at moderately acidic pH-values. In this case, the BSH+ strains could effectively profit from their activity in case they are associated with 7alpha-dehydroxylating bacteria which dehydroxylate the deconjugated bile salts. The dehydroxylated molecule has a low solubility and precipitates at moderately acidic pH.
Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.
The objective of this article is to review existing studies concerning the effects of probiotics and prebiotics on serum cholesterol concentrations, with particular attention on the possible mechanisms of their action. Although not without exception, results from animal and human studies suggest a moderate cholesterol-lowering action of dairy products fermented with appropriate strain(s) of lactic acid bacteria and bifidobacteria. Mechanistically, probiotic bacteria ferment food-derived indigestible carbohydrates to produce short-chain fatty acids in the gut, which can then cause a decrease in the systemic levels of blood lipids by inhibiting hepatic cholesterol synthesis and/or redistributing cholesterol from plasma to the liver. Furthermore, some bacteria may interfere with cholesterol absorption from the gut by deconjugating bile salts and therefore affecting the metabolism of cholesterol, or by directly assimilating cholesterol. For prebiotic substances, the majority of studies have been done with the fructooligosaccharides inulin and oligofructose, and although convincing lipid-lowering effects have been observed in animals, high dose levels had to be used. Reports in humans are few in number. In studies conducted in normal-lipidemic subjects, two reported no effect of inulin or oligofructose on serum lipids, whereas two others reported a significant reduction in serum triglycerides (19 and 27%, respectively) with more modest changes in serum total and LDL cholesterol. At present, data suggest that in hyperlipidemic subjects, any effects that do occur result primarily in reductions in cholesterol, whereas in normal lipidemic subjects, effects on serum triglycerides are the dominant feature.
Listeria monocytogenes must resist the deleterious actions of bile in order to infect and subsequently colonize the human gastrointestinal tract.
The molecular mechanisms used by the bacterium to resist bile and the influence of bile on pathogenesis are as yet largely
unexplored. This study describes the analysis of three genes—bsh, pva, and btlB—previously annotated as bile-associated loci in the sequenced L. monocytogenes EGDe genome (lmo2067, lmo0446, and lmo0754, respectively). Analysis of deletion mutants revealed a role for all three genes
in resisting the acute toxicity of bile and bile salts, particularly glycoconjugated bile salts at low pH. Mutants were unaffected
in the other stress responses examined (acid, salt, and detergents). Bile hydrolysis assays demonstrate that L. monocytogenes possesses only one bile salt hydrolase gene, namely, bsh. Transcriptional analyses and activity assays revealed that, although it is regulated by both PrfA and σB, the latter appears to play the greater role in modulating bsh expression. In addition to being incapable of bile hydrolysis, a sigB mutant was shown to be exquisitely sensitive to bile salts. Furthermore, increased expression of sigB was detected under anaerobic conditions and during murine infection. A gene previously annotated as a possible penicillin
V amidase (pva) or bile salt hydrolase was shown to be required for resistance to penicillin V but not penicillin G but did not demonstrate
a role in bile hydrolysis. Finally, animal (murine) studies revealed an important role for both bsh and btlB in the intestinal persistence of L. monocytogenes.
Epidemiologic studies have demonstrated variations in the incidence of colon cancer between populations and socioeconomic groups. Differences in dietary habits have been implicated in the risk of developing colon cancer. Diet appears to influence our colonic microflora. Such variations may allow for future utilization of the fecal flora as markers for screening and diagnosis of colon cancer. The composition of the diet not only dictates the available substrates for the flora but also helps to establish predictable and competitive relationships between intestinal bacteria. To appreciate the significance of populations deemed high and low risk based on host flora, an understanding of several dynamic microbial relationships and metabolites produced is necessary. In this review, we explore the critical relationships between bile acid 7 alpha-dehydroxylation, sulfidogenesis, methanogenesis, and how they relate to carbohydrate and bile acid metabolism. We summarize the chemopreventative, anticarcinogenic, and detoxifying activity of probiotics and prebiotics, as well as potential mechanisms for protection against colon cancer.
Two genes, bshA and bshB, encoding bile salt hydrolase enzymes (EC 3.5.1.24) were identified in the genome sequence of Lactobacillus acidophilus NCFM. Targeted inactivation of these genes via chromosomal insertion of an integration vector demonstrated different substrate
specificities for these two enzymes.
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.
Probiotics are defined as “living microorganisms, which upon ingestion in certain numbers exert health benefits on the host beyond inherent basic nutrition” (43). Various studies have indicated that probiotics may alleviate lactose intolerance; have a positive influence on the intestinal flora of the host; stimulate/modulate mucosal immunity; reduce inflammatory or allergic reactions; reduce blood cholesterol; possess anti-colon cancer effects; reduce the clinical manifestations of atopic dermatitis, Crohn's disease, diarrhea, constipation, candidiasis, and urinary tract infections; and competitively exclude pathogens (35, 67, 75, 80, 99). Considering this impressive list of potential health-promoting benefits, it is not surprising that there continues to be considerable interest in the use of probiotics as biotherapeutic agents (67, 75, 82). Furthermore, given a heightened awareness among consumers of the link between diet and health and the fact that probiotic-containing foods are generally perceived as “safe” and “natural,” the global market for such foods is on the increase, particularly dairy-based products marketed for the prophylaxis or alleviation of gastrointestinal disorders (84).
The selection of potential probiotic strains that would be capable of performing effectively in the gastrointestinal tract is a significant challenge. Strain selection has generally been based on in vitro tolerance of physiologically relevant stresses: e.g., low pH, elevated osmolarity, and bile (26, 53, 77, 100). In addition to these physiological assays, molecular investigations are now under way to determine the genetic basis of gastric survival and functionality (11, 78, 79) and inclusion of molecular markers identified by this approach into screening programs may lead to more well-defined and reliable results.
The ability of probiotic strains to hydrolyze bile salts has often been included among the criteria for probiotic strain selection, and a number of bile salt hydrolases (BSHs) have been identified and characterized. However, microbial BSH activity has also been mooted to be potentially detrimental to the human host, and thus it is as yet not completely clear whether BSH activity is in fact a desirable trait in a probiotic bacterium. We review here the available literature on the reaction catalyzed by BSH enzymes, explore the ecological significance of BSH production, and briefly examine the impact that bile hydrolysis may have on human physiology. We conclude with suggestions for future work and possible applications of BSH research.
Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine
or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating
the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are
regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain Lactobacillus plantarum WCFS1 was predicted to carry four bsh genes (bsh1, bsh2, bsh3, and bsh4), the functionality of these bsh genes was explored using Lactococcus lactis heterologous overexpression and multiple bsh deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in L. plantarum WCFS1. In addition, bsh1 of L. plantarum WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis
established that bsh1, bsh2, bsh3, and bsh4 are all expressed in L. plantarum WCFS1 during the exponential growth phase. Following biodiversity analysis, bsh1 appeared to be the only bsh homologue that was variable among L. plantarum strains; furthermore, the presence of bsh1 correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in L. plantarum strains. The fact that bsh2, bsh3, and bsh4 genes appeared to be conserved among L. plantarum strains suggests an important role of these genes in the physiology and lifestyle of the species L. plantarum. Analysis of these additional bsh-like genes in L. plantarum WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion
of substrates other than bile acids in the natural habitat.
A bile salt hydrolase (BSH) was purified from Lactobacillus plantarum CK 102 and its enzymatic properties were characterized. This enzyme was successfully purified using ion-exchange chromatography with Q-Excellose and hydrophobic interaction chromatography with Butyl-Excellose. The purified enzyme showed a single protein band of 37 kDa by SDS-polyacrylamide gel electrophoresis, which was similar to the molecular weight of known BSHs. The amino acid sequence of GLGLPGDLSSMSR, determined by MALDI-TOF, was identical to that of BSH of L. plantarum WCFS1. Although this BSH hydrolyzed all of the six major human bile salts, glycine-conjugated bile acid was the best substrate, based on its specificity and K m value. Among the various substrates, the purified enzyme maximally hydrolyzed glycocholate with apparent K m and V max values of 0.5 mM and 94 nmol/min/mg, respectively. The optimal pH of the enzyme ranged from 5.8 to 6.3. This enzyme was strongly inhibited by thiol enzyme inhibitors such as iodoacetate and periodic acid.
The purpose of this study was to isolate probiotic lactic acid bacteria (LAB) that produce bile salt hydrolase (BSH), and to evaluate its effects on serum cholesterol level. One-hundred-twenty bacterial colonies were initially isolated from human feces, and five strains were selected after screening based on their resistance to acids, tolerance against bile salts, and inhibitory activity on Escherichia coli. The Lactobacillus plantarum strain with the highest level of BSH activity was identified using 16S rRNA sequences, and was named L. plantarum CK 102. L. plantarum CK 102 at a level of 1.36× 108 cfu/ml survived in pH 2 buffer for 6 h and exhibited excellent tolerance for bile salt. Coculturing the strain with E. coli in MRS broth resulted in strong inhibition against growth of E. coli at 18 h. Furthermore, the potential effect of CK 102 on serum cholesterol level was evaluated in rats. Thirty-two rats [Sprague-Dawley (SD) male, 129±1 g, 5 weeks old] were divided into four groups of eight each. For six weeks, Group 1 was fed a normal diet (negative control); Group 2 was fed a cholesterol-enriched diet (positive control); Group 3 was fed a cholesterol-enriched diet plus L. plantarum CK 102 at 1.0× 107 cfu/ml; and Group 4 was fed a cholesterol-enriched diet plus L. plantarum CK 102 at 5.0×107 cfu/ml. Blood samples were collected, serum lipids were analyzed, and weights of the organs were measured. Total blood cholesterol level, triglyceride, LDL-cholesterol, and free-cholesterol values were lower in rats that were fed L. plantarum CK 102 than in those not fed L. plantarum CK 102. This cholesterol lowering effect implies that L. plantarum CK 102 could be utilized as an additive for health-assistance foods. In conclusion, these results suggest that the L. plantarum CK 102 isolated could be used commercially as a probiotic.
Taurine and glycine were given to human subjects and the bile acids studied. Taurine (0.5 g × 3 daily) markedly increased the proportion of taurine conjugated bile acids, whereas glycine (7 g × 3 daily) did not change the bile acid pattern of the normal subject. The findings have been discussed in relation to conjugation of bile acids under normal and pathological conditions.
Growth and bile salt hydrolase (BSH) activity of the isogenic Lactobacillus plantarum 80 (LP80) strains were studied in vitro. In pure culture experiments viability and growth performance of the BSH- LP80 strain was negatively affected by the presence of conjugated bile salts. The LP80 wild type (WT) and BSH overproducing LP80 (pCBH1) strains did not show a die-off upon supplementation of bile salts. The latter strains hydrolysed glyco-conjugated deoxycholate (GDCA) more readily than tauro-conjugated deoxycholate (TDCA), indicating substrate specificity of the enzyme. BSH activities towards TDCA of LP80 WT and LP80 (pCBH1) stationary phase cells were 017 and 1.02 μmol/mg CDW.h respectively; activities towards GDCA of the respective strains were 3.52 and 54.80 μmol/mg CDW.h respectively. The study of BSH activity as a function of growth revealed a marked difference in behaviour between LP80 WT and LP80 (pCBH1) with LP80 WT hydrolysing GDCA when reaching the exponential phase, whereas LP80 (pCBH1) immediately started to hydrolyse GDCA. TDCA hydrolysis increased after GDCA hydrolysis was completed. BSH activity of LP80 (pCBH1) in a mixed microbial association, resembling that of the small intestine, was comparable to that determined under pure culture conditions, indicating that BSH activity will probably not be influenced by the presence of the normal intestinal microbiota. Based on the BSH activity of LP80 (pCBHl) and on physiological data on the bile salt-cholesterol metabolism interrelationship, it was calculated that a daily intake of a realistic amount of highly BSH active Lactobacillus cells, e.g. in the form of yoghurt, might lead to a significant reduction of cholesterol. Hence, this in vitro study indicates that altering BSH activity can be a valid (micro) biological alternative treatment for patients with severe hypercholesterolaemia.
Bile salt hydrolase (BSH) activity was shown to be constitutive and substrate-specific: the BSH isogenic Lactobacillus plantarum wild type (LP80 WT) and BSH overproducing LP80 (pCBH1) strains preferentially hydrolysed glycodeoxycholic acid (GDCA), whereas the hamster Lact. animalis isolates H362 and H364 showed a higher affinity for taurodeoxycholic acid (TDCA). In viability studies in the presence of nutrients, it was demonstrated that GDCA exerted a higher toxicity than TDCA in a pH-dependent manner. This toxicity was inversely proportionate to the BSH activity level of the strains tested, indicating that BSH activity contributed towards bile salt resistance when appropriate nutrients were available. The high toxicity of GDCA relative to TDCA was suggested to be caused by their weak and strong acid properties respectively. It was therefore hypothesized that the protonated form of bile salts exhibited toxicity as it imported protons in the cell. This puts an energy-burden on BSH− lactobacilli which undergo intracellular acidification. BSH+ cells primarily protect themselves through the formation of the weaker DCA compound, which can help negate the pH-drop by recapturing and exporting the co-transported proton. However, since DCA is more toxic than its conjugated counterparts, an additional energy-dependent detoxification of DCA is suggested.
The bile salt hydrolase (BSH) of Lactobacillus reuteri CRL 1098 is a single, constitutive, intracellular enzyme which is only detectable in stationary phase cells. It has optimal activity at pH4.5–5.5 and 37–45C. The enzyme (80kDa apparent mass) has sulphydryl groups in the catalytic active site and hydrolyzes both glycine and taurine conjugated bile acids with higher affinity for glyco-conjugates.
Fourteen Lactobacillus strains of six species were investigated with their characteristics of bile salt tolerance, deconjugation of sodium taurocholate
and cholesterol removal in the spent broth. Meanwhile, a co-precipitation curve of cholesterol with cholic acid at concentrations
ranged 0.0–6.0 μM/ml was involved in the evaluation of cholesterol removal. Results demonstrated that both co-precipitation
and assimilation effects contributed to cholesterol removal during the incubation of these Lactobacillus strains. It was also indicated that the supplementation of bile salts influenced the cholesterol removal, not only as an
essential factor related to co-precipitation but also a critical condition for cholesterol assimilation. Out of all strains
tested, four L. plantarum strains LS12, LS31, Lp501 and Lp529 exhibited a high ability of cholesterol assimilation (maximum 20.76μg/ml), deconjugation
of sodium taurocholate (maximum 5.00 μM/ml) and bile tolerance. They could be further studied and used as potential probiotics
strains to reduce serum cholesterol in humans
Eleven strains of lactobacilli were screened for their bile salt deconjugation ability, bile salt hydrolase activity (BSH) and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation as determined by the release of cholic acid showed that more cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate, and Lactobacillus acidophilus strains had higher deconjugation ability than L. casei strains. BSH activity, as quantified by the amount of taurine or glycine liberated from conjugated bile salts, indicated that substrate specificity was more towards glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both conjugated bile, with more cholesterol being precipitated upon deconjugation of sodium glycocholate than upon that of sodium taurocholate. Cholesterol co-precipitation with deconjugated bile increased with decreasing pH. L. acidophilus ATCC 33200, 4356 and 4962 and L. casei ASCC 1521 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be promising candidates to exert beneficial bile deconjugation activity in vivo.
Printout. Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 1991. Vita. Includes bibliographical references (leaves 129-145). Available on microfilm from University Microfilms.
Autotrophic ammonia-oxidising bacteria (AOB) are a crucial component of the microbial communities of nitrifying wastewater treatment systems. Nitrification is known to occur in reactors of different configuration, but whether AOB communities are different in reactors of different design is unknown. We compared the diversity and community structure of the betaproteobacterial AOB in two full-scale treatment reactors - a biological aerated filter (BAF) and a trickling filter - receiving the same wastewater. Polymerase chain reaction (PCR) of 16S ribosomal RNA (rRNA) gene fragments with AOB-selective primers was combined with denaturing gradient gel electrophoresis (DGGE) to allow comparative analysis of the dominant AOB populations. The phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing PCR-amplified 16S rRNA gene fragments. DGGE profiles were compared using a probability-based similarity index (Raup and Crick). The use of a probability-based index of similarity allowed us to evaluate if the differences and similarities observed in AOB community structure in different samples were statistically significant or could be accounted for by chance matching of bands in DGGE profiles, which would suggest random colonisation of the reactors by different AOB. The community structure of AOB was different in different sections of each of the reactors and differences were also noted between the reactors. All AOB-like sequences identified, grouped within the genus Nitrosomonas. A greater diversity of AOB was detected in the trickling filters than in the BAF though all samples analysed appeared to be dominated by AOB most closely related to Nitrosococcus mobilis. Numerical analysis of DGGE profiles indicated that the AOB communities in depth profiles from the filter beds were selected in a non-random manner.
The Lowry method (G. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951, J. Biol. Chem.193, 265–275) for protein concentration measurement has been automated to permit assay of samples with concentrations from 1 to 400 μg/ml. Calibration with solutions of bovine serum albumin resulted in a nonlinear (quadratic) curve. The quantity of color developed in the assay was found to be strongly dependent on the concentration of the Folin-Ciocalteu phenol reagent. Color yield peaked sharply at a reagent concentration 40% lower than that used in the Lowry procedure. Optimization of the reagent concentration is necessary to obtain maximum sensitivity from the Lowry assay.
It is clear that a truly spectacular increase in our understanding of the biliary circulation has occurred during the past 5 years. A steady-state situation has now become dynamic. In a sense, we have clarified the anatomy and physiology of the biliary circulation, and the challenge now is to describe its biochemistry. Specifically, we remain totally ignorant of the mechanism of cell transport of bile acids, the regulation of their synthesis and the action of bile acids in bile formation. When we understand the biliary circulation, we can then clarify its diseases. That, in turn, should lead to the development of a new specialty of biliary therapy.
Bile salt hydrolase (BSH) activity was shown to be constitutive and substrate-specific: the BSH isogenic Lactobacillus plantarum wild type (LP80 WT) and BSH overproducing LP80 (pCBH1) strains preferentially hydrolysed glycodeoxycholic acid (GDCA), whereas the hamster Lact. animalis isolates H362 and H364 showed a higher affinity for taurodeoxycholic acid (TDCA). In viability studies in the presence of nutrients, it was demonstrated that GDCA exerted a higher toxicity than TDCA in a pH-dependent manner. This toxicity was inversely proportionate to the BSH activity level of the strains tested, indicating that BSH activity contributed towards bile salt resistance when appropriate nutrients were available. The high toxicity of GDCA relative to TDCA was suggested to be caused by their weak and strong acid properties respectively. It was therefore hypothesized that the protonated form of bile salts exhibited toxicity as it imported protons in the cell. This puts an energy-burden on BSH- lactobacilli which undergo intracellular acidification. BSH+ cells primarily protect themselves through the formation of the weaker DCA compound, which can help negate the pH-drop by recapturing and exporting the co-transported proton. However, since DCA is more toxic than its conjugated counterparts, an additional energy-dependent detoxification of DCA is suggested.
It has been shown that feces of patients with ulcerative colitis uniformly contain sulfate reducing bacteria. Sulfide produced by these bacteria interferes with butyrate-dependent energy metabolism of cultured colonocytes and may be involved in the pathogenesis of ulcerative colitis. Mucosal biopsies from the sigmoid rectum of 10 patients (no caner, polyps, inflammatory bowel disease) were incubated with either NaCl, sodium hydrogen sulfide (1 mmol/L), a combination of both sodium hydrogen sulfide and butyrate (10 mmol/L), or butyrate. Mucosal proliferation was assessed by bromodeoxyuridine labeling of cells in S-phase. Compared to NaCl, sulfide increased the labeling of the entire crypt significantly, by 19% (p < 0.05). This effect was due to an expansion of the proliferative zone to the upper crypt (compartments 3-5), where the increase in proliferation was 54%. Sulfide-induced hyperproliferation was reversed when samples were coincubated with sulfide and butyrate. The study shows that sodium hydrogen sulfide induces mucosal hyperproliferation. Our data support a possible role of sulfide in the pathogenesis of UC and confirm the role of butyrate in the regulation of colonic proliferation and in the treatment of UC.
Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDeltapH) was involved in Lactobacillus bile salt resistance.
Listeria monocytogenes is a bacterial pathogen causing severe food-borne infections in humans and animals. It can sense and adapt to a variety of harsh microenvironments outside as well as inside the host. Once ingested by a mammalian host, the bacterial pathogen reaches the intestinal lumen, where it encounters bile salts which, in addition to their role in digestion, have antimicrobial activity. Comparison of the L. monocytogenes and Listeria innocua genomes has revealed the presence of an L. monocytogenes-specific putative gene encoding a bile salt hydrolase (BSH). Here, we show that the bsh gene encodes a functional intracellular enzyme in all pathogenic Listeria species. The bsh gene is positively regulated by PrfA, the transcriptional activator of known L. monocytogenes virulence genes. Moreover, BSH activity increases at low oxygen concentration. Deletion of bsh results in decreased resistance to bile in vitro, reduced bacterial faecal carriage after oral infection of the guinea-pigs, reduced virulence and liver colonization after intravenous inoculation of mice. Taken together, these results demonstrate that BSH is a novel PrfA-regulated L. monocytogenes virulence factor involved in the intestinal and hepatic phases of listeriosis.
Bile salt hydrolases were purified to electrophoretic homogeneity from Bifidobacterium bifidum ATCC 11863, Bifidobacterium infantis KL412, Bifidobacterium longum ATCC 15708, Bifidobacterium longum KL507, and Bifidobacterium longum KL515. Three different types (A, B, and C) of bile salt hydrolase (BSH) were revealed during the purification study, exhibiting the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was around 35 kDa, and the native molecular mass in all five Bifidobacterium strains was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, an isoelectric point value of 4.45 was obtained with BSH (type B) from B. bifidum ATCC 11863 and the other BSH (types A and C) showed the similar pI values around 4.65. N-Terminal amino acid sequencing for the proteins of types A and C revealed that 6 out of 20 amino acid residues were different, and highly conserved residues were identified in both N-terminal sequences of types A and C. All BSH enzymes from five strains hydrolyzed six major human bile salts, and they showed a better deconjugation rate on glycine-conjugated bile salts than on taurine-conjugated forms.
Commensal and pathogenic microorganisms must resist the deleterious actions of bile in order to survive in the human gastrointestinal tract. Herein we review the current knowledge on the mechanisms by which Gram-positive and Gram-negative bacteria contend with bile stress. We describe the antimicrobial actions of bile, assess the variations in bile tolerance between bacterial genera and examine the interplay between bile stress and other stresses. The molecular mechanisms underlying bile tolerance are investigated and the relationship between bile and virulence is examined. Finally, the potential benefits of bile research are briefly discussed.
Probiotic utilization is becoming increasingly popular in veterinary medicine. However, only few probiotic products are available commercially for use in dogs in our market. Therefore, the aim of our study was to determine the properties of new potential probiotic Lactobacillus fermentum AD1 strain-own canine isolate and to investigate its effect on several microbiological and biochemical parameters in healthy dogs. The strain expressed in vitro survival by pH 3.0 after 3h (86.8%) and in the presence of 1% bile (75.4%). The AD1 strain adhered to the canine and human intestinal mucus. It was sensitive to commonly used antimicrobials. Fifteen healthy dogs were supplemented with 10(9)L. fermentum AD1 for 7 days. At the end of AD1 strain application, numbers of faecal lactobacilli and enterococci increased significantly in the canine faeces. Significant increase of total protein and total lipid and significant reduction of glucose in serum of dogs were noted. These data indicate that L. fermentum AD1 survive transit through the canine gastrointestinal tract, and populate the colon and probably increased absorption of some nutrients. Whether longer time of its application lead to the same results as well as its potential to improve immune function in dogs remains to be determined.
The bacterium Lactobacillus plantarum PH04 was isolated from infant feces and tested positive for bile/acid tolerance and bile salt hydrolase activity. It was evaluated as a potential probiotic with cholesterol-lowering effect. Bile salt hydrolase activity was nine times greater in stationary phase than in exponential phase cells and increased when the cells were exposed to conjugated bile salts. L. plantarum PH04 was resistant to seven of nine antibiotics tested and did not produce beta-glucuronidase. L. plantarum PH04 was fed to hypercholesterolemic mice at numbers of 10(7) CFU per mouse per day for 14 days. Compared with a control group, the serum cholesterol and triglycerides were respectively 7 and 10% lower in the group fed L. plantarum PH04, and fecal lactic acid bacteria increased while no any significant differences (P<0.05) in body weight, visceral weigh index or bacteria translocation between two groups were observed. The results indicated that L. plantarum PH04 might be effective as a probiotic with cholesterol-lowering activities.
A selection and optimization procedure for the synbiotic combination of probiotic and prebiotics was established to optimize its cholesterol removal in vitro. In light of fermentability, prebiotics utilization by probiotics was highly variable and interspecies differences existed. Based on the results of fermentability, L. plantarum LS12, Ls31, LP529 and L. ruminis La3 could be the better candidates for symbiotic research. The bile tolerance of all the tested strains could be improved by the strain-specific prebiotics comparing to the control carbon source (glucose). The strain LS12 was finally selected to form the symbiotic according to its better ability to ferment prebiotics and bile tolerance, while the five prebiotics (FOS, stachyose, GOS, IMO and mannitol) were selected to make their synbiotic combination because of their better enhancement of bile tolerance and growth support to LS12. The synbiotic combination for cholesterol removal was optimized by use of response surface methodology. The first-order model showed that the selected prebiotics mannitol and GOS were significant factors. Then through the second-order polynomial regression model, the optimum conditions of the two factors for cholesterol removal by the synbiotic were suggested.
Genetic analysis of two bile salt hydrolase activities in Lactobacillus acidophilus NCFM Diet, anaerobic bacterial metabolism, and colon cancer: a review of the literature Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli
- O Cano Rj
- Klaenhammer
- Mcgarr
- Se
- Jm
- Hylemon
- Moser
- Sa
- Savage
O, Cano RJ, Klaenhammer TR (2005) Genetic analysis of two bile salt hydrolase activities in Lactobacillus acidophilus NCFM. Appl Environ Microbiol 71:4925–4929 McGarr SE, Ridlon JM, Hylemon PB (2005) Diet, anaerobic bacterial metabolism, and colon cancer: a review of the literature. J Clin Gastroenterol 39:98–109 Moser SA, Savage DC (2001) Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli. Appl Environ Microbiol 67:3476–3480
Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects Optimization of folin-ciocalteu reagent concentration in an automated lowry protein assay
- Tdt
- Kang
- Jh
- Ms Lee
- Gm Oosta
- Mathewson Ns
- Gn
TDT, Kang JH, Lee MS (2007) Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects. Int J Food Microbiol 113:358– Oosta GM, Mathewson NS, Catravas GN (1978) Optimization of folin-ciocalteu reagent concentration in an automated lowry protein assay. Anal Biochem 89:31–34
Cholesterol lowering effect of Lactobacillus plantarum isolated from human feces The enterohepatic circulation of bile acids in man Bile acids The liver: biology and pathobiology. Raven Purification and characterization of three different types of bile salt hydrolases from Bifidobacterium strains
- Cg
- Cho
- Jk
- Ch Lee
- Chai
- Yg
- Ha
- Ya
- Shin Sh Arias Im
- Boyer
- Fausto N Jl
- Jackoby
- Wb
CG, Cho JK, Lee CH, Chai YG, Ha YA, Shin SH (2006b) Cholesterol lowering effect of Lactobacillus plantarum isolated from human feces. J Microbiol Biotechnol 16:1201–1209 Hofmann AF (ed) (1977) The enterohepatic circulation of bile acids in man. Saunders, Philadelphia Hofmann AF (1994) Bile acids. In: Arias IM, Boyer JL, Fausto N, Jackoby WB, Schachter DA, Shafritz DA (eds) The liver: biology and pathobiology. Raven, New York, pp 677–718 Kim GB, Yi SH, Lee BH (2004) Purification and characterization of three different types of bile salt hydrolases from Bifidobacterium strains. J Dairy Sci 87:258–266
Bile acids The liver: biology and pathobiology
- Af Hofmann
- Im Arias
- Jl Boyer
- N Fausto
- Wb Jackoby
- Da Schachter
- Da Shafritz