Bile salt deconjugation ability,bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains

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.     

三株植物乳杆菌降胆固醇机理的研究

测定了已筛选出的具有较高胆固醇去除能力和胆盐水解酶活性的3株植物乳杆菌(KLDS6.0330,KLDS6.0333,KLDS6.0335)的胆固醇同化作用,胆汁盐的去结合作用和胆固醇共沉淀作用。这3株植物乳杆菌能够同化较多的胆固醇(﹥43μg/mL);所有的菌株都表现出了对甘氨胆酸钠和牛磺胆酸钠的去结合作用,与牛磺胆酸钠相比,甘氨胆酸钠的去结合能够释放较多的胆酸;所有菌株都表现出了胆固醇与甘氨胆酸钠和牛磺胆酸钠去结合释放胆酸的共沉淀,与牛磺胆酸钠相比,甘氨胆酸钠去结合产生的胆酸表现出了与较多的胆固醇共沉淀;随着pH值的下降,解聚态的胆汁酸与胆固醇的共沉淀量增加,结合态胆盐与胆固醇的共沉淀量较少。结果表明这3株植物乳杆菌在体外可以通过3种机制去除胆固醇。     

Deconjugation and bile salts hydrolase activity by Bifidobacterium strains with acquired resistance to bile

Deconjugation by bile salts hydrolases in probiotics has been related to reduction of serum cholesterol levels in mammals. We compared the susceptibility to conjugated primary (glycocholate and taurocholate) and secondary (glycodeoxycholate and taurodeoxycholate) salts and the level of hydrolase activity of Bifidobacterium strains with acquired resistance to bile and of their more sensitive original strains. Minimum inhibitory concentrations against conjugated salts of the resistant strains were higher than that of the corresponding originals. None of the strains displayed deconjugation against primary salts, whereas most of them deconjugated secondary salts. Salts of cholic acid were more toxic than that of deoxycholic acid. Derivatives showed higher hydrolase activity than their originals. These results suggested a relationship between bile resistance and deconjugation. Finally, the resistance of bifidobacteria against glycodeoxycholate increased in the presence of maltose and cellobiose as compared with glucose, which could be related to a more efficient energy procurement from disaccharides.     

酸菜来源植物乳杆菌体外去除胆固醇特性

对4株酸菜来源具有潜在益生特性的植物乳杆菌降胆固醇特性进行研究.研究发现其降胆固醇能力受胆盐浓度和胆盐种类的影响,当牛胆盐的添加浓度为3g/L时,降胆固醇能力达到最高(9.67mg/g～21.32mg/g[干重]);在培养基中添加不同种类的胆盐,降胆固醇能力不同,依次为牛磺胆酸钠＞混合胆盐＞甘氨胆酸钠.其产生的游离胆酸的量与胆固醇移除能力呈正相关,且以在牛磺胆酸钠存在下最佳.     

Bile salt deconjugation activity of Propionibacterium strains and their cholesterol co‐precipitation abilities

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.     

Bile salt hydrolase activity of three strains of Lactobacillus acidophilus

Three strains of Lactobacillus acidophilus, two from human intestinal origin (016 and L1) and one from porcine intestinal origin (ATCC 43121), were tested for their bile salt deconjugation activity. The L. acidophilus ATCC 43121 had more deconjugating activity of both sodium glycocholate and sodium taurocholate at pH 6.5 than did either L. acidophilus 016 or L1. The activity of intracellular bile salt hydrolase found in strain ATCC 43121 was 14-fold higher than that in either of the other two strains. The optimum pH for deconjugation of sodium glycocholate was between 4 and 5.5 for all three strains. For deconjugation of sodium taurocholate, the optimum pH was between 3.5 and 4.5 for strains L1 and ATCC 43121 and was between pH 5 and 6 for strain O16. The molecular mass of the enzyme in all three strains of L. acidophilus was estimated to be 126 kDa by Sephadex G-200 gel filtration. All three strains exhibited more bile salt hydrolase activity towards sodium glycocholate than towards sodium taurocholate.     

Bile tolerance, taurocholate deconjugation, and binding of cholesterol by Lactobacillus gasseri strains

Bile tolerance, deconjugation of sodium taurocholate, and the cholesterol-binding ability of 28 strains of Lactobacillus gasseri were examined. There was significant variation among strains in growth in media containing bile and also variation in the ability to bind cholesterol. Cultures grown for 12 h at 37 degrees C bound significantly more cholesterol than did cells from a 48-h incubation. Variation among strains in the ability to deconjugate sodium taurocholate was not significantly different. Maximal deconjugation of sodium taurocholate was achieved with the cells during the stationary phase of growth (12 h). Statistical analysis showed no significant correlation between bile tolerance and sodium taurocholate deconjugation, bile tolerance and cholesterol-binding ability, or sodium taurocholate deconjugation and cholesterol-binding ability.     

Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract

Five strains of lactobacilli were studied for their ability to remove cholesterol in vitro under conditions that mimic the human gastrointestinal tract. The highest assimilation of cholesterol was observed in media supplemented with oxgall and the lowest in the presence of taurocholic acid. Scanning electron micrographs showed that cholesterol was adhered to the cellular surface of lactobacilli cells. Resting and dead cells were able to remove cholesterol although in small amounts. Additionally, inhibition of cholesterol micelles formation was observed in the presence of bile salts. All strains were able to deconjugate bile salts, where higher deconjugation was observed in the presence of sodium glycocholate compared with other bile salts studied. All strains also exhibited bile salt hydrolase activity and most strains showed higher substrate specificity towards glycine-conjugated bile than towards taurine-conjugated bile. The results indicated that lactobacilli could remove cholesterol in vitro via various mechanisms, and may exert such hypocholesterolaemic effects in vivo.     

Bile salt deconjugation and cholesterol removal from media by Lactobacillus strains used as probiotics in chickens

BACKGROUND: Bile salt deconjugation by Lactobacillus strains is often closely linked to bile tolerance and survival of the strains in the gut and lowering of cholesterol in the host. The present study investigated the deconjugation of bile salts and removal of cholesterol by 12 Lactobacillus strains in vitro. The 12 strains were previously isolated from the gastrointestinal tract of chickens. RESULTS: The 12 Lactobacillus strains could deconjugate sodium glycocholate (GCA, 16.87–100%) and sodium taurocholate (TCA, 1.69–57.43%) bile salts to varying degrees, with all strains except L. salivarius I 24 having a higher affinity for GCA. The 12 Lactobacillus strains also showed significant (P < 0.05) differences in their ability to remove cholesterol from the growth medium (26.74–85.41%). Significant (P < 0.05) correlations were observed between cholesterol removal and deconjugation of TCA (r = 0.83) among the L. reuteri strains (C1, C10 and C16) and between cholesterol removal and deconjugation of TCA (r = 0.38) and GCA (r = 0.70) among the L. brevis strains (I 12, I 23, I 25, I 211 and I 218). In contrast, although L. gallinarum I 16 and I 26 and L. panis C 17 showed high deconjugating activity, there was no correlation between cholesterol removal and deconjugation of bile salts in these strains. CONCLUSION: The results showed that the 12 Lactobacillus strains were able to deconjugate bile salts and remove cholesterol in vitro, but not all strains with high deconjugating activity removed cholesterol effectively. Copyright © 2009 Society of Chemical Industry     

Binding of Bile Salts by Soluble Fibers and Its Effect on Deconjugation of Glycocholate by Lactobacillus acidophilus and Lactobacillus casei.

ABSTRACT: Binding of sodium cholate, sodium taurocholate, and sodium glycocholate by guar gum, soluble oat fiber, xanthan gum, and inulin was studied. All soluble fibers were able to bind sodium cholate, sodium taurocholate, and sodium glycocholate from a mixture of the three, but when tested individually, the fibers bound little or no sodium cholate. In general, the fibers bound higher concentrations of conjugated bile salts than they did free bile salts. Soluble oat fiber, which bound as much or more sodium cholate and sodium glycocholate as did the other 3 fibers, was tested for its effect on deconjugation of glycocholate by Lactobacillus acidophilus and Lactobacillus casei. The presence of the fiber significantly increased (P < 0.05) deconjugation by all cultures.     

Deconjugation of bile salts by Lactobacillus acidophilus isolates

Deconjugation of bile salts by Lactobacillus acidophilus strains isolated from men, fermented milk, and pigs resulted in precipitate halo, opaque granular white colonies, shiny precipitate halo or clear zone around the microbial colonies on bile salt–MRS agar plates depending on the type of bile salts added. None of the L. acidophilus cultures tested exhibited 7α-dehydroxylase activity that transforms cholate into deoxycholate. L. acidophilus SNUL020 and SNUL01 deconjugated both taurocholate (TCA) and glycocholate (GCA) at similar rates, while L. acidophilus FM01 deconjugated GCA more rapidly than TCA. Most L. acidophilus strains tested precipitated more soluble cholesterol in the media containing taurochenodeoxycholate (TCDCA) and taurodeoxycholate (TDCA) than with TCA and GCA. In the medium containing TDCA, L. acidophilus SNUL020, SNUL01, and FM01 precipitated more than 50% of soluble cholesterol. All L. acidophilus strains tested were more resistant to taurine- than to glycine-conjugated bile salts. Fecal excretions of cholesterol and deoxycholate increased slightly with acidophilus milk intake containing live cells of L. acidophilus SNUL01.     

Effects of bile salt deconjugation by probiotic strains on the survival of antibiotic-resistant foodborne pathogens under simulated gastric conditions

This study was designed to evaluate the effects of bile acid deconjugation by probiotic strains on the antibiotic susceptibility of antibiotic-sensitive and multiple antibiotic-resistant Salmonella Typhimurium and Staphylococcus aureus. Eight probiotic strains, Bifidobacterium longum B6, Lactobacillus acidophilus ADH, Lactobacillus brevis KACC 10553, Lactobacillus casei KACC 12413, Lactobacillus paracasei ATCC 25598, Lactobacillus rhamnosus GG, Leuconostoc mesenteroides KACC 12312, and Pediococcus acidilactici KACC 12307, were used to examine bile acid tolerance. The ability to deconjugate bile acids was evaluated using both thin-layer chromatography and high-performance liquid chromatography. The antibiotic susceptibility testing was carried out to determine the synergistic inhibitory activity of deconjugated bile acids. L. acidophilus, L. brevis, and P. acidilactici showed the most tolerance to the conjugated bile acids. P. acidilactici deconjugated glycocholic acid and glycodeoxycholate from 3.18 and 3.09 mM to the detection limits, respectively. The antibiotic susceptibility of selected foodborne pathogens was increased by increasing the concentration of deconjugated bile acids. The study results are useful for understanding the relationship between bile acid deconjugation by probiotic strains and antibiotic susceptibility in the presence of deconjugated bile acids, and they may be useful for designing new probiotic-antibiotic combination therapy based on bile acid deconjugation.     

Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts

Bile salt hydrolase activity of Lactobacillus acidophilus was measured based on the disappearance of sodium glycocholate and sodium taurocholate from the reaction mixture using HPLC. The amount of sodium glycocholate and sodium taurocholate that disappeared was proportional to the amount of sodium cholate that appeared in the mixture as detected by HPLC. Sodium glycocholate did not precipitate at the enzyme reaction conditions (37 degrees C and pH 5.4) for determining bile salt hydrolase activity. The bile salt hydrolase assay was insensitive to low oxidation-reduction potential when measuring bile salt hydrolase from L. acidophilus, an intestinal microorganism. However, EDTA and freezing temperatures were necessary to maintain stability of the partially purified enzyme during storage.     

Survival of bifidobacteria in the presence of bile salt

The ability of Bifidobacterium species to survive in the presence of sodium glycocholate, at concentrations ranging from 0 to 3 g liter^?1, was tested. Bacterial growth was monitored by measuring the optical density for up to 48 h. All the Bifidobacterium strains tested showed growth limitation in the presence of sodium glycocholate. However, after a series of propagations in fresh trypticase-peptone-yeast extract medium devoid of bile salt all bacterial strains retained their activity. It was concluded that Bifidobacterium species are able to survive physiological and higher bile salt levels and that bifidobacteria can be considered for use as dietary adjuncts.     

A sensitive method for qualitative screening of bile salt hydrolase-active lactobacilli based on thin-layer chromatography

A sensitive protocol based on thin-layer chromatography (TLC) was developed to screen qualitatively bile salt hydrolase (BSH)-active lactobacilli. The sodium salts of glycocholic acid and taurocholic acid were used as substrates, and bacterial BSH activity was confirmed by detecting cholic acid as a product of the bile conjugates using a TLC assay with direct visual observation. Forty-five lactobacilli isolated from human fecal samples were tested for BSH activity by the TLC assay, a conventional plate assay, and a quantitative colorimetric assay. With the TLC and quantitative colorimetric assays, the same 24 BSH-positive strains were detected. No false-positive or false-negative results were detected by the TLC assay. However, only 20 BSH-positive strains were detected with the conventional plate assay. Compared with the conventional plate assay, the TLC assay is more sensitive for the detection of BSH activity of lactobacilli and, thus, more suitable for screening of BSH-active lactobacilli of human origin.     

具有胆盐水解酶活性乳酸菌的筛选及影响其活性因素分析

具有胆盐水解酶活性的乳酸菌有潜在的降低血液胆固醇的作用。对分离自酸马奶的10株乳杆菌的胆盐水解酶活性进行了定性及定量分析,在此基础上对影响胆盐水解酶的一些因素进行了研究。结果表明,3株乳杆菌具有胆盐水解酶活性,其中以MG13-3为最高,其次为XB4-1及MG16-3;当pH值为6时,超声波破碎时间在12～15min之间,培养基中加入巯基乙酸钠并在厌氧条件下培养时,3株乳杆菌均体现出较高的胆盐水解酶活性。     

Short communication: Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression

Bile salt hydrolase (BSH) plays an essential role in the cholesterol-removing effect of lactic acid bacteria, which hydrolyze conjugated bile salts to amino acid and deconjugated bile salts. However, Lactobacillus casei lacks the bsh gene, which may make it highly sensitive to bile salt stress. We wanted to improve the BSH activity of L. casei for various food-industry applications (e.g., milk fermentation). Plate assay testing indicated that Lactobacillus plantarum AR113 has the highest BSH activity. We cloned and sequenced 4 bsh genes from the genome of L. plantarum AR113. Structure modeling and molecular docking of BSH indicated that BSH1 and BSH3 could react efficiently with bile salts, so we selected BSH1 and BSH3 for heterologous expression in L. casei. Compared with single expression of BSH1 or BSH3, co-expression of both protein sequences showed the highest hydrolysis activity by HPLC analysis. Our results suggested that heterologous expression of BSH in L. casei can significantly improve host activity against bile salts, and in silico molecular docking could be an efficient method of rapid screening for BSH with high activity.     

一株乳酸乳球菌的降胆固醇特性及其作用机制

探讨了乳酸乳球菌乳亚种HUCM 201的降胆固醇特性及其体外去除胆固醇的机制。乳酸乳球菌乳亚种HUCM 201菌株可从培养基中去除33.1%的胆固醇,其中14.3%的胆固醇发生共沉淀并重新溶解在洗涤液中,18.1%的胆固醇被吸收到菌体细胞内。HUCM 201菌株对5种结合型胆酸盐分别表现出了不同的胆盐水解酶活性,其中对甘氨胆酸钠的水解能力最强,总酶活为0.279 U/mL,比酶活为0.076 U/mg。     

In vitro binding of bile salts by lentil flours, lentil protein concentrates and lentil protein hydrolysates

The binding capacity of bile salts by lentil flours produced from two varieties, Blaze and Laird and their protein concentrates and hydrolysates were studied. Sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate and sodium chenodeoxycholate were tested individually, and their binding interactions with the lentil products were analyzed using the Trinity Biotech Bile Acids Kit 450-10 and compared to cholestyramine. All tested samples bound the bile salts investigated, and the amount of bile salts bound (> 70%) was sometimes greater than that bound by cholestyramine. Overall, there were no major differences in the bile salt binding capacities of similar samples prepared from the two varieties of lentil. In vitro digestion of the lentil proteins by pepsin/trypsin/??-chymotrypsin, alcalase/flavourzyme and papain significantly reduced the bile salt binding capacity compared to the undigested samples except in the case of sodium deoxycholate where no significant differences in bile salt binding were observed before and after hydrolysis. Binding of bile salts has been linked to cholesterol reduction, thus, the ability of the lentil products to bind bile salts is of interest as it may suggest that lentils could potentially have cholesterol-reducing properties.     

Purification and characterization of three different types of bile salt hydrolases from Bifidobacterium strains

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.