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.     

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.     

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.     

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.     

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 BSH activity of five bifidobacterial strains and their cholesterol co-precipitating properties

Five strains of bifidobacteria were screened for their bile salt deconjugation ability, bile salt hydrolase (BSH) activity and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation was determined by the release of cholic acid. All strains exhibited deconjugation of both sodium glycocholate and sodium taurocholate. More cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate. BSH activity was quantified by determining the amount of glycine or taurine liberated from conjugated bile salts by bifidobacteria strains. There was higher substrate specificity for glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both sodium glycocholate and sodium taurocholate, and by all bifidobacteria strains studied. More cholesterol was precipitated with cholic acid when sodium glycocholate was used compared to sodium taurocholate. Increased cholesterol co-precipitation with deconjugated bile was observed with decreasing pH levels. Bifidobacterium infantis 17930 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be a promising candidate to exert beneficial bile deconjugation activity in vivo.     

Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use

A set of simple in vitro tests (identification by species-specific PCR, genetic diversity, phage sensitivity, growth and viability in milk, resistance to salts and flavor compounds, bacterial interactions, tolerance to simulated gastric juice and bile, bile salts deconjugation, hydrophobicity and β-galactosidase and antibacterial activities), that can be carried out in almost every laboratory of microbiology, mainly in developing countries where there is often limited access to sophisticated techniques, allowed us to identify, among 19 intestinal human isolates, a potential candidate for new probiotic dairy foods for the local market. Lactobacillus gasseri LgF_37/1 performed well in the culture media used for the enumeration of probiotic bacteria in argentinian dairy products. This strain showed also high tolerance to the technological challenges assessed, bile salts resistance, the capacity to produce bacteriocin-like metabolites, to inhibit pathogenic bacteria, to deconjugate bile salts and high hydrophobicity. Further in vivo research should be carried out with this strain before claiming probiotic properties for it. However, the use of a set of simple in vitro techniques proved to be important to determine which strains should undergo future and more complex studies.     

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.     

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.     

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.     

In vitro characterisation of probiotic properties of Enterococcus faecium and Enterococcus durans strains isolated from raw milk and traditional dairy products

In this study, some probiotic characteristics such as antimicrobial activity, vancomycin resistance, growth ability at pH, resistance to bile salts, bile salt deconjugation and hydrophobicity of 30 Enterococcus faecium and Enterococcus durans strains isolated and identified from raw milk and various dairy products were investigated. According to the study results, antimicrobial activity profiling, pH and bile salt resistance and bile salt deconjugation ability of Enterococcus strains varied depending on the species and strains and all the strains showed resistance to the tested bile salt concentrations. It was concluded that the E. faecium and E. durans strains tested showed probiotic characteristics and have the potential to be used in food production.     

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。     

Assessment of probiotic characteristics of lactic acid bacteria isolated from fermented yak milk products of Sikkim,India: Chhurpi,Shyow, and Khachu

The present study documents the probiotic attributes of indigenous lactic acid bacteria (LAB) isolated from local fermented Yak milk products namely Chhurpi, Shyow and Khachu prepared in the northern and eastern region of Sikkim in the Himalayas. Samples were collected aseptically and a total of 170 LAB was isolated and screened for putative probiotic properties like hypocholesteromic effect, acid tolerance, bile tolerance, bile salt hydrolase (BSH) activity and cell surface hydrophobicity. It was observed that 70 LAB isolates showed cholesterol lowering activity, out of which 35 isolates were selected that showed 50% and less cholesterol reducing effect in vitro. Acid tolerance test revealed good tolerance of 12 isolates at pH 2.5 and pH 2.0 for up to 2 hours. The tolerance to 0.5% and 1% of three bile salts acid revealed more growth in MRS broth containing taurocholic acid with the isolates revealing good BSH activity leading to bile acid deconjugation. The cell surface hydrophobicity ranged from 20–95%. Furthermore, 16S rRNA gene sequencing revealed Lactobacillus plantarum YD5S and YD9S, L. pentosus YD8S, L. paraplantarum YD11S, Enterococcus lactis YHC20 and E. faecium YY1 as the best isolates with technological properties. The isolates may serve as potential probiotic candidates with potential for hypocholesteromic benefits in the future.     

Assimilation of cholesterol and probiotic characterisation of yeast strains isolated from raw milk and fermented foods

In this study, cholesterol assimilation ratios and some probiotic characteristics of yeasts were investigated. For this purpose, yeasts were isolated from milk and foods that were naturally fermented and not containing starter culture. In vitro cholesterol assimilation properties were determined in media. The Cholesterol assimilation by yeast strains ranged between 1.36 and 73.33%. Twenty‐one yeast strains showing high assimilation percentage were selected, and their acid tolerance, bile tolerance, bile salt deconjugation activity and survival in simulated gastrointestinal conditions were investigated. Among the strains assessed, 12 of them showed probiotic characteristics.     

In vitro comparison of probiotic properties of Lactobacillus casei Zhang, a potential new probiotic, with selected probiotic strains

Since 2004, our research group has isolated 240 Lactobacillus strains from Koumiss, a traditional fermented alcoholic beverage prepared from mare's milk in Inner Mongolia, Xinjiang of China and Mongolia. Among these Lactobacillus strains a novel strain with potential probiotic properties, Lactobacillus casei Zhang, was screened out and studied extensively for its probiotic properties, health-promoting effects and fermentation characteristics. In vitro tests indicated that L. casei Zhang had high tolerance to simulated gastric, intestine juices and bile salts, similar to commercial probiotic strains such as Lactobacillus acidophilus NCFM, Lactobacillus rhamnosus GG, L. casei Shirota and Bifidobacterium animalis Bb12. Higher acid-production activity and proteolytic activity was observed in the fermented milk inoculated with L. casei Zhang during the refrigerated storage than in the samples inoculated with the selected commercial probiotics. The yogurt samples fermented with L. casei Zhang exhibited similarer viable count (1.0 × 10⁹ cfu/mL) as the other samples after 28 d of refrigerated storage. The results suggested that L. casei Zhang showed good potential for application in functional foods and health-related products.     

A bile salt hydrolase gene of Lactobacillus plantarum BBE7 with high cholesterol-removing activity

Sixteen Lactobacillus strains were screened using a plate agar containing glycodeoxycholic acid or taurodeoxycholic acid and quantitative BSH assay method, along with cholesterol-removing properties in vitro. Most BSH-positive strains tested were more efficient in hydrolyzing glycoconjugated bile salts than tauroconjugated bile salts. Among these strains, Lactobacillus plantarum isolate BBE7 had a relatively high BSH activity toward both bile salts and a higher cholesterol-removing activity (about 72.8?%) from cholesterol (100???g/mL)-containing MRS culture, as compared with other tested strains. The bsh gene of this strain composed of about 43?kDa protein was cloned, sequenced, and overexpressed in Escherichia coli BL21 (DE3). The bsh gene contained a single ORF of 975 nucleotides flanked by a methionine start codon and translational termination codon and encoded a 324-amino-acid protein.     

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.     

Influence of Homofermentative Lactobacilli on the Microflora and Soluble Nitrogen Components in Cheddar Cheese

Cheddar cheese was manufactured by inoculation with and without cultures of homofermentative Lactobacillus strains (L. casei-subsp-casei, L. casei-subsp-pseudoptantarum, L. plantarum). Growth rate of microflora, the incidence of heterofermentative lactobacilli and the rate of proteolysis were then studied during aging. The total number of psychotrophs, mesophiles and lactic streptococci reached a maximum at 5 months but maximum numbers attained were dependent upon the curing temperature (7°C or 15°C). The acceleration of cheese ripening by Lactobacillus cultures was accompanied by a greater degree of protein hydrolysis which was detectable after 8 months aging as soluble nitrogen in TCA extracts (360 mg/100g at 15°C and 240 mg at 7°C, versus 170 mg and 75 mg, respectively, for the controls).     

Probiotic potential and biochemical and technological properties of Lactococcus lactis ssp. lactis strains isolated from raw milk and kefir grains

Lactococcus lactis ssp. lactis is one of the most important starter bacteria used in dairy technology and it is of great economic importance because of its use in the production of dairy products, including cheese, butter, cream, and fermented milks. Numerous studies have evaluated the biochemical and probiotic properties of lactococci; however, limited studies on the probiotic characteristics of lactococci were conducted using strains originating from raw milk and dairy products. Characterizing the probiotic properties of strains isolated from raw milk and fermented milk products is important in terms of selecting starter culture strains for the production of functional dairy products. In this study, biochemical properties (including antibiotic sensitivity, lipolytic activity, amino acid decarboxylation, antioxidant activity) and probiotic properties (including antimicrobial activity, growth in the presence of bile salts, bile salts deconjugation, and hydrophobicity) of 14 Lactococcus lactis strains isolated from raw milk and kefir grains were investigated. Strains originating from kefir grains had better characteristics in terms of antimicrobial activity and bile salt deconjugation, whereas strains from raw milk had better hydrophobicity and antioxidant activity characteristics. None of the strains were able to grow in the presence of bile salt and did not show amino acid decarboxylation or lipolytic activities. Biochemical and probiotic properties of L. lactis strains varied depending on the strain and some of these strains could be used as functional cultures depending on their properties. However, these strains did not possess all of the properties required to meet the definition of a probiotic.     

Molecular cloning,expression and characterization of bile salt hydrolase from Lactobacillus rhamnosus E9 strain

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.