Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

Susceptibility and Adaptive Response to Bile Salts in Propionibacterium freudenreichii: Physiological and Proteomic Analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

Flow cytometric assessment of viability of lactic acid bacteria

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells; in exponential-phase cultures almost all cells were labeled, while 70 degrees C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications.     

Development of a novel probiotic delivery system based on microencapsulation with protectants

The establishment of the health-promoting benefits of probiotics is challenged by the antimicrobial bio-barriers throughout the host’s gastrointestinal (GI) tract after oral administration. Although microencapsulation has been frequently utilised to enhance the delivery of probiotics, microcapsules of sub-100 μm were found to be ineffective and therefore questioned as an effective delivery vehicle for viable probiotics despite the sensory advantage. In this study, four probiotics strains were encapsulated in chitosan-coated alginate microcapsules of sub-100 μm. Only a minor protective effect was observed from this original type of microcapsule. In order to enhance the survival of these probiotics, sucrose, a metabolisable sugar, and lecithin vesicles were added to the wall material. Both of the ingredients could be readily encapsulated with the probiotics, and protected them from stresses in the simulated GI fluids. The metabolisable sugar effectively increased the survival of the probiotics in gastric acid, mainly through energizing the membrane-bound F₁F₀-ATPases. The lecithin vesicles proved to alleviate the bile salt stress, and hence notably reduced the viability loss at the elevated bile salt concentrations. Overall, three out of the total four probiotics in the reinforced sub-100 μm microencapsules could significantly survive through an 8-h sequential treatment of the simulated GI fluids, giving less than 1-log drop in viable count. The most vulnerable strain of bifidobacteria also yielded a viability increase of 3-logs from this protection. In conclusion, the sub-100 μm microcapsules can be a useful vehicle for the delivery of probiotics, as long as suitable protectants are incorporated in the wall matrix.     

Flow Cytometric Assessment of Viability of Lactic Acid Bacteria

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells; in exponential-phase cultures almost all cells were labeled, while 70°C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications.     

Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 [1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3'-trimethylammoniumpropyl)-pyridinium tetraiodide] for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 10(5) cells ml(-1). The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products.     

Multiparametric flow cytometry and cell sorting for the assessment of viable,injured, and dead bifidobacterium cells during bile salt stress

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.     

Development of a Flow Cytometric Method To Analyze Subpopulations of Bacteria in Probiotic Products and Dairy Starters

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 {1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3′-trimethylammoniumpropyl)-pyridinium tetraiodide} for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 10⁵ cells ml⁻¹. The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products.     

Influence of total lipid concentration, bile salt:lecithin ratio, and cholesterol content on inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile.

We modified classic equilibrium dialysis methodology to correct for dialysant dilution and Donnan effects, and have systematically studied how variations in total lipid concentration, bile salt (taurocholate):lecithin (egg yolk) ratio, and cholesterol content influence inter-mixed micellar/vesicular (non-lecithin-associated) concentrations (IMC) of bile salts (BS) in model bile. To simulate large volumes of dialysant, the total volume (1 ml) of model bile was exchanged nine times during dialysis. When equilibrium was reached, dialysate BS concentrations plateaued, and initial and final BS concentrations in the dialysant were identical. After corrections for Donnan effects, IMC values were appreciably lower than final dialysate BS concentrations. Quasielastic light scattering was used to validate these IMC values by demonstrating that lipid particle sizes and mean scattered light intensities did not vary when model biles were diluted with aqueous BS solutions of the appropriate IMC. Micelles and vesicles were separated from cholesterol-supersaturated model bile, utilizing high performance gel chromatography with an eluant containing the IMC. Upon rechromatography of micelles and vesicles using an identical IMC, there was no net transfer of lipid between micelles and vesicles. To simulate dilution during gel filtration, model biles were diluted with 10 mM Na cholate, the prevailing literature eluant, resulting in net transfer of lipid between micelles and vesicles, the direction of which depended upon total lipid concentration and BS/lecithin ratio. Using the present methodology, we demonstrated that inter-mixed micellar/vesicular concentrations (IMC) values increased strongly (5 to 40 mM) with increases in both bile salt (BS):lecithin ratio and total lipid concentration, whereas variations in cholesterol content had no appreciable effects. For model biles with typical physiological biliary lipid compositions, IMC values exceeded the critical micellar concentration of the pure BS, implying that in cholesterol-supersaturated biles, simple BS micelles coexist with mixed BS/lecithin/cholesterol micelles and cholesterol/lecithin vesicles. We believe that this methodology allows the systematic evaluation of IMC values, with the ultimate aim of accurately separating micellar, vesicular, and potential other cholesterol-carrying particles from native bile.     

Different probiotic properties for Lactobacillus fermentum strains isolated from swine and poultry

Systematic procedures were used to evaluate the probiotic properties of Lactobacillus fermentum (L. fermentum) strains isolated from swine and poultry. The major properties included their capabilities to adhere to the intestinal epithelium of swine and poultry, the inhibition on pathogenic bacteria, and their tolerance to the gastric juice and bile salts. Results showed that L. fermentum strains from poultry digestive tract showed better adherence to the swine intestine and chicken crop epithelial cells as compared to those strains from the swine origin. In addition, six strains from poultry and one strain from swine showed adhesion specificity to their own intestinal epithelium. Four poultry isolates and one swine isolate were able to adhere to the epithelial cells from both swine and chicken. For gastric juice and bile tolerance, most of the strains isolated from swine or poultry were acid tolerant but less strains were bile intolerant. The spent culture supernatant (SCS) of these L. fermentum strains showed antagonistic effect against the indicator bacteria, such as Escherichia coli, Salmonella spp., Shigella sonnei and some enterotoxigenic Staphylococcus aureus. From the above studies, some L. fermentum strains isolated from poultry were found to have the probiotic properties required for use in animal feed supplement. This study suggested that poultry digestive tract may serve as potential source for the isolation of probiotic lactic acid bacteria.     

Characterization of Intestinal Lactobacillus reuteri Strains as Potential Probiotics

This study was conducted to evaluate the probiotic properties of Lactobacillus reuteri isolated from human infant feces (less than 3?months). Out of thirty-two representative L. reuteri strains isolated from the infant human feces, nine isolates (i.e. LR5, LR6, LR9, LR11, LR19, LR20, LR25, LR26 and LR34) showed survival in acid, bile and simulated stomach?Cduodenum passage conditions, indicating their high tolerance to gastric juice, duodenal juice and bile environments. The nine isolates did not show strong hydrophobic properties because the percentages of adhesion to the apolar solvent, n-hexadecane, did not exceed 40%, showing that their surfaces were rather hydrophilic. Functionality of these nine probiotic isolates was supported by their antagonistic activity and their ability to deconjugate bile salts. The safety of the nine indigenous L. reuteri isolates was supported by the absence of transferable antibiotic resistance determinants, DNase activity, gelatinase activity and hemolysis. The results obtained so far suggest that the nine strains are resistant to low pH, bile salts and duodenum juice, so they could survive when passing through the upper part of the gastrointestinal tract and fulfill their potential probiotic action in the host organism. According to these results, the L. reuteri strains isolated from human infant feces possess interesting probiotic properties that make them potentially good candidates for probiotics.     

Probiotic Potential of Pediococcus pentosaceus CRAG3: A New Isolate from Fermented Cucumber

A novel strain of lactic acid bacterium isolated from fermented cucumber was selected due to its high glucansucrase activity. It was identified on the basis of 16S rRNA sequence analysis as Pediococcus pentosaceus CRAG3 (GenBank accession number JX679020). The isolate was round shaped, Gram positive, and catalase negative displaying typical features of lactic acid bacterium. It produced 145 ± 3.27 mg lactic acid per ml of cell-free supernatant. It showed ability to ferment carbohydrates such as sucrose, dextrose, and arabinose; showed resistance to antibiotics such as ciprofloxacin, kanamycin, vancomycin; displayed acid production in triple sugar iron agar test and non-motile nature. Interestingly, the isolate also displayed potential probiotic properties such as hydrophobicity, autoaggregation, coaggregation, and in vitro cell adhesion ability. It exhibited resistance against lysozyme and simulated gastric juice at pH 3.0 with 75 and 58 % survival, respectively. It also showed tolerance toward 0.3 %, w/v bile salts with 73 % survival and ability to deconjugate bile salts. The isolate exhibited antibacterial activity and ability to utilize prebiotics such as inulin and raffinose. These results indicate both probiotic property and glucansucrase-producing ability of P. pentosaceus CRAG3.     

Mathematical model of biliary lipid secretion: a quantitative analysis of physiological and biochemical data from man and other species

We propose a simple mathematical model to account for the coupling of secretion rates of bile salts, lecithin, and cholesterol into bile. The model assumes that: 1) molecules of "biliary" lecithin and cholesterol enter a functional compartment located in the endoplasmic reticulum of the hepatocyte from which they are secreted into bile, and in the case of cholesterol, also catabolized to bile salts; 2) the rates at which lecithin and cholesterol enter the "secretory" compartment are regulated independently by feedback loops that control their synthesis and/or uptake; 3) lecithin secretion is coupled by an unknown transport mechanism, possibly micellar or vesicular, to the flux of bile salts passing through the compartment; 4) cholesterol secretion is coupled by a similar mechanism to lecithin secretion and not to bile salt secretion directly; and 5) bile salt synthesis is proportional to the cholesterol content of the compartment. The model predicts that in the steady state the dependences, lecithin secretion vs bile salt secretion; cholesterol secretion vs lecithin secretion; and cholesterol secretion vs bile salt secretion, will all have the form of rectangular hyperbolae. Four independent parameters related to the postulated mechanisms of biliary lipid synthesis, uptake, and transport determine the quantitative features of these hyperbolae. These four "secretion parameters" also determine how the biliary lipid composition of hepatic and "fasting" gallbladder bile varies with bile salt secretion rate. A quantitative analysis of biochemical and physiological data on biliary lipid secretion in rat, dog, and man confirms the general predictions of the model. Deductions of the secretion parameters are made for each species and are compared with other relevant data on biliary lipid metabolism. From this analysis, we offer new insights into: i) the species differences in biliary lipid secretion and bile composition; ii) the influence of obesity on biliary lipid secretion in man; and iii) the causes of cholesterol super-saturation in fasting gallbladder bile.     

Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC₄(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.     

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

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

Cholesterol gallstone dissolution in bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms

Using compressed discs and microcrystals of cholesterol monohydrate, we evaluated the mechanisms and kinetics of dissolution in conjugated bile salt-lecithin solutions. In stirred conjugated ursodeoxycholate-lecithin and cheno-deoxycholate-lecithin solutions, dissolution of 10,000-psi discs was micellar and linear with time for 10 hours. The dissolution rate constants (k) decreased in proportion to the lecithin content and dissolution rates and k values were appreciably smaller in conjugated ursodeoxycholate-lecithin solutions. After dissolution for 5 to 10 days the discs incubated with ursodeoxycholate-lecithin systems became progressively transformed into macroscopic liquid crystals. Unstirred dissolution of 3,000-psi discs in "simulated" human bile containing physiological lecithin concentrations gave apparent k values that decreased in the following order: ursodeoxycholate-rich >/= chenodeoxycholate-rich > normal. In most cases the discs incubated with ursodeoxycholate-rich bile became covered with a microscopic liquid-crystalline layer. With 20-25 moles % lecithin, these layers eventually dispersed into the bulk solution as microscopic vesicles. During dissolution of microcrystalline cholesterol in conjugated ursodeoxycholate-lecithin systems, a bulk liquid-crystalline phase formed rapidly (within 12 hours) and the final cholesterol solubilities were greater than those in conjugated chenodeoxycholate-lecithin micellar systems. Prolonged incubation of cholesterol microcrystals with pure lecithin or lecithin plus bile salt liposomes did not reproduce these effects. Condensed ternary phase diagrams of conjugated ursodeoxycholate-lecithin-cholesterol systems established that cholesterol-rich liquid crystals constituted an equilibrium precipitate phase that coexisted with cholesterol monohydrate crystals and saturated micelles under physiological conditions. Similar phase dissolution-relationships were observed at physiological lecithin-bile salt ratios for a number of other hydrophilic bile salts (e.g., conjugated ursocholate, hyocholate, and hyodeoxycholate). In contrast, liquid crystals were not observed in conjugated chenodeoxycholate-lecithin-cholesterol systems except at high (nonphysiological) lecithin contents. Based on these and other results we present a molecular hypothesis for cholesterol monohydrate dissolution by any bile salt-lecithin system and postulate that enrichment of bile with highly hydrophilic bile salts will induce crystalline cholesterol dissolution by a combination of micellar and liquid crystalline mechanisms. Since bile salt polarity can be measured and on this basis the ternary phase diagram deduced, we believe that the molecular mechanisms of cholesterol monohydrate dissolution as well as the in vivo cholelitholytic potential of uncommon bile salts can be predicted.-Salvioli, G., H. Igimi, and M. C. Carey. Cholesterol gallstone dissolution in bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms.     

Active efflux of bile salts by Escherichia coli.

Enteric bacteria such as Escherichia coli must tolerate high levels of bile salts, powerful detergents that disrupt biological membranes. The outer membrane barrier of gram-negative bacteria plays an important role in this resistance, but ultimately it can only retard the influx of bile salts. We therefore examined whether E. coli possessed an energy-dependent efflux mechanism for these compounds. Intact cells of E. coli K-12 appeared to pump out chenodeoxycholate, since its intracellular accumulation increased more than twofold upon deenergization of the cytoplasmic membrane by a proton conductor. Growth inhibition by bile salts and accumulation levels of chenodeoxycholate increased when mutations inactivating the acrAB and emrAB gene clusters were introduced. The AcrAB system especially appeared to play a significant role in bile acid efflux. However, another efflux system(s) also plays an important role, since the accumulation level of chenodeoxycholate increased strongly upon deenergization of acrA emrB double mutant cells. Everted membrane vesicles accumulated taurocholate in an energy-dependent manner, apparently consuming delta pH without affecting delta psi. The efflux thus appears to be catalyzed by a proton antiporter. Accumulation by the everted membrane vesicles was not decreased by mutations in acr and emrB genes and presumably reflects activity of the unknown system seen in intact cells. It followed saturation kinetics with Vmax and Km values in the neighborhood of 0.3 nmol min(-1) mg of protein(-1) and 50 microM, respectively.     

Effect of skim milk-alginate beads on survival rate of bifidobacteria

In this study, an attempt was made to increase the survival rate of bifidobacteria entrapped in alginate in the gastrointestinal tract, and to investigate the potential industrial applications, for example lyophilized capsules and yogurt. First, the protective effect of various food additives on bifidobacterial survivability was determined after exposure to simulated gastric juices and bile salts. The additives used in this study were skim milk (SM), poly dextrose (PD), soy fiber (SF), yeast extract (YE), chitosan (CS), κ-carageenan (κ-C) and whey, which were added at 0.6% concentration (w/v) to 3% alginate-bifidobacterial solution. In the simulated gastric juices and bile salts, the protective effect of 0.6% skim milk-3% alginate (SM-A) beads on the survival rate of bifidobacteria proved to be higher than the other additives. Second, the hydrogen ion permeation was detected through SM-A vessel without bifidobacterial cells at different SM concentrations (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%). There were no differences in terms of the pH decrease in SM-A vessels at 0.6%, 0.8%, and 1.0% (w/v) SM concentrations. The survival rate of bifidobacteria in SM-A beads would appear to be related to the SM buffering capacity against hydrogen ions and its tendency to reduce the pore size of bead. In this experiment, the survival rate of bifidobacteria entrapped in beads containing 0.6% SM showed the highest viability after exposure to simulated gastric juices for 3 h, thereby indicating that 0.6% SM is the optimum concentration for 3% alginate bead preparation. Third, the effect of SM-A beads on the freeze-drying and yogurt storage for 10 days was investigated. SM-A beads were found to be more efficient for freeze drying and yogurt storage than untrapped cells and the alginate bead. Consequently, the survival rate of bifidobacteria entrapped in SM-A beads was increased in simulated gastric juices, bile salts and probiotic products such as lyophilized capsules and yogurt, SM-A beads can be expected to produce high value probiotic products.