Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties

This study evaluated the use of a bile-salt-hydrolyzing Lactobacillus fermentum strain as a probiotic with potential hypocholesterolemic properties. The effect of L. fermentum on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the Lactobacillus strain was evaluated. Administration of L. fermentum resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial, Lactobacillus, Bacteroides, and clostridial populations through L. fermentum supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the L. fermentum strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that L.Fermentum KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.

Bile salt composition is secondary to bile salt concentration in determining hydrocortisone and progesterone solubility in intestinal mimetic fluids.

Simulated intestinal fluids (SIFs) used to assay the solubility of orally administered drugs are typically based on a single bile salt; sodium taurocholate (STC). The aim of this study was to develop mimetic intestinal fluids with a closer similarity to physiological fluids than those reported to date by developing a mixed bile salt (MBS) system (STC, sodium glycodeoxycholate, sodium deoxycholate; 60:39:1) with different concentrations of lecithin, the preponderant intestinal phospholipid. Hydrocortisone and progesterone were used as model drugs to evaluate systematically the influence of SIF composition on solubility. Increasing total bile salt concentration from 0 to 30 mM increased hydrocortisone and progesterone solubility by 2- and ∼25-fold, respectively. Accordingly, higher solubilities were measured in the fed-state compared to the fasted-state SIFs. Progesterone showed the greatest increases in solubility in STC and MBS systems (2-7-fold) compared to hydrocortisone (no significant change; P>0.05) as lecithin concentration was increased. Overall, MBS systems gave similar solubility profiles to STC. In conclusion, the addenda of MBS and lecithin were found to be secondary to the influence of BS concentration. These data provide a foundation for the design of more bio-similar media for pivotal decision-guiding assays in drug development and quality control settings.

Solution calorimetry as a tool for investigating drug interaction with intestinal fluid

Solution calorimetry offers a reproducible technique for measuring the enthalpy of solution (ΔsolH) of a solute dissolving into a solvent. The ΔsolH of two solutes, propranolol HCl and mannitol were determined in simulated intestinal fluid (SIF) solutions designed to model the fed and fasted states within the gut, and in Hanks’ balanced salt solution (HBSS) of varying pH. The bile salt and lipid within the SIF solutions formed mixed micelles. Both solutes exhibited endothermic reactions in all solvents. The ΔsolH for propranolol HCl in the SIF solutions differed from those in the HBSS and was lower in the fed state than the fasted state SIF solution, revealing an interaction between propranolol and the micellar phase in both SIF solutions. In contrast, for mannitol the ΔsolH was constant in all solutions indicating minimal interaction between mannitol and the micellar phases of the SIF solutions. In this study, solution calorimetry proved to be a simple method for measuring the enthalpy associated with the dissolution of model drugs in complex biological media such as SIF solutions. In addition, the derived power–time curves allowed the time taken for the powdered solutes to form solutions to be estimated.

Microencapsulation of probiotics for gastrointestinal delivery

The administration of probiotic bacteria as nutraceuticals is an area that has rapidly expanded in recent years, with a global market worth $32.6 billion predicted by 2014. Many of the health promoting claims attributed to these bacteria are dependent on the cells being both viable and sufficiently numerous in the intestinal tract. The oral administration of most bacteria results in a large loss of viability associated with passage through the stomach, which is attributed to the high acid and bile salt concentrations present. This loss of viability effectively lowers the efficacy of the administered supplement. The formulation of these probiotics into microcapsules is an emerging method to reduce cell death during GI passage, as well as an opportunity to control release of these cells across the intestinal tract. The majority of this technology is based on the immobilization of bacteria into a polymer matrix, which retains its structure in the stomach before degrading and dissolving in the intestine, unlike the diffusion based unloading of most controlled release devices for small molecules. This review shall provide an overview of progress in this field as well as draw attention to areas where studies have fallen short. This will be followed by a discussion of emerging trends in the field, highlighting key areas in which further research is necessary.

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.

Capillary flow behavior of worm-like micelles studied by small-angle X-ray scattering and small angle light scattering

A novel capillary flow device has been developed and applied to study the orientation of worm-like micelles, among other systems. Small-angle X-ray scattering (SAXS) data from micelles formed by a Pluronic block copolymer in aqueous salt solution provides evidence for the formation of worm-like micelles, which align under flow. A transition from a rod-like form factor to a less persistent conformation is observed under flow. Flow alignment of worm-like micelles formed by the low molar mass amphiphile system cetyl pyridinium chloride+sodium salicylate is studied for comparative purposes. Here, inhomogenous flow at the micron scale is revealed by streaks in the small-angle light scattering pattern perpendicular to the flow direction. Copyright (c) 2006 John Wiley & Sons, Ltd.

Molecular dynamics simulations of threadlike cetyltrimethylammonium chloride micelles: effects of sodium chloride and sodium salicylate salts

We use atomistic molecular dynamics simulations to probe the effects of added sodium chloride (NaCl) and sodium salicylate (NaSal) salts on the spherical-to-threadlike micelle shape transition in aqueous solutions of cetyltrimethylammonium chloride (CTAC) surfactants. Long threadlike micelles are found to be unstable and break into spherical micelles at low concentrations or NaCl, but remain stable for 20 ns above a threshold value of [NaCl] approximate to 3.0 M, which is about 2.5 times larger than the experimental salt concentration at which the transition between spherical and rodlike micelles occurs. The chloride counterions associate weakly oil the surface of the CTAC micelles with the degree of counterion dissociation decreasing slightly with increasing [NaCl] on spherical micelles, but dropping significantly on the threadlike micelles tit high [NaCl]. This effect indicates that the electrolyte ions drive the micellar shape transition by screening the electrostatic repulsions between the micellar headgroups, The aromatic salicylate counterions, on the other hand, penetrate inside the micelle with their hydrophilic groups staying in the surfactant headgroup region and the hydrophobic groups partially embedded into the hydrophobic core of the micelle. The strong association of the salicylate ions with the surfactant headgroups leads to dense packing of the surfactant molecules, which effectively reduces the surface area per surfactant, and increases intramicellar ordering of the surfactant headgroups, favoring the formation of long threadlike micelles. Simulation predictions of the geometric and electrostatic properties of the spherical and threadlike micelles are in good agreement with experiments.

Salt-induced hydrogelation of functionalised-dipeptides at high pH

Addition of divalent cations to a solution of a naphthalene-diphenylalanine that forms worm-like micelles at high pH results in the formation of a rigid, self-supporting hydrogel

Self-assembly of a peptide amphiphile: transition from nanotape fibrils to micelles

A thermal transition is observed in the peptide amphiphile C16-KTTKS (TFA salt) from nanotapes at 20 degrees C to micelles at higher temperature (the transition temperature depending on concentration). The formation of extended nanotapes by the acetate salt of this peptide amphiphile, which incorporates a pentapeptide from type I procollagen, has been studied previously [V. Castelletto et al., Chem. Commun., 2010, 46, 9185]. Here, proton NMR and SAXS provide evidence for the TFA salt spherical micelles at high temperature. The phase behavior, with a Krafft temperature separating insoluble aggregates (extended nanotapes) at low temperature from the high temperature micellar phase resembles that for conventional surfactants, however this has not previously been reported for peptide amphiphiles.

Salt marsh morphodynamics: An investigation of tidal flows and marsh channel equilibrium

Although numerous field studies have evaluated flow and transport processes in salt marsh channels, the overall role of channels in delivering and removing material from salt marsh platforms is still poorly characterised. In this paper, we consider this issue based on a numerical hydrodynamic model for a prototype marsh system and on a field survey of the cross-sectional geometry of a marsh channel network. Results of the numerical simulations indicate that the channel transfers approximately three times the volume of water that would be estimated from mass balance considerations alone. Marsh platform roughness exerts a significant influence on the partitioning of discharge between the channel and the marsh platform edge, alters flow patterns on the marsh platform due to its effects on channel-to-platform transfer and also controls the timing of peak discharge relative to marsh-edge overtopping. Although peak channel discharges and velocities are associated with the flood tide and marsh inundation, a larger volume of water is transferred by the channel during ebb flows, a portion of which transfer takes place after the tidal height is below the marsh platform. Detailed surveys of the marsh channels crossing a series of transects at Upper Stiffkey Marsh, north Norfolk, England, show that the total channel cross-sectional area increases linearly with catchment area in the inner part of the marsh, which is consistent with the increase in shoreward tidal prism removed by the channels. Toward the marsh edge, however, a deficit in the total cross-sectional area develops, suggesting that discharge partitioning between the marsh channels and the marsh platform edge may also be expressed in the morphology of marsh channel systems.

Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in European eels Anguilla anguilla from United Kingdom estuaries

A total of 94 European eels (Anguilla anguilla) were collected from five estuaries in the UK. The deconjugated metabolites of polycyclic aromatic hydrocarbons (PAHs) in the bile of the eels were separated using HPLC. Six PAH metabolites were identified: 1-hydroxy (1-OH) metabolites of phenanthrene, pyrene and chrysene; and the 1-OH, 3-OH and 7,8 dihydrodiol metabolites of benzo[a]pyrene (BaP). The mean concentration of the six metabolites was greatest in eels from the Tyne (49 muM) followed by the Wear (33 muM), Tees (19 muM), Thames (4 muM) and Severn (2 muM) estuaries. Although 1-OH pyrene was always the dominant compound, there were significant differences (P<0.05) between sites and between estuaries for some metabolites. Normalising the molar concentration of the bile metabolites to the bile biliverdin absorbance reduced sample variation. When the metabolites identified were-each expressed as a percentage of the total detected, the metabolite profile was characteristic for each estuary. (C) 2002 Elsevier Science B.V. All rights reserved.

Hydrolysis of N2O5 on sub-micron mineral salt aerosols

The kinetics of reactive uptake of gaseous N2O5 on sub-micron aerosol particles composed of aqueous ammonium sulfate, ammonium hydrogensulfate and sodium nitrate has been investigated. Uptake was measured in a laminar flow reactor, coupled with a differential mobility analyser (DMA) to obtain the aerosol size distribution, with N2O5 detection using NO chemiluminescence. FTIR spectroscopy was used to obtain information about the composition and water content of the aerosol particles under the conditions used in the kinetic measurements. The aerosols were generated by the nebulisation of aqueous salt solutions. The uptake coefficient on the sulfate salts was in the range [gamma]=0.0015 to 0.033 depending on temperature, humidity and phase of the aerosol. On sodium nitrate aerosols the values were much lower, [gamma]<0.001, confirming the inhibition of N2O5 hydrolysis by nitrate ions. At high humidity (>50% r.h.) the uptake coefficient on liquid sulfate aerosols is independent of water content, but at lower humidity, especially below the efflorescence point, the reactivity of the aerosol declines, correlating with the lower water content. The lower uptake rate on solid aerosols may be due to limitations imposed by the liquid volume in the particles. Uptake on sulfate aerosols showed a negative temperature dependence at T>290 K but no significant temperature dependence at lower temperatures. The results are generally consistent with previous models of N2O5 hydrolysis where the reactive intermediate is NO2+ produced by autoionisation of nitrogen pentoxide in the condensed phase.

Reactive uptake coefficients for heterogeneous reaction of N2O5 with submicron aerosols of NaCl and natural sea salt

The kinetics of uptake of gaseous N2O5 on submicron aerosols containing NaCl and natural sea salt have been investigated in a flow reactor as a function of relative humidity (RH) in the range 30-80% at 295±2K and a total pressure of 1bar. The measured uptake coefficients, γ, were larger on the aerosols containing sea salt compared to those of pure NaCl, and in both cases increased with increasing RH. These observations are explained in terms of the variation in the size of the salt droplets, which leads to a limitation in the uptake rate into small particles. After correction for this effect the uptake coefficients are independent of relative humidity, and agree with those measured previously on larger droplets. A value of γ=0.025 is recommended for the reactive uptake coefficient for N2O5 on deliquesced sea salt droplets at 298K and RH>40%.