Emulsions stabilised by whey protein microgel particles: towards food-grade Pickering emulsions.

We have investigated a new class of food-grade particles, whey protein microgels, as stabilisers of triglyceride-water emulsions. The sub-micron particles stabilized oil-in-water emulsions at all pH with and without salt. All emulsions creamed but exhibited exceptional resistance to coalescence. Clear correlations exist between the properties of the microgels in aqueous dispersion and the resulting emulsion characteristics. For conditions in which the particles were uncharged, fluid emulsions with relatively large drops were stabilised, whereas emulsions stabilized by charged particles contained smaller flocculated drops. A combination of optical microscopy of the drops and spectrophotometry of the resolved aqueous phase allowed us to estimate the interfacial adsorption densities of the particles using the phenomenon of limited coalescence. We deduce two classes of particle arrangement. Complete adsorption of the particles was obtained when they were neutral or when their charges were screened by salt resulting in at least one particle monolayer at the interface. By contrast, only around 50% of the particles adsorbed when they were charged with emulsion drops being covered by less than half a monolayer. These findings were supported by direct visualization of drop interfaces using cryo-scanning electron microscopy. Uncharged particles were highly aggregated and formed a continuous 2-D network at the interface. Otherwise particles organized as individual aggregates separated by particle-free regions. In this case, we suggest that some particles spread at the interface leading to the formation of a continuous protein membrane. Charged particles displayed the ability to bridge opposing interfaces of neighbouring drops to form dense particle disks protecting drops against coalescence; this is the main reason for the flocculation and stability of emulsions containing sparsely covered drops.

Foams stabilized by multilamellar polyglycerol ester self-assemblies.

The importance of surfactant self-assemblies in foam stabilization is well-known. The aim of the current study was to investigate the self-assemblies of the nonionic surfactant polyglycerol ester (PGE) in bulk solutions, at the interface and within foams, using a combined approach of small-angle neutron scattering, neutron reflectivity, and electron microscopy. PGE bulk solutions contain vesicles as well as open lamellar structures. Upon heating of the solutions the lamellar spacing increases, with significant differences in the presence of NaCl or CaCl(2) as compared to the standard solution. The adsorption of the multilamellar structures present in the bulk solutions lead to a multilayered film at the air-water interface. The ordering within this film was increased as a result of a 20% area compression mimicking a coalescence event. Finally, PGE foams were shown to be stabilized not only by strong interfacial films but also by agglomerated self-assemblies within the interstitial areas of the foams.

Multiscale characterization of individualized beta-lactoglobulin microgels formed upon heat treatment under narrow pH range conditions.

Aqueous dispersions of demineralized beta-lactoglobulin (beta-lg) were held at 85 degrees C for 15 min at a constant protein concentration of 1 wt % in the pH range of 3.0-7.0. This led to denatured protein content ranging from 20% (pH 3.0) to 90% (pH 5.0). The protein aggregates formed were characterized as to their stability to sedimentation (turbidity), morphology, size, surface charge, ANS surface hydrophobicity, and content in accessible thiol groups. Additionally, the changes in secondary structures of the protein upon heating were followed by Fourier transform infrared spectroscopy (FTIR). Stable dispersions (no sedimentation for 10 min) of individualized beta-lg microgels were obtained at specific pH 4.6 and 5.8, corresponding to an aggregation yield of about 80%. The width of the pH region leading to these microgels was 0.3 pH unit below or above the two specific pH values. Microgels were characterized by a spherical shape and remarkably low polydispersity in size (<0.2). Their z-average hydrodynamic diameter determined by dynamic light scattering (DLS) was between 160 and 220 nm, and their zeta-potential was +30 or -40 mV, depending on the initial pH before heating. Microgels obtained at pH 4.6 displayed a lower binding capacity for ANS and a lower content of accessible thiol groups as compared to those obtained at pH 5.8. Both types of microgels might therefore differ in their internal and interfacial structures. Between pH 4.6 and 5.8, large sedimenting protein particulates were obtained, whereas soluble aggregates were formed at pH <4.6 or >5.8. Interestingly, DLS experiments showed that before heating, beta-lg was mainly present in an oligomeric state at pH 4.6 and 5.8. This result was confirmed by FTIR measurements indicating the stronger contribution of the 1616-1624 cm(-1) spectral band corresponding to intermolecular beta-sheets in the pH range of 4.0-6.0. Upon heating, FTIR spectroscopy revealed that individualized microgels were obtained under pH conditions where a balance between attractive forces arising from protein unfolding leading to the formation of intermolecular beta-sheets (1616-1624 cm(-1 )band) and the repulsive electrostatic forces due to the initial protein net charge was achieved.

Monocomponent hexa- and dodecaethylene glycol succinyl-tocopherol esters: self-assembly structures, cellular uptake and sensitivity to enzyme hydrolysis.

We have chemically synthesized two water-soluble forms of tocopherol succinate linked via an ester bond to hexaethylene glycol and dodecaethylene glycol. The self-assembly structure of the former in water is vesicular, whereas the latter forms elongated micelles. We treated Caco-2 cells with these compounds in these physical forms, in addition to a mixed micelle form. The intact compounds were taken up into the cells, influenced by both the chain length and the physical structure. In addition, the tocopherol derivatives were also metabolized into tocopherol succinate and tocopherol inside the cell. The total hydrolysis and uptake into the cells was two-fold higher from tocopherol hexaethylene glycol succinate in the form of mixed micelles than in vesicular form as assessed by analyzing intracellular tocopherol and tocopherol succinate. The longer polyethylene glycol chain gave a higher intracellular tocopherol succinate/tocopherol ratio. The major intracellular esterase in Caco-2 cells is carboxyl esterase 1 (EC 3.1.1.1), and in silico modelling studies show that the position of docking and hence the site of hydrolysis is influenced by the chain length. The in silico prediction is consistent with the in vitro data, since a longer chain length is predicted to favour hydrolysis of the ester bond between the succinate and polyethylene glycol moieties.

Whey protein soluble aggregates from heating with NaCl: physicochemical, interfacial, and foaming properties.

Whey protein isolate was heat-treated at 85 degrees C for 15 min at pH ranging from 6.0 to 7.0 in the presence of NaCl in order to generate the highest possible amount of soluble aggregates before insolubility occurred. These whey protein soluble aggregates were characterized for composition, hydrodynamic diameter, apparent molecular weight, zeta-potential, surface hydrophobicity index, activated thiol group content, and microstructure. The adsorption kinetics and rheological properties (E', etad) of these soluble aggregates were probed at the air/water interface. In addition, the gas permeability of a single bubble stabilized by the whey protein soluble aggregates was determined. Finally, the foaming and foam-stabilizing properties of these aggregates were measured. The amount of whey protein soluble aggregates after heat treatment was increased from 75% to 95% from pH 6.0 to pH 7.0 by addition of 5 mM to 120 mM NaCl, respectively. These soluble aggregates involved major whey protein fractions and exhibited a maximum of activated thiol group content at pH > 6.6. The hydrodynamic radius and the surface hydrophobicity index of the soluble aggregates increased from pH 6.0 to 7.0, but the molecular weight and zeta-potential decreased. This loss of apparent density was clearly confirmed by microscopy as the soluble aggregates shifted from a spherical/compact structure at pH 6.0 to a more fibrillar/elongated structure at pH 7.0. Surface adsorption was faster for soluble aggregates formed at pH 6.8 and 7.0 in the presence of 100 and 120 mM NaCl, respectively. However, interfacial elasticity and viscosity measured at 0.01 Hz were similar from pH 6.0 to 7.0. Single bubble gas permeability significantly decreased for aggregates generated at pH > 6.6. Furthermore, these aggregates exhibited the highest foamability and foam liquid stability. Air bubble size within the foam was the lowest at pH 7.0. The coarsening exponent, alpha, fell within predicted values of 1/3 and 1/2, except for very dry foams where it was 1/5.

Interactions of water with roasted and ground coffee in the wetting process investigated by a combination of physical determinations.

Three complementary techniques were used in this study to investigate the physical changes during wetting of roasted and ground coffee. Scanning electron microscopy (SEM) was found to provide indirect evidence of the presence of liquid water in the coffee particles. The effect of wetting on coffee closed porosity was studied by helium pycnometry, and finally, particle sizing was used to determine the swelling kinetics of coffee after wetting. Due to the solubilization of compounds, the presence of liquid water could be detected in the coffee cells by SEM. The technique was then used to investigate different water contents; for example, for roasted and ground coffee containing 1 g of water per gram of coffee on a dry basis, liquid water was present in cells only at the periphery of approximately 1.0 mm diameter particles. Coffee closed porosity decreased with increasing water content, as evidenced by pycnometry. For roasted and ground coffee containing 1 g of water per gram of coffee, results showed a closed porosity lower that 0.1 cm3/g ( approximately 20% of the closed porosity measured in dry particles). The decrease of closed porosity may be attributed to both (1) water filling cells' lumen and (2) plasticization of cell wall polymers, resulting in the matrix relaxation and increase of helium accessibility to the pores. Water binding to the matrix polymers was further investigated by calorimetric measurements. The integration of the endothermic peak of freezing water showed that approximately 0.15 g of water/g of coffee is nonfreezable water, that is, water bound to the matrix polymers. Finally, the use of particle sizing showed that the average volume of the coffee particles with 1 g of water/g of coffee increased by up to 20-23% at 10-15 min following wetting. Moisture diffusion coefficients in coffee particles [( approximately 2-3) x 10(-11) m2 s(-1)] were approximated by fitting the swelling curves with a model of diffusion. The observed results may give information about homogeneity and the physical state of water in wetted roasted coffee and thus increase the understanding of the mechanisms of molecular mass transfer during extraction.

Cell surface-associated elongation factor Tu mediates the attachment of Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins.

The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.

The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533.

Lactobacillus johnsonii NCC 533 is a member of the acidophilus group of intestinal lactobacilli that has been extensively studied for their "probiotic" activities that include, pathogen inhibition, epithelial cell attachment, and immunomodulation. To gain insight into its physiology and identify genes potentially involved in interactions with the host, we sequenced and analyzed the 1.99-Mb genome of L. johnsonii NCC 533. Strikingly, the organism completely lacked genes encoding biosynthetic pathways for amino acids, purine nucleotides, and most cofactors. In apparent compensation, a remarkable number of uncommon and often duplicated amino acid permeases, peptidases, and phosphotransferase-type transporters were discovered, suggesting a strong dependency of NCC 533 on the host or other intestinal microbes to provide simple monomeric nutrients. Genome analysis also predicted an abundance (>12) of large and unusual cell-surface proteins, including fimbrial subunits, which may be involved in adhesion to glycoproteins or other components of mucin, a characteristic expected to affect persistence in the gastrointestinal tract (GIT). Three bile salt hydrolases and two bile acid transporters, proteins apparently critical for GIT survival, were also detected. In silico genome comparisons with the >95% complete genome sequence of the closely related Lactobacillus gasseri revealed extensive synteny punctuated by clear-cut insertions or deletions of single genes or operons. Many of these regions of difference appear to encode metabolic or structural components that could affect the organisms competitiveness or interactions with the GIT ecosystem.

Contribution of aggregation-promoting factor to maintenance of cell shape in Lactobacillus gasseri 4B2.

Aggregation-promoting factor (APF) was originally described as a protein involved in the conjugation and autoaggregation of Lactobacillus gasseri 4B2, whose corresponding apf gene was cloned and sequenced. In this report, we identified and sequenced an additional apf gene located in the region upstream of the previously published one. Inactivation of both apf genes was unsuccessful, indicating that APF function may be essential for the cell. Overproduction of APF proteins caused drastic alteration in the cell shape of this strain. These cells were irregular, twisted, enlarged, and tightly bound in unbreakable clumps of chains. Down-regulation of APF synthesis was achieved by cloning of the apf2 promoter region on a high-copy-number plasmid, which recruited a putative apf activator. As a consequence, the shape of the corresponding recombinant cells was elongated (filamentous) and cell division sites were no longer visible. None of the induced changes in APF production levels was clearly correlated with modifications of the aggregation phenotype. This report shows, for the first time, that APF proteins are mainly critical for L. gasseri 4B2 cell shape maintenance.