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. © 2014 the Partner Organisations

Inhibition of <i>Giardia intestinalis</i> by extracellular factors from Lactobacilli: an in vitro study

The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G1 phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimento

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

Internal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment

Covalently cross-linked whey protein microgels (WPM) were produced without the use of a chemical cross-linking agent. The hierarchical structure of WPM is formed by a complex interplay of heat denaturation, aggregation, electrostatic repulsion, and formation of disulfide bonds. Therefore, well-defined spherical particles with a diameter of several hundreds of nanometers and with relatively low polydispersity are formed in a narrow pH regime (5.8–6.2) only. WPM production was carried out on large scale by heating a protein solution in a plate-plate heat exchanger. Thereafter, the microgels were concentrated by microfiltration and spray dried into a powder. The spherical structure of the WPM was conserved in the powder. After re-dispersion, the microgel dispersions fully recovered their initial structure and size distribution. Due to the formation of disulfide bonds the particles were internally covalently cross-linked and were remarkably stable in a large pH range. Because of the pH dependent charge of the constituents the particles underwent significant size changes upon shifting the pH. Small angle X-ray scattering experiments were used to reveal their internal structure, and we report on the pH-induced structural changes occurring on different length scale. Our experiments showed that close analogies could be drawn to internally cross-linked and pH-responsive microgels based on weak polyelectrolytes. WPM also exhibited a pronounced swelling at pH values below the isoelectric point (IEP), and a collapse at the IEP. However, in contrast to classical microgels, WPM are not build up by simple polymer chains but possess a complex hierarchical structure consisting of strands formed by clusters of aggregated denatured proteins that act as primary building blocks. They were flexible enough to respond to changes of the environment, and were stable enough to tolerate pH values where the proteins were highly charged and the strands were stretched

Lactobacillus johnsonii La1 shares carbohydrate-binding specificities with several enteropathogenic bacteria

The carbohydrate-binding specificities of the probiotic lactic acid bacterium Lactobacillus johnsonii La1 (a health-beneficial bacterial strain able to be incorporated into the human intestinal microflora) were investigated in vitro. First various soluble complex carbohydrates were tested as potential inhibitors of the strain adhesion onto Caco-2 intestinal epithelial cells, and then bacterial binding to glycolipids immobilized on TLC plates was probed. Two major carbohydrate-binding specificities of Lactobacillus johnsonii La1 were identified. A first one for an Endo-H treated yeast cell wall mannoprotein carrying mainly O-linked oligomannosides, and a second one for the gangliotri- and gangliotetra-osylceramides (asialo-GM1). Similar carbohydrate-binding specificities are known to be expressed on cell surface adhesins of several enteropathogens, enabling them to adhere to the host gut mucosa. These findings corroborate the hypothesis that selected probiotic bacterial strains could be able to compete with enteropathogens for the same carbohydrate receptors in the gu

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

Inhibition of <i>Giardia intestinalis</i> by extracellular factors from Lactobacilli: an in vitro study

The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G1 phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimento