Effect of calcium ions on in vitro pellicle formation from parotid and whole saliva.

The salivary pellicle is a protein-rich, bacteria-free, self-assembling film that adsorbs to all surfaces within the oral cavity. The pellicle has numerous functions that are vital for maintaining oral health. Currently however, there are no commercially available artificial salivas that accurately mimic the complex film forming properties (i.e. film thickness and viscoelasticity) of human saliva. To understand these properties further we have examined the in vitro formation of the salivary pellicle, by adsorbing stimulated parotid saliva (PS) and whole mouth saliva (WMS) from 14 healthy volunteers, onto oxidised silicon surfaces, using a quartz crystal microbalance with dissipation monitoring (QCMD) and a dual polarisation interferometer (DPI). A dramatic impact on the hydrated mass, polymer mass, thickness and polymer concentration of the pellicle for both WMS and PS was observed when the natural calcium concentration of the respective salivas was increased from 0 mM to 10mM. In addition, QCMD data showed that on addition of 10mM calcium the salivary pellicle formed by both PS and WMS became more predominantly elastic. The results presented here also suggest that calcium can easily diffuse in and out of the pellicle, permitting free calcium exchange between the saliva and the adsorbed pellicle under physiological conditions, which may potentially facilitate the mineralisation of enamel.

The effect of physiological conditions on the surface structure of proteins: setting the scene for human digestion of emulsions.

Understanding and manipulating the interfacial mechanisms that control human digestion of food emulsions is a crucial step towards improved control of dietary intake. This article reports initial studies on the effects of the physiological conditions within the stomach on the properties of the film formed by the milk protein (ß-lactoglobulin) at the air-water interface. Atomic force microscopy (AFM), surface tension and surface rheology techniques were used to visualize and examine the effect of gastric conditions on the network structure. The effects of changes in temperature, pH and ionic strength on a preformed interfacial structure were characterized in order to simulate the actual digestion process. Changes in ionic strength had little effect on the surface properties. In isolation, acidification reduced both the dilatational and the surface shear modulus, mainly due to strong repulsive electrostatic interactions within the surface layer and raising the temperature to body temperature accelerated the rearrangements within the surface layer, resulting in a decrease of the dilatational response and an increase of surface pressure. Together pH and temperature display an unexpected synergism, independent of the ionic strength. Thus, exposure of a pre-formed interfacial ß-lactoglobulin film to simulated gastric conditions reduced the surface dilatational modulus and surface shear moduli. This is attributed to a weakening of the surface network in which the surface rearrangements of the protein prior to exposure to gastric conditions might play a crucial role.

Disruption of viscoelastic beta-lactoglobulin surface layers at the air-water interface by nonionic polymeric surfactants.

Nonequilibrium interfacial layers formed by competitive adsorption of beta-lactoglobulin and the nonionic triblock copolymer PEO99-PPO65-PEO99 (F127) to the air-water interface were investigated in order to explain the influence of polymeric surfactants on protein film surface rheology and foam stability. Surface dilatational and shear rheological methods, surface tension measurements, dynamic thin-film measurements, diffusion measurements (from fluorescence recovery after photo bleaching), and determinations of foam stability were used as methods. The high surface viscoelasticity, both the shear and dilatational, of the protein films was significantly reduced by coadsorption of polymeric surfactant. The drainage rate of single thin films, in the presence of beta-lactoglobulin, increased with the amount of added F127, but equilibrium F127 films were found to be thicker than beta-lactoglobulin films, even at low concentration of the polymeric surfactant. It is concluded that the effect of the nonionic triblock copolymer on the interfacial rheology of beta-lactoglobulin layers is similar to that of low molecular weight surfactants. They differ however in that F127 increases the thickness of thin liquid films. In addition, the significant destabilizing effect of low molecular weight surfactants on protein foams is not found in the investigated system. This is explained as due to long-range steric forces starting to stabilize the foam films at low concentrations of F127.

In situ measurement of the displacement of protein films from the air/water interface by surfactant.

The displacement of spread protein films from the air/water interface by surfactant was followed using Brewster angle microscopy (BAM) and interfacial rheology. The displacement of beta-lactoglobulin and beta-casein by a nonionic surfactant was monitored as a function of both surface pressure and time. In both cases, protein displacement occurred over the same surface pressure range that had been observed previously by atomic force microscopy (AFM). In the case of the beta-lactoglobulin, surfactant domains grew large enough in the protein film to be visible in the BAM images. The shapes of the domains were very similar to those seen previously by AFM in the late stages of displacement. The results from both proteins confirm the results published previously while highlighting some implications for the application of the "orogenic" model of displacement for large protein films. The surface rheological data showed that the beta-lactoglobulin/surfactant mixed film retained much of its elasticity until the latter stages of displacement. This indicates that at least in the early stages of displacement, the mixed film was dominated by the behavior of the protein in the film.

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin.

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.

Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy.

The molecular weight of the extracellular polysaccharide (CR1/4) produced by Acetobacter xylinum strain CR1/4 has been shown to be dependent upon growth conditions. Under normal growth conditions a high molecular weight polysaccharide ( > 1 x 10(6) Da) is produced. Maintaining the pH at 5 results in an order of magnitude increase in the total yield of polysaccharide, but also an order of magnitude decrease in molecular weight. Analysis of the CR1/4 polysaccharides by the techniques of atomic force microscopy and static light scattering suggests that they are double helices. In solution the molecules behave as stiff coils with a Kuhn statistical segment length of 325 nm.

Evidence for intermolecular binding between deacetylated acetan and the glucomannan konjac mannan.

Binary mixtures of deacetylated acetan and konjac mannan form thermoreversible gels under conditions for which the individual components do not gel. Such synergistic behaviour is normally attributed to intermolecular binding between the two polysaccharides. X-ray diffraction data obtained from oriented fibres prepared from deacetylated acetan-konjac mannan gels provides direct evidence for intermolecular binding between the two polysaccharides. The novel heterotypic junction zones appear to be six-fold helices with a pitch of 5.6 +/- 0.1 nm.

Effect of o-acyl substituents on the functional behaviour of Rhizobium meliloti succinoglycan.

The effects of selective removal of acetyl or succinyl substituents on the functionality of succinoglycan polysaccharide have been studied by comparing the behaviour of the polysaccharides isolated from native Rhizobium meliloti strain Rm1021, and genetically modified R. meliloti species. Removal of the succinyl groups was found to dramatically improve pseudoplasticity of the aqueous succinoglycan samples and also increase the cooperativity of the order-disorder transition exhibited by the polysaccharide. Removal of the acetyl substituent led to a decrease in the order-disorder transition temperature, whereas the removal of the succinyl groups led to an increase.

Mixed iota-kappa carrageenan gels.

Studies have been made of the gelation of mixtures of iota and kappa carrageenan. Differential scanning calorimetry (DSC) studies suggest that the iota and kappa components gel independently of each other. The DSC data conflict with evidence presented for the currently accepted Domain model for carrageenan gelation (Morris, E.R., Rees, D.A. and Robinson, G.J. J. Mol. Biol. 1980; 138:349). In particular, the present data suggest that iota carrageenan gels show thermoreversible setting and melting behaviour associated with the coil-helix transition. Analysis of rheological data favours an interpenetrating network (IPN) model for gelation of the mixtures.

Heterologous protein secretion by Aspergillus niger growing in submerged culture as dispersed or aggregated mycelia.

The secreted production of a heterologous enzyme, hen egg-white lysozyme, by Aspergillus niger was studied in shake flasks containing media of different initial viscosities. Raising the viscosity of the medium by addition of polyvinylpyrrolidone (PVP) brought about a transition in the form of growth from aggregated mycelia (pellets) to dispersed mycelia. The specific yield of lysozyme in cultures containing an initial concentration of 5% (w/v) starch was 8 mg lysozyme/g dry weight. Addition of 2% (w/v) PVP to the medium resulted in a specific yield of 14 mg lysozyme/g dry weight.

Observation of the helical structure of the bacterial polysaccharide acetan by atomic force microscopy.

A method has been developed that has been found to give reproducible images of uncoated polysaccharides by Atomic Force Microscopy (AFM). Aqueous solutions of the polysaccharide are deposited as drops onto freshly cleaved mica surfaces, air dried, and then imaged under butanol. The method has been used to obtain images of the bacterial polysaccharide acetan. In regions within the deposited sample, where the molecules are aligned side-by-side, it has been possible to observe a periodic structure along the polysaccharide chain, attributable to the helical structure of acetan.

Physicochemical characterization of an acetan variant secreted by Acetobacter xylinum strain CR1/4.

Chemical mutagenesis has been used to produce mutants of Acetobacter xylinum NRRL B42 that are cellulose-negative and that produce variants of the acetan structure deficient in the side-chain sugar residues. The product of A. xylinum strain CR1/4 has been shown to possess a tetrasaccharide repeat unit with the side chain terminating in glucuronic acid. X-ray diffraction studies of oriented fibres suggest that the polysaccharide CR1/4 forms a fivefold helix with a pitch of 4.8 nm. Light-scattering studies on CR1/4 solutions suggest a molecular weight of 1.2 x 10(6) with radii of gyration values of 86 nm (aqueous solution) and 67 nm (0.1 M NaCl solution). The magnitude of the measured radii of gyration and the shape of the Holtzer plots suggest that CR1/4 can be described as a stiff coil. Preliminary differential scanning calorimetry data show melting behaviour consistent with order-disorder transitions of a charged helical structure. Rheological studies have revealed new synergistic interactions of CR1/4 with locust bean gum. Comparative studies of acetan and CR1/4 show that decreasing the length of the side chain enhances the solution viscosity.