Pectin Conformation in Solution.

The interplay of degree of methylesterification (DM), pH, temperature, and concentration on the macromolecular interactions of pectin in solution has been explored. Small-angle X-ray scattering complemented by atomic force microscopy and molecular dynamics was employed to probe chain dimensions and solution structure. Two length scales have been observed with the first level of structure characterising chain clusters with sizes ranging between 100-200 nm. The second level of structure arises from single biopolymer chains with a radius of gyration between ∼6 and 42 nm. The development of a range of macromolecular dimensions in vitro and in silico shows that the chain flexibility increases with DM and at acidic pH, whereas hydrogen bonding is the responsible thermodynamic driving force for cluster formation. High methyl pectins create structures of lower fractal dimension with less efficient packing. This work unveils pectin conformations covering most of its industrially and biologically relevant environments, enabling rational design of advanced biomaterials based on pectin.

Structural modifications of the salivary conditioning film upon exposure to sodium bicarbonate: implications for oral lubrication and mouthfeel.

The salivary conditioning film (SCF) that forms on all surfaces in the mouth plays a key role in lubricating the oral cavity. As this film acts as an interface between tongue, enamel and oral mucosa, it is likely that any perturbations to its structure could potentially lead to a change in mouthfeel perception. This is often experienced after exposure to oral hygiene products. For example, consumers that use dentifrice that contain a high concentration of sodium bicarbonate (SB) often report a clean mouth feel after use; an attribute that is clearly desirable for oral hygiene products. However, the mechanisms by which SB interacts with the SCF to alter lubrication in the mouth is unknown. Therefore, saliva and the SCF was exposed to high ionic strength and alkaline solutions to elucidate whether the interactions observed were a direct result of SB, its high alkalinity or its ionic strength. Characteristics including bulk viscosity of saliva and the viscoelasticity of the interfacial salivary films that form at both the air/saliva and hydroxyapatite/saliva interfaces were tested. It was hypothesised that SB interacts with the SCF in two ways. Firstly, the ionic strength of SB shields electrostatic charges of salivary proteins, thus preventing protein crosslinking within the film and secondly; the alkaline pH (≈8.3) of SB reduces the gel-like structure of mucins present in the pellicle by disrupting disulphide bridging of the mucins via the ionization of their cysteine's thiol group, which has an isoelectric point of ≈8.3.

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.

Interfacial & colloidal aspects of lipid digestion.

Amongst the main issues challenging the food manufacturing sector, health and nutrition are becoming increasingly important. Global concerns such as obesity, the ageing population and food security will have to be addressed. Food security is not just about assuring food supply, but is also about optimising nutritional delivery from the food that is available [1]. Therefore one challenge is to optimise the health benefits from the lipids and lipid soluble nutrients. Colloid scientists have an affinity for lipids because they are water insoluble, however this presents a challenge to the digestive system, which has to convert them to structures that are less insoluble so they are available for uptake. Despite this, the human digestive system is remarkably effective at digesting and absorbing most lipids. This is primarily driven through maximising energy intake, as lipids possess the highest calorific value, which was a survival trait to survive times of famine, but is now an underlying cause of obesity in developed countries with high food availability. The critical region here is the lipid-water interface, where the key reactions take place to solubilise lipids and lipid soluble nutrients. Digestive lipases have to adsorb to the oil water interface in order to hydrolyse triacylglycerols into fatty acids and mono glycerides, which accumulate at the interface [2], and inhibit lipase activity. Pancreatic lipase, which is responsible for the majority of lipid hydrolysis, also requires the action of bile salts and colipase to function effectively. Bile salts both aid the adsorption of co-lipase and lipase, and help solubilise the lipolysis products which have accumulated at the interface, into mixed micelles composing bile salts and a range of other lipids, to facilitate transport to the gut mucosal surface prior to uptake and absorption. The process can be affected by the lipid type, as shorter chain, fatty acids are more easily absorbed, whereas the uptake of longer chain fatty acids, particularly the very long chain n-3 fatty acids from fish oils are dependent on source and so may depend on food microstructure for optimal uptake [3]. The uptake of some poorly water soluble nutrients are enhanced by the presence of lipids, but the mechanisms are not clear. In addition, controlling the digestion of lipids can be beneficial as slower release of lipids into the bloodstream can reduce risk of cardiovascular disease, and can promote gut feedback processes that reduce appetite. This presents an opportunity to colloid and interfacial science, as there are many unanswered questions regarding the specific physicochemical mechanisms underlying the process of lipid digestion and uptake. I will review our current knowledge of lipid digestion and present examples of how fundamental research in colloidal and interface science is beginning to address these issues. These include the adsorption behaviour of physiological surfactants such as bile salts; interfacial processes by which different polar lipids can influence lipolysis; and the effect of emulsion based delivery systems on cellular uptake of lipid soluble nutrients. A fundamental understanding of these processes is required if we are to develop intelligent design strategies for foods that will deliver optimal nutrition and improved health benefits in order to address the global challenges facing the food sector in the future.

Comparison of the orogenic displacement of sodium caseinate with the caseins from the air-water interface by nonionic surfactants.

Displacement of sodium caseinate from the air-water interface by nonionic surfactants Tween 20 and Tween 60 was observed by atomic force microscopy (AFM). The interfacial structure was sampled by Langmuir-Blodgett deposition onto freshly cleaved mica substrates. Protein displacement occurred through an orogenic mechanism: it involved the nucleation and growth of surfactant domains within the protein network, followed by failure of the protein network. The surface pressure at which failure of the protein network occurred was essentially independent of the type of surfactant. The major component of sodium caseinate is beta-casein, and previous studies at the air-water interface have shown that beta-casein networks are weak, failing at surface pressures below that observed for sodium caseinate. The other components of sodium caseinate are alpha(s)- and kappa-caseins. Studies of the displacement of alpha(s)-caseins from air-water interfaces show that these proteins also form weak networks that fail at surface pressures below that observed for sodium caseinate. However, kappa-casein was found to form strong networks that resisted displacement and failed at surface pressures comparable to those observed for sodium caseinate. The AFM images of the displacement suggest that, despite kappa-casein being a minor component, it dominates the failure of sodium caseinate networks: alpha(s)-casein and beta-casein are preferentially desorbed at lower surface pressures, allowing the residual kappa-casein to control the breakdown of the sodium caseinate network at higher surface pressures.

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.

Effect of hydrocarbon chain and pH on structural and topographical characteristics of phospholipid monolayers.

Structural characteristics (structure, elasticity, topography, and film thickness) of dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylcholine (DOPC) monolayers were determined at the air-water interface at 20 degrees C and pH values of 5, 7, and 9 by means of surface pressure (pi)-area (A) isotherms combined with Brewster angle microscopy (BAM) and atomic force microscopy (AFM). From the pi-A isotherms and the monolayer elasticity, we deduced that, during compression, DPPC monolayers present a structural polymorphism at the air-water interface, with the homogeneous liquid-expanded (LE) structure; the liquid-condensed structure (LC) showing film anisotropy and DPPC domains with heterogeneous structures; and, finally, a homogeneous structure when the close-packed film molecules were in the solid (S) structure at higher surface pressures. However, DOPC monolayers had a liquid-expanded (LE) structure under all experimental conditions, a consequence of weak molecular interactions because of the double bond of the hydrocarbon chain. DPPC and DOPC monolayer structures are practically the same at pH values of 5 and 7, but a more expanded structure in the monolayer with a lower elasticity was observed at pH 9. BAM and AFM images corroborate, at the microscopic and nanoscopic levels, respectively, the same structural polymorphism deduced from the pi-A isotherm for DPPC and the homogeneous structure for DOPC monolayers as a function of surface pressure and the aqueous-phase pH. The results also corroborate that the structural characteristics and topography of phospholipids (DPPC and DOPC) are highly dependent on the presence of a double bond in the hydrocarbon chain.

Atomic force microscopy of emulsion droplets: probing droplet-droplet interactions.

A method has been developed for attaching oil (tetradecane) droplets to the end of an atomic force microscopy (AFM) cantilever and for immobilizing droplets on a glass substrate. This approach has permitted the monitoring of droplet-droplet interactions in aqueous solution as a function of interdroplet separation. Coating the droplet surfaces with added proteins or surfactants has allowed the production of model emulsions. We demonstrate that AFM measurements of droplet deformability are sensitive to interfacial rheology by modifying the interfacial film on a pair of droplets in situ. For droplets coated with the anionic surfactant sodium dodecyl sulfate, screening of the double layer has been found to facilitate coalescence. Direct imaging of the droplets has revealed the presence of regularly spaced concentric rings on the droplet surfaces. Careful experimental studies suggest that these structures may be imaging artifacts and are not perturbations of the droplet surface determined by the composition of the interface.

Role of beer lipid-binding proteins in preventing lipid destabilization of foam.

The negative effect of fatty acids on the foam stability of beer has been assessed. Long-chain fatty acids are far more damaging than short-chain fatty acids on the foam stability of beer at the concentrations employed. Polypeptides have been isolated from an all malt beer by hydrophobic interaction chromatography. Using this technique five groups of polypeptides were isolated, group 1 being the least hydrophobic and group 5 the most hydrophobic, all of which exhibited similar polypeptide compositions by SDS-PAGE. All five hydrophobic polypeptide groups bound [(14)C]linoleic acid; however, group 5, the most hydrophobic group, bound the most linoleic acid. Groups 1 and 5 were titrated with cis-parinaric acid (CPA) to produce binding curves, which were compared with a binding curve obtained for bovine serum albumin (BSA). Groups 1 and 5 both produced binding curves that saturated at approximately 5.5 microM and 4 microM CPA and had association constants (K(a)) of 6.27 x 10(7) and 1.62 x 10(7) M(-1), respectively. In comparison, BSA produced a binding curve that saturated at 6 microM CPA and had a K(a) of 3.95 x 10(7) M(-1). Further investigation has shown that group 1 is pH sensitive and group 5 pH insensitive with respect to lipid binding. The lipid-binding activity of group 5 was also shown to be unaffected by ethanol concentration. Linoleic acid (5 microM) when added to beer resulted in unstable foam. Group 5 was added to the lipid-damaged beer and was shown to restore the foam stability to values that were obtained for the control beer. It has therefore been demonstrated that proteins isolated from beer have a lipid-binding capacity and that they can convey a degree of protection against lipid-induced foam destabilization.

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.

Adsorbed protein secondary and tertiary structures by circular dichroism and infrared spectroscopy with refractive index matched emulsions.

The secondary structure of protein adsorbed at the emulsion interface has been studied in refractive index matched emulsions using the techniques of circular dichroism (CD) and Fourier transform infrared spectroscopy. Bovine serum albumin (BSA) and bovine beta-lactoglobulin (betalg) stabilized emulsions were studied, and the refractive index was altered by the addition of glycerol or polyethylene glycol. The effect of additive on the solution and adsorbed protein structure in addition to the effect of adsorption time was considered. Both adsorption and glycerol addition alter protein secondary structure; however, the majority of secondary structure remains. Small changes are observed in the secondary structure of adsorbed protein with time. Near-ultraviolet CD studies showed the effect of glycerol and adsorption on the aromatic groups. BSA showed small changes both upon the addition of glycerol to protein in solution and upon adsorption. betalg showed slightly larger changes upon the addition of glycerol to protein in solution and a larger change upon adsorption.

Molecular mobility in the monolayers of foam films stabilized by porcine lung surfactant.

Certain physical properties of a range of foam film types that are believed to exist in vivo in the lung have been investigated. The contribution of different lung surfactant components found in porcine lung surfactant to molecular surface diffusion in the plane of foam films has been investigated for the first time. The influence of the type and thickness of black foam films, temperature, electrolyte concentration, and extract composition on surface diffusion has been studied using the fluorescence recovery after photobleaching technique. Fluorescent phospholipid probe molecules in foam films stabilized by porcine lung surfactant samples or their hydrophobic extracts consisting of surfactant lipids and hydrophobic lung surfactant proteins, SP-B and SP-C, exhibited more rapid diffusion than observed in films of its principal lipid component alone, L-alpha-phosphatidylcholine dipalmitoyl. This effect appears to be due to contributions from minor lipid components present in the total surfactant lipid extracts. The minor lipid components influence the surface diffusion in foam films both by their negative charge and by lowering the phase transition temperature of lung surfactant samples. In contrast, the presence of high concentrations of the hydrophillic surfactant protein A (SP-A) and non-lung-surfactant proteins in the sample reduced the diffusion coefficient (D) of the lipid analog in the adsorbed layer of the films. Hysteresis behavior of D was observed during temperature cycling, with the cooling curve lying above the heating curve. However, in cases where some surface molecular aggregation and surface heterogeneity were observed during cooling, the films became more rigid and molecules at the interfaces became immobilized. The thickness, size, capillary pressure, configuration, and composition of foam films of lung surfactant prepared in vitro support their investigation as realistic structural analogs of the surface films that exist in vivo in the lung. Compared to other models currently in use, foam films provide new opportunities for studying the properties and function of physiologically important alveolar surface films.